AU706417B2 - Method and reagent for inhibiting the expression of disease related genes - Google Patents

Description

0 95'2322.

PC 1 IIS1101lo 1 METl D AND REA'GENT F"R I.11 h I TH i lXPRESIoI DISEASE RELATED GENE kgO of th invention This invention relates to reagents useful as inhibitors of gene expression relating to diseases such as inflammatory or autoimmune disorders, chronic myelogenous leukemia, or respiratory tract illness.

Summary of the lnVoention The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting the expression of disease related genes, ICAM-1, IL-5, relA, TNF-o., p210 bcr-abl, and respiratory syncytlal virus genes. Such ribozymes can be used in a method for treatment of diseases caused by the expression of these genes in man and other animals, including other primates.

Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro, Kim et al,, 84 Proc, Natl, Acad, Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al,, 17 Nucleic Acids Research 1371, 1989, Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table 1 summarizes some of the characteristics of these ribozymes, Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA, Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.

After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymnatic nature of a rihozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply hinds to a nucleic acid target to block its translation) since thile effective concentration of riboyvine necessary to etfect a therapeutic treatment is lower than that of anll antisense oligonucleotide. T1he a advantage reflects the ability of thile riozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, tilhe ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is lo caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site. With their catalytic activity and increased site specificity, ribozymes represent more potent and sale therapeutic molecules than antisense oligonucleotides.

The present invention relates to 5'-C-alkylnucleotides, 2'deoxy-2' -alkylnucleotides and 3' and/or 5'-dihalophosphonate-substituted nucleotides which are useful in oligonucleotides capable of enzymatic cleavage of RNA or single-stranded D)NA, and also 20 as antisense oligonucleotides.

In one embodiment, the invention provides an enzymatic nucleic acid molecule comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotid. 2'deoxy-2'-alkylnucleotide, 5'-deoxy-5'-dihalo-methyl nucleotide, 5'-deoxy-5'-dil1uoromethylnucleotide, 3'-deoxy-3'-.dihalo-methylnucleotide, and 5',3'-dideoxy-5'.3'bis(dilhalo)-methylphosphonate.

In another embodiment, the invention provides method for producing an enzymatic Snucleic acid molecule having activity to cleave an RNA or single-stranded D)NA *molecule, comprising the step of forming said enzymatic molecule with at least one nucleotide having an alkyl group at its 5'-position or 2'-position.

This invention also relates to ribozymes, or enzymatic RNA molecules, directed to Scleave RNA species encoding ICAM-1, IL-5, rielA TNI-u., p2 1 0ber-ahl, or RSV proteins.

i In particular, applicant describes the selection and ilunction of ribozymes capable of cleaving these RNAs and their use to reduce levels of' ICAM-1 IL-5, relA, TNF-P p210b)Cr-abl or RSV proteins in various tissues to treat the diseases discussed herein, Such ribozymes are also useful for diagnostic uses.

Applicant indicates that these ribozymes are able to inhibit expression of ICAM-1, rel A, TNF-a, p 2 O0bcr-abl, or RSV genes and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in thile art, will find that it is clear from the examples described that other ribozymes that cleave target

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ICAM-1 IL-5, rel A, TNF-(A, 20)rai or RSV enICOdin~g niRNAs may lie readily dleSigned an1d ae0 withinl the inlvention.- Th'lese emliically or enzymatical ly synthesized RN A molecules Contain Subst rate binding domains that bind to accessible regions of' their target mRNAs. The RNA Molecules also contain domains that cataly/e the

~-II-

J'2322? 31 I11')5'll 3 cleavage of RNA, Upon binding, the ribozymes cleave the target encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.

By "gene" is meant to refer to either the protein coding regions of the cognate mRNA, or any regulatory regions in the RNA which regulate synthesis of the protein or stability of the mRNA; the term also refers to those regions of an mRNA which encode the ORF of a cognate polypeptide product, and the proviral genome.

By "enzymatic RNA molecule" it is meant an RNA molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave RNA in that target. That is, the enzymatic RNA molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule.

This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur.

One hundred percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention, By "equivalent" RNA to a virus is meant to include those naturally occurring viral encoded RNA molecules associated with viral caused diseases in various animals, including humans, cats, simians, and other primates, These viral or viralencoded RNAs have similar structures and equivalent genes to each other.

By "complementarity" it is meant a nucleaic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson- Crick or other non-traditional types (for examplke, Hoogsteen type) of basepaired interactions.

In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in associateion with an RNA guide sequence) or Neurospora VS RNA, Examples of such hammerhead motifs are described by Rossi et al,, 1992, Aids Research and Human Retroviruses 8,183, of hairpin motifs by Hampel and Tritz, 1989 Biochemistry, 28, 4929, EP 0360257 and Hampel et al,, 1990, Nucleic Acids Res. 18,299 and an example of the hepatitis delta virus motif is described by Perotta and Been, 1992 Biochemistry, 31 16 of the RNaseP motif by Guerrier-Takada et al., 1983 Cell, 35 849,

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~C V 0 9)523225 P(I 4t Neurospora VS RNA ribozyme motif is described by Collins (Seville and Collins, 1990 el 61, 685-696; Saville and Collins, 1991 Proc. Natl. Ac Sci.. JSA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795- 2799 Guo and Collins, 1995 EMBOJ., 14, 368) and of the Group I intron by Cech et al,, U.S. Patent 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it has nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target I CAM-1, IL-5, reLA, TNF-a, p210 bcr-abl or RSV proteins) encoding mRNA such that specific treatment of a disease or condition can be provided with either one or several enzymatic nucleic acids, Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required., Alternatively, the ribozymes can be expressed from vectors that are delivered to specific cells, By "vectors" is meant any nucleic acid and/or viral-based technique used to deliver a desired nucleic acid.

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive, In this invention small enzymatic nucleic acid motifs of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structrure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters Scanion, K.J. et al., 1991, Proc.

Natl, Acad. Sci,. USA, 88, 10591-5; Kashani-Sabet, et al,,1992, Antisense Res. Dev., 2, 3-15; Dropoulic, et al,, 1992, J. Virol, 66, 1432- 41; Weerasinghe, et al., 191, J. Virol, 65, 5531-4; Ojwang, et al., 1992, Proc, Natl. Acad. Sci., USA. 89 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver, et al,, 1990 Science, 247, 1222- 1225). Those skilled in the art would realize that any ribozyme can be I 5/23225 IN( I 1lli5 (0l151o expressed in eukaryotic cells from the appropriate DNA or RNA vector. The activity of such ribozymes can be augmented by their release from t;,e primary transcript by a second ribozyme (Draper et al., PCT WO93123569, and Sullivan et al., PCT W094/02595, both hereby incorporated in their totality by reference herein; Ohkawa, et al., 1992, Nucleic Acids Svmp.

Ser, 27, 15-6; Taira, K. et al., Nucleic Acids Res., 19, 5125-30; Ventura, M., et al,, 1993, Nucleic Acids Res., 21, 3249-55, Chowrira et al., 1994 J. B3i Chem., 269, 25856 By "inhibit" is meant that the activity or level of ICAM-1,Rel A, TNF-a, p210 b c r a b l or RSV encoding mRNA is reduced below that observed in the absense of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.

Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of ICAM-1, IL-5, Rel A, TNF-a, p 2 10bcr-abl or RSV protein or activity in a cell or tissue. By "related" is meant that the inhibition of ICAM-1, IL-5, Rel A, TNF-a, p21obcrabl or RSV mRNA translation, and thus reduction in the level of, ICAM-1, IL-5, Rel A, TNF-a, p210 b c r a b l or RSV proteins will relieve to some extent the symptoms of the disease or condition, Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.

The RNA or RNA complexes can be locally administered to relevant tissues through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers, In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 2,3,6-9, 11, 13, 15-23, 27, 28, 31, 33, 34, 36 and 37.

Examples of such ribozymes are shown in Tables 4-8, 10, 12, 14-16, 19-22, 24, 26-28, 30, 32, 34 and 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.

No 0 95 2322t NC I 11195 il ,i Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity.

For example, stem-loop II sequence of hammerhead ribozymes listed in the above identified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two basepaired stem structure can form, Similarly, stem-loop IV sequence of hairpin ribozymes listed in the above idei:ified Tables can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequence listed in the above identified Tables may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables, In another aspect of the invention, ribozymes that cleave target molecules and inhibit ICAM-1, IL-5, Rel A, TNF-c, p210bcr-abl or RSV gene expression are expressed from transcription units inserted into DNA, RNA, or viral vectors. Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA or RNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc,) present nearby, Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl.

Acad, Sci, USA, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21 2867-72; Lieber et al,, 1993 Methods Enzymol,, 217, 47-66; Zhou et al,, 1990 Mol. Cell. Biol,, 10, 4529-37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad, Sci. USA, 90, 6340-4; L'Huiller et al,, 1992 EMBO J. 11, 4411-8; Lisziewicz et al., 1993 Proc. Natl.

Acad, Sci, 90 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors 0 ~bBI-~ 0 95,23225 P(I 1 1139111i% 7 (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors).

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description Of The Preferred Embodiments The drawings will first briefly be described.

Drawings: Figure 1 is a diagrammatic representation of the hammerhead ribozyme domain known in the art. Stem II can be 2 base-pair long.

Figure 2(a) is a diagrammatic representation of the hammerhead ribozyme domain known in the art; Figure 2(b) is a diagrammatic representation of the hammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into a substrate and enzyme portion; Figure 2(c) is a similar diagram showing the hammerhead divided by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two portions; and Figure 2(d) is a similar diagram showing the hammerhead divided by Jeffries and Symons (1989, Nucl, Acids. Res,, 17, 1371-1371) into two portions.

Figure 3 is a diagrammatic representation of the general structure of a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs n is 1,2,3 or 4) and helix 5 can be optionally provided of length 2 or more bases (preferably 3-20 bases, ie,, m is from 1-20 or more), Helix 2 and helix 5 may be covalently linked by one or more bases r is 1 base).

Helix 1, 4 or 5 may also be extended by 2 or more base pairs 4-20 base pairs) to stabilize the ribozyme structure, and preferably is a protein binding site, In each instance, each N and N' independently is any normal or modified base and each dash represents a potential base-pairing interaction. These nucleotides may be modified at the sugar, base or phosphate, Complete base-pairing is not required in the helices, but is preferred, Helix 1 and 4 can be of any size o and p is each independently from 0 to any number, e.g. 20) as long as some base-pairing is maintained, Essential bases are shown as specific bases in the structure, but those in the art will recognize that one or more may be C IIII~ICIII- \WO '95/23225 'T/I lniUii0015 8 modified chemically (abasic, base, sugar and/or phosphate modifications) or replaced with another base without significant effect. Heiix 4 can be formed from two separate molecules, without a connecting loop, The connecting loop when present may be a ribonucleotide with or without modifications to its base, sugar or phosphate, is 2 bases, The connecting loop can also be replaced with a non-nucleotide linker molecule. H refers to bases A, U, or C, Y refers to pyrimidine bases, refers to a covalent bond, Figure 4 is a representation of the general structure of the hepatitis delta virus ribozyme domain known in the art, Figure 5 is a representation of the general structure of the selfcleaving VS RNA ribozyme domain.

Figure 6 is a diagrammatic representation of the genetic map of RSV strain A2.

Figure 7 is a diagrammatic representation of the solid-phase synthesis of RNA, Figure 8 is a diagrammatic representation of exocyclic amino protecting groups for nucleic acid synthesis, Figure 9 is a diagrammatic representation of the deprotection of RNA.

Figure 10 is a graphical representation of the cleavage of an RNA substrate by ribozymes synthesized, deprotected and purified using the improved methods described herein.

Figure 11 is a schematic representation of a two pot deprotection protocol. Base deprotection is carried out with aqueous methyl amine at °C for 10 min, The sample is dried in a speed-vac for 2-24 hours depending on the scale of RNA synthesis, Silyl protecting group at the 2'hydroxyl position is removed by treating the sample with 1,4 M anhydrous HF at 650C for 1.5 hours, Figure 12 is a schematic representation of a one pot deprotection of RNA synthesized using RNA phosphoramidite chemistry. Anhydrous methyl amine is used to deprotect bases at 650C for 15 min. The sample is allowed to cool for 10 min before adding TEA-3HF reagent, to the same 95/23225 P("I I 1119'51) pot, to remove protecting groups at the 2'-hydroxyl position. The deprotection is carried out for 1.5 hours.

Figs. 13a b is a HPLC profile of a 36 nt long ribozyme, targeted to site B, The RNA is deprotected using either the two pot or the one pot deprotection protocol, The peaks corresponding to full-length RNA is indicated. The sequence for site B is CCUGGGCCAGGGAUUA

AUGGAGAUGCCCACU.

Figure 14 is a graph comparing RNA cleavage activity of ribozymes deprotecte. two pot vs one pot deprotection protocols.

Figure 15 is a schematic representation of an improved method of synthesizing RNA containing phosphorothioate linkages.

Figure 16 shows RNA cleavage reaction catalyzed by ribozymes containing phosphorothioate linkages Hammerhead ribozyme targeted to site C is synthesized such that 4 nts at the 5' end contain phosphorothioate linkages. P=O refers to ribozyme without phosphorothioate linkages. P=S refers to ribozyme with phosphorothioate linkages. The sequence for site C is UCAUUUUGGCCAUCUC UUCCUUCAGGCGUGG, Figure 17 is a schematic representation of synthesis of 2'-Nphtalimido-nucleoside phosphoramidite.

Figure 18 is a diagrammatic representation of a prior art method for the solid-phase synthesis of RNA using silyl ethers, and the method of this invention using SEM as a 2'-protecting group, Figure 19 is a diagrammatic representation of the synthesis of 2'- SEM-protected nucleosides and phosphoramidites useful for the synthesis of RNA. B is any nucleotide base as exemplified in the Figure, P is purine and I is inosine. Standard abbreviations are used throughout this application, well known to those in the art.

Figure 20 is a diagrammatic representation of a prior art method for deprotection of RNA using TBDMS protection of the 2'-hydroxyl group, Figure 21 is a diagrammatic representation of the deprotection of RNA having SEM protection of the 2'-hydroxyl group.

SUBSTITUTE SHEET (RULE 256 4 232.1;2 I Il\llI 95015f ;igure 22 is a representation of an HPLC chromatogram of a fully deprotected 10-mer of uridylic acid, Figs. 23 25 are diagrammatic representations of hammerhead, hairpin or hepatitis delta virus ribozyme containing self-processing RNA transcript. Solid arrows indicate self-processing sites, Boxes indicate the sites of nucleotide substitution. Solid lines are drawn to show the binding sites of primers used in a primer-extension assay. Lower case letters indicate vector sequence present in the RNA when transcribed from a Hindlll-linearized plasmid. (23) HH Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hammerhead ribozyme. The structure of the hammerhead ribozyme is based on phylogenetic and mutational analysis (reviewed by Symons, 1992 supra).

The trans ribozyme domain extends from nucleotide 1 through 49. After 3'end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (UC at positions 50 and 51) at its 3' end, The 3' processing ribozyme is comprised of nucleotides 44 through 96. Roman numerals I, II and III, indicate the three helices that contribute to the structure of the 3' cis-acting hammerhead ribozyme (Hertel et al., 1992 Nucleic Acids Res, 20, 3252), Substitution of G70 and A71 to U and G respectively, inactivates the hammerhead ribozyme (Ruffner et al,, 1990 Biochemistry 29, 10695) and generates the HH(mutant) construct, (24) HP Cassette, transcript containing the hammerhead trans-acting ribozyme linked to a 3' cis-acting hairpin ribozyme. The structure of the hairpin ribozyme is based on phylogenetic and mutational analysis (Berzal-Herranz et al,, 1993 EMBO. J 12, 2567). The trans-ribozyme domain extends from nucleotide 1 through 49. After 3'-end processing, the trans-ribozyme contains 5 non-ribozyme nucleotides (UGGCA at positions 50 to 54) at its 3' end, The 3' cis-acting ribozyme is comprised of nucleotides 50 through 115. The transcript named HP(GU) was constructed with a potential wobble base pair between G52 and U77; HP(GC) has a Watson-Crick base pair between G52 and C77. A shortened helix 1 (5 base pairs) and a stable tetraloop (GAAA) at the end of helix 1 was used to connect the substrate with the catalytic domain of the hairpin ribozyme (Feldstein Bruening, 1993 Nucleic Acids Res. 21, 1991; Altschuler et al,, 1992 supra). (25) HDV Cassette, transcript containing the trans-acting hammerhead ribozyme linked to a 3' cis-acting hepatitis delta virus (HDV) ribozyme. The secondary structure of the HDV ribozyme is as proposed by Been and LI I WO 9')523225 P CI ISi 0IK SIS 11 coworkers (Been et al,, 1992 Biochemistry 31, 118.1 .lo trans-ribozyme domain extends from nucleotides 1 through 48. Ater 3'-end processing, the trans-ribozyme contains 2 non-ribozyme nucleotides (AA at positions 49 to 50) at its 3' end. The 3' cis-acting HDV ribozyme is comprised of nucleotides 50 through 114, Roman numerals I, II, 111 IV, indicate the location of four helices within the 3' cis-acting HDV ribozymo (Perrota Been, 1991 Nature 350, 434), The AHDV transcript contains a 31 nucleotide deletion in the HDV portion of the transcript (nucleotides 84 through 115 deleted).

Fig, 26 is a schematic representation of a plasmid containing the insert encoding self-processing cassette. The figure is not drawn to scale.

Fig, 27 demonstrates the effect of 3' flanking sequences on RNA selfprocessing in vitro, H, Plasmid templates linearized with Hindlll restriction enzyme. Transcripts from H templates contain four non-ribozyme nucleotides at the 3' end, N, Plasmid templates linearized with Ndel restriction enzyme. Transcripts from N templates contain 220 nonribozyme nucleotides at the 3' end, R, Plasmid templates linearized with Rcal restriction enzyme. Transcripts from R templates contain 450 nonribozyme nucleotides at the 3' end, Fig. 28 shows the effect of 3' flanking sequences on the transcleavage reaction catalyzed by a hammerhead ribozyme, A 622 nt internally-labeled RNA (<10 nM) was incubated with ribozyme (1000 nM) under single turn-over conditions (Herschlag and Cech, 1990 Biochemistry 29, 10159). HH+2, HH+37, and HH+52 are trans-acting ribozymes produced by transcription from the HH, AHDV, and HH(mutant) constructs, respectively, and that contain 2, 37 and 52 extra nucleotides on the 3' end, The plot of the fraction of uncleaved substrate versus time was fit to a double exponential curve using the KaleidaGraph graphing program (Synergy Software, Reading, PA). A double exponential curve fit was used because the data points did not fall on a single exponential curve, presumably due to varying conformers of ribozyme and/or substrate RNA.

Fig. 29 shows RNA self-processing in OST7-1 cells. In vitro lanes contain full-length, unprocessed transcripts that were added to cellular lysates prior to RNA extraction. These RNAs were either pre-incubated with MgCI2 or with DEPC-treated water prior to being hybridized

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I \O 95 23225 A 11(5 I 15(1 1? with 5' end-labeled primers. Cellular lanes contain total cellular RNA from cells transfected with one of the four self-processing constructs. Cellular RNA are probed for ribozyme expression using a sequence specific primorextension assay. Solid arrows indicate the location of primer extension bands corresponding to Full-Length RNA and 3' Cleavage Products, Figs, 30,31 are diagrammatic representations of self-processing cassettes that will release trans-acting ribozymes with defined, stable stemloop structures at the 5' and the 3' end following self-processing. shows various permutations of a hammerhead self-processing cassette. 31, shows various permutations of a hairpin self-processing cassette.

Figs. 32a-b Schematic representation of RNA polymerse III promoter structure, Arrow indicates the transcription start site and the direction of coding region, A, B and C, refer to consensus A, B and C box promoter sequences, I, refers to intermediate cis-acting promoter sequence. PSE, refers to proximal sequence element. DSE, refers to distal sequence element, ATF, refers to activating transcription factor binding element. refers to cis-acting sequence element that has not been fully characterized, EBER, Epstein-Barr-virus-encoded-RNA. TATA is a box well known in the art, Figs. 33a-e Sequence of the primary tRNAlmet and A3-5 transcripts.

The A and B box are internal promoter regions necessary for pol III transcription, Arrows indicate the sites of endogenous tRNA processing.

The A3-5 transcript is a truncated version of tRNA wherein the sequence 3' of B box has been deleted (Adeniyi-Jones et al., 1984 supra). This modification renders the A 3-5 RNA resistant to endogenous tRNA processing, Figure 34. Schematic representation of RNA structural motifs inserted into the A3-5 RNA. A3-5/HHI- a hammerhead (HHI) ribozyme was cloned at the 3' region of A3-5 RNA; S3- a stable stem-loop structure was incorporated at the 3' end of the A3-5/HHI chimera; S5- stable stem-loop structures were incorporated at the 5' and the 3' ends of A3-5/HHI ribozyme chimera; S35- sequence at the 3' end of the A3-5/HHI ribozymo chimera was altered to enable duplex formation between the 5' end and a complementary 3' region of the same RNA; S35Plus- in addition to structural alterations of S35, sequences were altered to facilitate additional SUBSTITUTE SHEET (RULE 26) 1 4 1 111 95/23225 11( 1i I 1 ,19 1 1?( 13 duplex formation within the non-ribozyme sequence of the chimera, Figures 35 and 36. Northern analysis to quantitate ribozyme expression in T cell lines transduced with A3-5 vectors, 35) A3-5/HHI and its variants were cloned individually into the DC retroviral vector (Sullenger et al,, 1990 supra). Northern analysis of ribozyme chimeras expressed in MT-2 cells was performed. Total RNA was isolated from cells (Chomczynski Sacchi, 1987 Analytical Biochemistry 162, 156-159), and transduced with various constructs described in Fig. 34, Northern analysis was carried out using standard protocols (Curr, Protocols Mol, Biol. 1992, ed, Ausubel et al,, Wiley Sons, NY). Nomenclature is same as in Figure 34, This assay measures the level of expression from the type 2 pol III promoter. 36) Expression of S35 constructs in MT2 cells, S35 (+ribozyme), construct containing HHI ribozyme. S35 (-ribozyme), S35 construct containing no ribozyme, Figure 37, Ribozyme activity in total RNA extracted from transduced MT-2 cells, Total RNA was isolated from cells transduced with constructs described in Figs, 35 and 36 In a standard ribozyme cleavage reaction, 5 pg total RNA and trace amounts of 5' terminus-labeled ribozyme target RNA were denatured separately by heating to 90C0 for 2 min in the presence of 50 mM Tris-HCI, pH 7.5 and 10 mM MgCl 2 RNAs were renatured by cooling the reaction mixture to 37°C for 10-15 min, Cleavage reaction was initiated by mixing the labeled substrate RNA and total cellular RNA at 370C, The reaction was allowed to proceed for 18h, following which the samples were resolved on a 20 urea-polyacrylamide gel, Bands were visualized by autoradiography.

Figures 38 and 39. Ribozyme expression and activity levels in transduced clonal CEM cell lines, 38) Northern analysis of transduced clonal CEM cell lines. Standard curve was generated by spiking known concentrations of in vitro transcribed S5 RNA into total cellular RNA isolated from non-transduced CEM cells, Pool, contains RNA from pooled cells transduced with S35 construct. Pool (-G418 for 3 Mo), contains RNA from pooled cells that were initially selected for resistance to G418 and then grown in the absence of G418 for 3 months. Lanes A through N contain RNA from individual clones that were generated from the pooled cells transduced with S35 construct, tRNAimet, refers to the 0 95/23225 'V(J)PCI 1N O I$5: ()I 14 endogenous tRNA. S35, refers to the position of the ribozyme band. M, marker lane. 39) Activity levels in S35-transduced clonal CEM cell lines.

RNA isolation and cleavage reactions were as described in Fig.37.

Nomenclature is same as in Figs. 35 and 36 except, S, 5' terminus-labeled substrate RNA. P, 8 nt 5' terminus-labeled ribozyme-mediated RNA cleavage product.

Figures 40 and 41 are proposed secondary structures of S35 and containing a desired RNA (HHI), respectively, The position of HHI ribozyme is indicated in figure 41. Intramolecular stem refers to the stem structure formed due to an intramolecular base-paired interaction between the 3' sequence and the complementary 5' terminus. The length of the stem ranges from 15-16 base-pairs. Location of the A and the B boxes are shown, Figures 42 and 43 are proposed secondary structures of S35 plus and S35 plus containing HHI ribozyme.

Figures 44, 45, 46 and 47 are the nucleotide base sequences of S35 Plus, and HHIS35 Plus respectively.

Figs. 48a-b is a general formula for pol III RNA of this invention.

Figure 49 is a digrammatic representation of 5T construct. In this construct the desired RNA is located 3' of the intramolecular stem.

Figures 50 and 51 contain proposed secondary structures of construct alone and 5T contruct containing a desired RNA (HHI ribozyme) respectively.

Figure 52 is a diagrammatic representation of TRZ-tRNA chimeras, The site of desired RNA insertion is indicated.

Figure 53 shows the general structure of HHITRZ-A ribozyme chimera, A hammerhead ribozyme targeted to site I is inserted into the stem II region of TRZ-tRNA chimera.

Figure 54 shows the general structure of HPITRZ-A ribozyme chimera, A hairpin ribozyme targeted to site I is cloned into the indicated region of TRZ-tRNA chimera.

SUBSTITUTE SHEET (RULE 26) q_ W\O 95/23225 P( 'l/IW'0i5il(156 Figure 55 shows a comparison of RNA cleavage activity of HHITRZ-A, HHITRZ-B and a chemically synthesized HHI hammerhead rbozymes.

Figure 56 shows expression of ribozymes in T cell lines that are stably transduced with viral vectors, M, markers; lane 1, non-transduced CEM cells; lanes 2 and 3, MT2 and CEM cells transduced with retroviral vectors; lanes 4 and 5, MT2 and CEM cells transduced with AAV vectors, Figs, 57a-b Schematic diagram of adeno-associated virus and adenovirues vectors for ribozyme delivery, Both vectors utilize one or more ribozyme encoding transcription units (RZ) based on RNA polymerase II or RNA polymerase III promoters, A. Diagram of an AAV-based vector containing minimal AAV sequences comprising the inverted terminal repeats (ITR) at each end of the vector genome, an optional selectable marker (Neo) driven by an exogenous promoter (Pro), a ribozyme transcription unit, and sufficient additional sequences (stuffer) to maintain a vector length suitable for efficient packaging. B. Diagram of ribozyme expressing adenovirus vectors containing deletions of one or more wild type adenoviorus coding regions (cross-hatched boxes marked as El, plX, E3, and E4), and insertion of the ribozyme transcription unit at any or several of those regions of deletions, Fig, 58 is a graph showing the effect of arm length variation on the activity of ligated hammerhead (HH) ribozymes. Nomenclature 5/5, 6/6, 7/7, 8/8 and so on refers to the number of base-pairs being formed between the ribozyme and the target, For example, 5/8 means that the HH ribozyme forms 5 bp on the 5' side and 8 bp on the 3' side of the cleavage site for a total of 13 bp. -AG refers to the free energy of binding calculated for basepaired interactions between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Ann, Rev. Biophys. Chem. 17, 167). RPI A is a HH ribozyme with 6/6 binding arms.

Figs, 59 and 60 and 61 show cleavage of long substrate (622 nt) by ligated HH ribozymes.

Fig. 62 is a diagrammatic representation of a hammerhead ribozyme (HH-H) targeted against a site termed H. Variants of HH-H are also shown that contain either a 2 base-paired stem II (HH-H1 and HH-H2) or a 3 basepaired stem II (HH-H3 and HH-H4).

SUBSTITUTE SHEET (RULE 26) WO 95/23225 P( 1I 11195 (ll 16 Figs, 63 and 64 show RNA cleavage activity of HH-I and its variants (see Fig.62). 63) cleavage of matched substrate RNA (15 nt). 64) cleavage of long substrate RNA (613 nt), Figs, 65a-b is a schematic representation of a method of this invention to synthesize a full length hairpin ribozyme. No splint strand is required for ligation but rather the two fragments hybridize together at helix 4 prior to ligation, The only prerequisite is that the 3' fragment is phosphorylated at its 5' end and that the 3' end of the 5' fragment have a hydroxyl group, The hairpin ribozyme is targeted against site J, H1 and H2 are intermolecular helices formed between the ribozyme and the substrate, H3 and H4 are intramolecular helices formed within the hairpin ribozyme motif. Arrow indicates the cleavage site, Fig, 66 shows RNA cleavage activity of ligated hairpin ribozymes targeted against site J.

Figs. 67a-b is a diagrammatic representation of a Site K Hairpin Ribozyme (HP-K) showing the proposed secondary structure of the hairpin ribozyme -substrate complex as described in the art (Berzal-Herranz et al., 1993 EMBO, J.12, 2567), The ribozyme has been assembled from two fragments (bimolecular ribozyme; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835); #H1 and H2 represent intermolecular helix formation between the ribozyme and the substrate. H3 and H4 represent intramolecular helix formation within the ribozyme (intermolecular helix in the case of bimolecular ribozyme), Left panel (HP-K1) indicates 4 basepaired helix 2 and the right panel (HP-K2) indicates 6 base-paired helix 2, Arrow indicates the site of RNA cleavage, All the ribozymes discussed herein were chemically synthesized by solid phase synthesis using RNA phosphoramadite chemistry, unless otherwise indicated, Those skilled in the art will recognize that these ribozymes could also be made transcriptionally in vitro and in vivo, Figure 68 is a graph showing RNA cleavage by hairpin ribozymes targeted to site K. A plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown. HP-K2 (6 bp helix 2) cleaves a 422 target RNA to a greater extent than the HP-K1 (4 bp helix 2).

SUBSTITUTE SHEET (RULE 26) C WO 95/23225 PC I 'Il.lll 150 17 To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 422 nt region (containing hairpin site A) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed in a standard transcription buffer in the presence of 3 2 P]CTP (Chowrira Burke, 1991 supra). The reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol precipitated with isopropanol and washed with 70% ethanol, The dried pellet was resuspended in 20 .1 DEPC-treated water and stored at Unlabeled ribozyme (lp.M) and internally labeled 422 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 10 mM MgCI2) by heating to 90°C for 2 min, and slow cooling to 37"C for 10 min. The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 370C. Aliquots of 5 p1 were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5% polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager (Molecular Dynamics, Sunnyvale,

CA).

Figs. 69a-b is the Site L Hairpin Ribozyme (HP-L) showing proposed secondary structure of the hairpin ribozyme*substrate complex. The ribozyme was assembled from two fragments as described above. The nomenclature is the same as above.

Figure 70 shows RNA cleavage by hairpin ribozymes targeted to site L. A, plot of fraction of the target RNA uncleaved (fraction uncleaved) as a function of time is shown, HP-L2 (6 bp helix 2) cleaves a 2 KB target RNA to a greater extent than the HP-L1 (4 bp helix To make internallylabeled substrate RNA for trans-ribozyme cleavage reactions, a 2 kB region (containing hairpin site L) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. The cleavage reactions were carried out as described above.

SUBSTITUTE SHEET (RULE 26) ~a I _111 0 95;23225 PCTill)5'()I1156 18 Figs, 71a-b shows a Site M Hairpin Ribozyme (HP-M) with the proposed secondary structure of the hairpin ribozymesubstrate complex.

The ribozyme was assembled from two fragments as described above.

Figure 72 is a graph showing RNA cleavage by hairpin ribozymes targeted to site M. The ribozymes were tested at both 20 C and at 26"C.

To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 1,9 KB region (containing hairpin site M) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence, Cleavage reactions were carried out as described above except that 20"C and at 26°C temperatures were used, Figs. 73a-d shows various structural modifications of the present invention, A) Hairpin ribozyme lacking helix 5. Nomenclature is same as described under figure 3, B) Hairpin ribozyme lacking helix 4 and helix Helix 4 is replaced by a nucleotide loop wherein q is 2 bases, Nomenclature is same as described under figure 3. C) Hairpin ribozyme lacking helix 5, Helix 4 loop is replaced by a linker 103"L", wherein L is a non-nucleotide linker molecule (Benseler et al,, 1993 J, Am. Chem. Soc.

115, 8483; Jennings et al., WO 94/13688). Nomenclature is same as described under figure 3. D) Hairpin ribozyme lacking helix 4 and helix Helix 4 is replaced by non-nucleotide linker molecule (Benseler et al., 1993 supra; Jennings et al., supra), Nomenclature is same as described under figure 3.

Figs, 74a-b shows Hairpin ribozymes containing nucleotide spacer region at the indicated location, wherein s is 1 base. Hairpin ribozymes containing spacer region, can be synthesized as one fragment or can be assembled from multiple fragments. Nomenclature is same as described under figure 3.

Figs. 75a-e shows the structures of the nucleotides. R 1 is as defined above. R is OH, H, O-protecting group, NH, or any group described by the publications discussed above, and those described below. B is as defined in the Figure or any other equivalent nucleotide base, CE is cyanoethyl, DMT is a standard blocking group.

Other abbreviations are standard in the art.

SUBSTITUTE SHEET (RULE 26)

I

I I 0 95/23225 PCI 1195/10 19 Figure 76 is a diagrammatic representation of the synthesis of alkyl-D-allose nucleosides and their phosphoramidites.

Figure 77 is a diagrammatic representation of the synthesis of alkyl-L-talose nucleosides and their phosphoramidites.

Figure 78 is a diagrammatic representation of hammerhead ribozymes targeted to site 0 containing 5'-C-methyl-L-talo modifications at various positions.

Figure 79 shows RNA cleavage activity of HH-O ribozymes. Fraction of target RNA uncleaved as a function of time is shown, Figure 80 is a diagrammatic representation of a position numbered hammerhead ribozyme (according to Hertel et al. Nucleic Acids Res. 1992, 3252) showing specific substitutions, Figs. 81a-j shows the structures of various 2'-alkyl modified nucleotides which exemplify those of this invention. R groups are alkyl groups, Z is a protecting group, Figure 82 is a diagrammatic representation of the synthesis of 2'-Callyl uridine and cytidine.

Figure 83 is a diagrammatic representation of the synthesis of 2'-Cmethylene and 2'-C-difluoromethylene uridine, Figure 84 is a diagrammatic representation of the synthesis of 2'-Cmethylene and 2'-C-difluoromethylene cytidine.

Figure 85 is a diagrammatic representation of the synthesis of 2'-Cmethylene and 2'-C-difluoromethylene adenosine, Figure 86 is a diagrammatic representation of the synthesis of 2'-Ccarboxymethylidine uridine, 2'-C-methoxycarboxymethylidine uridine and derivatized amidites thereof. X is CH 3 or alkyl as discussed above, or another substituent.

Figure 87 is a diagrammatic representation of a synthesis of nucleoside SUBSTITUTE SHEET (RULE 26) ~BPLIIIII II 0 95,23225 (N 15t 23225!( 1') Figure 88 is a diagrammatic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylphosphonate 3'-phosphoramidites, dimers and solid supported dimers.

Figure 89 is a diagrammatic representation of the synthesis of nucleoside 5'-deoxy-5'-difluoromethylene triphosphates.

Figures 90 and 91 are diagrammatic representations of the synthesis of 3'-deoxy-3'-difluoromethylphosphonates and dimers.

Figure 92 is a schematic representation of synthesizing RNA phosphoramidite of a nucleotide containing a 2'-hydroxyl group modification of the present invention.

Figs. 93a-b describes a method for deprotection of oligonucleotides containing a 2'-hydroxyl group modification of the present invention.

Figure 94 is a diagrammatic representation of a hammerhead ribozyme targeted to site N. Positions of 2'-hydroxyl group substitution is indicated, Figure 95 shows RNA cleavage activity of ribozymes containing a 2'hydroxyl group modification of the present invention, All RNA, represents hammerhead ribozyme (HHN) with no 2'-hydroxyl group modifications. U7ala, represents HHN ribozyme containing 2'-NH-alanine modification at the U7 position. U4/U7-ala, represents HHA containing 2'-NH-alanine modifications at U4 and U7 positions, U4 lys, represents HHA containing 2'-NH-lysine modification at U4 position. U7 lys, represents HHA containing 2'-NH-lysine modification at U7 position, U4/U7-lys, represents HHN containing 2'-NH-lysine modification at U4 and U7 positions.

Figures 96 and 97 are schematic representations of synthesizing (solid-phase synthesis) 3' ends of RNA with modification of the present invention, B, refers to either a base, modified base or an H.

Figure 98 and 99 are schematic representations of synthesizing (solid-phase synthesis) 5' ends of RNA with modification of the present invention, B, refers to either a base, modified base or an H, Figures 100 and 101 are general schematic representations of the invention.

SUBSTITUTE SHEET (RULE 26) 95!23225 l( 119I5/00 Fig. 102a-d :s a schematic representation of a method of the invention.

Fig. 103 is a graph of the results of the experiment diagrammed in figure 104, Figure 104 is a diagrammatic representation of a fusion mRNA used in the experiment diagrammed in Fig, 102, Figure 105 is a diagrammatic representation of a method for selection of useful ribozymes of this invention.

Figure 106 generally shows R-loop formation, and an R-loop complex. In addition, it indicates the location at which ligands can be provided to target the R-loop complex to cells using at least three different procedures, such as ligand receptor interaction, lipid or calcium phosphate mediated delivery, or electroporation.

Figure 107 shows a method for use of self-processing ribozymes to generate therapeutic ribozymes of unit length. This method is essentially described by Draper et al., PCT WO 93/23509, Figure 108 shows a method of linking ligands like folate, carbohydrate or peptides to R-loop forming RNA, Ribozymes of this invention block to some extent ICAM-1, IL-5, rel A, TNF-u, p210 b c r a b l, or RSV genes expression and can be used to treat diseases or diagnose such diseases. Ribozymes will be delivered to cells in culture and to tissues in animal models, Ribozyme cleavage of ICAM-1, 11-5, rel A, TNF-a ,p21bcr-abl, or RSV mRNA in these systems may prevent or alleviate disease symptoms or conditions, I. Target sites Targets for useful ribozymes can be determined as disclosed in Draper et al PCT W093/23509, Sullivan et al., PCT W094/02595 as well as by Draper et al., PCT/US94/13129 and hereby incorporated by reference herein in totality. Rather than repeat the guidance provided in those documents here, below are provided specific examples of such methods, not limiting to those in the art. Ribozymes to such targets are designed as described in those applications and synthesized to be tested in vitro and in vivo, as also described. Such ribozymes can also be SUBSTITUTE SHEET (RULE 26) SIBlb-a ~p I 0 '5,23225 P( *1711i)5(i( ISO 22 optimized and delivered as described therein. While specific examples to animal and human RNA are provided, those in the art will recognize that the equivalent human RNA targets described can be used as described below. Thus, the same target may be used, but binding arms suitable for targeting human RNA sequences are present in the ribozyme, Such targets may also be selected as described below.

It must be established that the sites predicted by the computer-based RNA folding algorithm correspond to potential cleavage sites, Hammerhead or hairpin ribozymes are designed that could bind and are individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl, Acad. Sci., USA, 86 7706-7710) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration, Varying binding arm lengths can be chosen to optimize activity, Generally, at least 5 bases on each arm are able to bind to, or otherwise interact with, the target RNA, mRNA is screened for accessible cleavage sites by the method described generally in Draper et al,, PCT W093/23569 hereby incorporated by reference herein, Briefly, DNA oligonucleotides representing potential hammerhead or hairpin ribozyme cleavage sites are synthesized. A polymerase chain reaction is used to gernrate a substrate for T7 RNA polymerase transcription from cDNA clones, Labeled RNA transcripts are synthesized in vitro from DNA templates. The oligonucleotides and the labeled trascripts are annealed, RNaseH is added and the mixtures are incubated for the designated times at 370C, Reactions are stopped and RNA separated on sequencing polyacrylamide gels. The percentage of the substrate cleaved is determined by autoradiographic quantitation using a phosphor imaging system. From these data, hammerhead or hairpin ribozynme sites are chosen as the most accessible.

Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences desribed above. The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990 I~l~a~ 0 ')5/23225 1 I 51)!150, 23 Nucleic Acids Res., 18, 5433 and made use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the phosphoramidites at the 3'-end. The average stepwise coupling yeilds are Inactive ribozymes are synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al,, 1992 Nucleic Acids Res,, 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840), Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbach, 1989, Methods Enzymol, 180, 51), Ai ribozymes are modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'H (for a review see Usman and Cedergren, 1992 TIBS 17,34). Ribozymes are purified by gel electrophoresis using heneral methods or are purified by high pressure liquid chromatography and are resuspended in water.

Example 1: ICAM-1 Ribozymes that cleave ICAM-1 mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders, ICAM-1 function can be blocked therapeutically using monoclonal antibodies, Ribozymes have the advantage of being generally immunologically inert, whereas significant neutralizing anti-lgG responses can be observed with some monoclonal antibody treatments.

The following is a brief description of the physiological role of ICAM-1, The discussion is not meant to be complete and is provided only for understanding of the invention that follows, This summary is not an admission that any of the work described below is prior art to the claimed invention.

Intercellular adhesion molecule-1 (ICAM-1) is a cell surface protein whose expression is induced by inflammatory mediators. ICAM-1 is required for adhesion of leukocytes to endothelial cells and for several immunological functions including antigen presentation, immunoglobulin production and cytotoxic cell activity. Blocking ICAM-1 function prevents immune cell recognition and activity during transplant rejection and in animal models of rheumatoid arthritis, asthma and reperfusion injury.

I

~SI~SBII~Brm~ 23225 I "111*0) Uri(, 24 Cell-cell adhesion plays a pivotal role in inflammatory and immune responses (Springer et al., 1987 Ann, Rev. Immunol. 5, 223-252), Cell adhesion is required for leukocytes to bind to and migrate through vascular endothelial cells. In addition, cell-cell adhesion is required for antigen presentation to T cells, for B cell induction by T cells, as well as for the cytotoxicity activity of T cells, NK cells, monocytes or granulocytes.

Intercellular adhesion molecule-1 (ICAM-1) is a 110 kilodalton member of the immunoglobulin superfamily that is involved in all of these cell-cell interactions (Simmons et al., 1988 Nature (London) 331, 624-627).

ICAM-1 is expressed on only a limited number of cells and at low levels in the absence of stimulation (Dustin et al., 1986 J, Immunol. 137, 245-254). Upon treatment with a number of inflammatory mediators (lipopolysaccharide, y-interferon, tumor necrosis factor-a, or interleukin-1), a variety of cell types (endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface (Sringer et. al. supra; Dustin et al,, supra; and Rothlein et al,, 1988 J, Immunol. 141, 1665-1669). Induction occurs via increased transcription of ICAM-1 mRNA (Simmons et al., supra). Elevated expression is detectable after 4 hours and peaks after 16 24 hours of induction.

ICAM-1 induction is critical for a number of inflammatory and immune responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to cytokine-activated endothelial cells (Boyd,1988 Proc. Natl. Acad, Sci, USA 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331), Thus, ICAM-1 expression may be required for the extravasation of immune cells to sites of inflammation, Antibodies to ICAM-1 also block T cell killing, mixed lymphocyte reactions, and T cell-mediated B cell differentiation, suggesting that ICAM-1 Is required for these cognate cell interactions (Boyd et al., supra). The Importance of ICAM-1 in antigen presentation is underscored by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell proliferation (Dang et al., 1990 J.

Immunol. 144, 4082-4091). Conversely, murine L cells require transfection with human ICAM-1 in addition to HLA-DR in order to present antigen to human T cells (Altmann et al,, 1989 Nature (London) 338, 512-514). In summary, evidence in vitro indicates that ICAM-1 is required for cell-cell interactions critical to inflammatory responses, cellular immune responses, and humoral antibody responses.

I 'I C ar W\O 95,23225 1,1 B 011.% By engineering ribozyme motifs we have designed several ribozymes directed against ICAM-1 mRNA sequences, These have been synthesized with modifications that improve their nuclease resistance. These ribozymes cleave ICAM-1 target sequences in vitro.

The sequence of human, rat and mouse ICAM-1 mRNA can be screened for accessible sites using a compter folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Tables 2, 3, and 6-9. (All sequences are 5' to 3' in the tables) While rat, mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility, The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 4 8 and 10. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity and may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables, The ribozymes will be tested for function in vivo by exogenous delivery to human umbilical vein endothelial cells (HUVEC), Ribozymes will be delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA or RNA vectors described above, Cytokine-induced ICAM-1 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. ICAM-1 mRNA levels will be assessed by Northern, by RNAse protection, by primer extension or by quantitative RT-PCR analysis.

Ribozymes that block the induction of ICAM-1 protein and mRNA by more than 90% will be identified, As disclosed by Sullivan et al,, PCT W094/02595, incorporated by reference herein, ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of ICAM- 1 mRNA and protein. The effect of the anti-ICAM-1 ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced I I 95;23225 PC IIB9') 1i0I ISo 26 into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis, Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used, One dose (or a few infrequent doses) of a stable anti-ICAM-1 ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases, Uses ICAM-1 plays a central role in immune cell recognition and function, Ribozyme inhibition of ICAM-1 expression can reduce transplant rejection and alleviate symptoms in patients with rheumatoid arthritis, asthma or other acute and chronic inflammatory disorders, We have engineered several ribozymes that cleave ICAM-1 mRNA. Ribozymes that efficiently inhibit ICAM-1 expression in cells can be readily found and their activity measured with regard to their ability to block transplant rejection and arthritis symptoms in animal models, These anti-ICAM-1 ribozymes represent a novel therapeutic for the treatment of immunological or inflammatory disorders.

The therapeutic utility of reduction of activity of ICAM-1 function is evident in the following disease targets, The noted references indicate the role of ICAM-1 and the therapeutic potential of ribozymes described herein, Thus, these targets can be therapeutically treated with agents that reduce ICAM-1 expression or function. These diseases and the studies that support a critical role for ICAM-1 in their pathology are listed below. This list is not meant to be complete and those in the art will recognize further conditions and diseases that can be effectively treated using ribozymes of the present invention.

STransplant rejection ICAM-1 is expressed on venules and capillaries of human cardiac biopsies with histological evidence of graft rejection (Briscoe et al,, 1991 Transplantation 51, 537-539).

Antibody to ICAM-1 blocks renal (Cosimi et al,, 1990J. Immunol, 144, 4604- 4612) and cardiac (Flavin et al., 1991 Transplant. Proc. 23, 533-534) graft rejection in primates.

L- I I 95,23225 PICTl 3l'W 27 A Phase I clinical trial of a monoclonal anti-ICAM-1 antibody showed significant reduction in rejection and a significant increase in graft function in human kidney transplant patients (Haug, et al., 1993Transplantation 55, 766-72).

SRheumatoid arthritis ICAM-1 overexpression is seen on synovial fibroblasts, endothelial cells, macrophages, and some lymphocytes (Chin et al., 1990 Arthritis Rheum 33, 1776-86; Koch et al., 1991 Lab Invest 64, 313-20).

Soluble ICAM-1 levels correlate with disease severity (Mason et al., 1993 Arthritis Rheum 36, 519-27).

Anti-ICAM antibody inhibits collagen-induced arthritis in mice (Kakimoto et al., 1992 Cell Immunol 142, 326-37).

Anti-lOAM antibody Inhibits adjuvant-induced arthritis in rats (ligo et al., 1991 J Immunol 147, 4167-71), Myocardial ischemia, stroke, and reperfusion injury Anti-ICAM-1 antibody blocks adherence of neutrophils to anoxic endothelial cells (Yoshida et al., 1992 Am J Physiol 262, H1891-8), Anti-ICAM-1 antibody reduces neurological damage in a rabbit model of cerebral stroke (Bowes et al., 1993 Exp Neurol 119, 215-9), Anti-ICAM-1 antibody protects against reperfusion injury in a cat model of myocardial ischemia (Ma et al., 1992Circulation 86, 937-46), *Asthma Antibody to ICAM-1 partially blocks eosinophil adhesion to endothelial cells and is overexpressed on inflamed airway endothelium and epithelium in vivo (Wegner et al,, 1990 Science 247, 456-9).

In a primate model of asthma, anti-ICAM-1 antibody blocks airway eosinophilia (Wegneret al., supra) and prevents the resurgence of airway inflammation and hyper-responsiveness after dexamethosone treatment (Gundel et al., 1992 Clin Exp Allergy 22, 569-75), Psoriasis 0 ~ll~deL I WO 95/23225 PCT/IBm;l5s1115 28 Surface ICAM-1 and a clipped, soluble version of ICAM-1 is expressed in psoriatic lesions and expression correlates with inflammation (Kellner et al., 1991 Br J Dermatol 125, 211-6; Griffiths 1989 J Am Acad Dermatol 20, 617-29; Schopf et al., 1993 BrJ Dermatol 128, 34-7).

Anti-ICAM antibody blocks keratinocyte antigen presentation to T cells (Nickoloff et al., 1993J Immunol 150, 2148-59).

SKawasaki disease Surface ICAM-1 expression correlates with the disease and is reduced by effective immunoglobulin treatment (Leung, et al., 1989Lancet 2, 1298-302).

Soluble ICAM levels are elevated in Kawasaki disease patients; particularly high levels are observed in patients with coronary artery lesions (Furukawa et al., 1992Arthritis Rheum 35, 672-7; Tsuji, 1992 Arerugi 41, 1507-14).

Circulating LFA-1 T cells are depleted (presumably due to ICAM-1 mediated extravasation) in Kawasaki disease patients (Furukawa et al., 1993Scand J Immunol 37, 377-80).

Example 2: Ribozymes that cleave IL-5 mRNA represent a novel therapeutic approach to inflammatory disorders like asthma. The invention features use of ribozymes to treat chronic asthma, by inhibiting the synthesis of IL-5 in lymphocytes and preventing the recruitment and activation of eosinophils.

A number of cytokines besides IL-5 may also be involved in the activation of inflammation in asthmatic patients, including platelet activating factor, IL-1, IL-3, IL-4, GM-CSF, TNF-a, gamma interferon, VCAM, ILAM-1, ELAM-1 and NF-iB. In addition to these molecules, it is appreciated that any cellular receptors which mediate the activities of the cytokines are also good targets for intervention in inflammatory diseases. These targets include, but are not limited to, the IL-1R and TNF-aR on keratinocytes, epithelial and endothelial cells in airways, Recent data suggest that certain neuropeptides may play a role in asthmatic symptoms. These peptides include substance P, neurokinin A and calcitonin-gene-related peptides, These target genes may have more general roles in inflammatory diseases, but are currently assumed to have a role only in asthma.

1 WO 95/23225 PI'T/I B95/00 150 29 Ribozymes of this invention block to some extent IL-5 expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of asthma (Clutterbuck et al., 1989 supra: Garssen et al,, 1991 Am. Rev.

Respir. Dis. 144, 931-938; Larsen et al., 1992 J. Clin. Invest. 89, 747-752; Mauser et al,, 1993 supra), Ribozyme cleavage of IL-5 mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms.

The sequence of human and mouse IL-5 mRNA were screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 11, 13, and 14, 15. (All sequences are 5' to 3' in the tables,) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme, (In Table 12, lower case letters indicate positions that are not conserved between the Human and the Mouse IL-5 sequences.) The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 12, 14 16. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity, For example, stem loop II sequence of hammerhead ribozymes listed in Tables 12 and 14 can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form.

Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables and 16 (5'-CACGUUGUG-3') can be altered (substitution, deletion and/or insertion) to contain any sequence provided, a minimum of two basepaired stem structure can form, The sequences listed in Tables 12, 14 16 may be formed of ribonucleotides or other nucleotides or non-nucleotides, Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

By engineering ribozyme motifs we have designed several ribozymes directed against IL-5 mRNA sequences. These ribozymes are synthesized ~i~ll~ils I- PClI 1195100 156 WO 95/23225 with modifications that improve their nuclease resistance, The ability of ribozymes to cleave IL-5 target sequences in vitro is evaluated.

The ribozymes will be tested for function in vivo by analyzing expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA or RNA vectors. IL-5 expression will be monitored by biological assays, ELISA, by indirect immunofluoresence, and/or by FACS analysis. IL-5 mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR.

Ribozymes that block the induction of IL-5 activity and/or IL-5 mRNA by more than 90% will be identified.

Uses Interleukin 5 a cytokine produced by CD4+ T helper cells and mast cells, was originally termed B cell growth factor II (reviewed by Takatsu et al., 1988 Immunol. Rev. 102, 107). It stimulates proliferation of activated B cells and induces production of IgM and IgA. IL-5 plays a major role in eosinophil function by promoting differentiation (Clutterbuck et al., 1989 Blood 73, 1504-12), vascular adhesion (Walsh et al., 1990 Immunology 71, 258-65) and in vitro survival of eosinophils (Lopez et al., 1988 J. Exp. Med. 167, 219-24), This cytokine also enhances histamine release from basophils (Hirai et al., 1990 J. Exp, Med. 172, 1525-8). The following summaries of clinical results support the selection of IL-5 as a primary target for the treatment of asthma: Several studies have shown a direct correlation between the number of activated T cells and the number of eosinophils from asthmatic patients vs. normal patients (Oehling et al,, 1992 J. Investig. Allergol. Clin. Immunol.

2, 295-9), Patients with either allergic asthma or intrinsic asthma were treated with corticosteroids. The bronchoalveolar lavage was monitored for eosinophils, activated T helper cells and recovery of pulmonary function over a 28 to 30 day period. The number of eosinophils and activated T helper cells decreased progressively with subsequent improvement in pulmonary function compared to intrinsic asthma patients with no corticosteroid treatment.

Bronchoalveolar lavage cells were screened for production of cytokines using in situ hybridization for mRNA. In situ hybridization signals L~IIEERI~ I WO 95/23225 PC"r'T/B95/l00115( 31 were detected for IL-2, IL-3, IL-4, IL-5 and GM-CSF, Upregulation of mRNA was observed for IL-4, IL-5 and GM-CSF (Robinson et al,, 1993 J. Allergy Clin. Immunol. 92, 313-24). Another study showed that upregulation of transcripts from allergen challenged vs. saline challenged asthmatic patients (Krishnaswamy et al., 1993 Am. J. Respir. Cell. Mol, Biol. 9, 279- 86).

An 18 patient study was performed to determine a mechanism of action for corticosteroid improvement of asthma symptoms, Improvement was monitored by methacholine responsiveness. A correlation was observed between the methacholine responsiveness, a reduction in the number of eosinophils, a reduction in the number of cells expressing IL-4 and IL-5 mRNA and an increase in number of cells expressing interferongamma.

Bronchial biopsies from 15 patients were analyzed 24 hours after allergen challenge (Bentley et al,, 1993 Am. J, Respir. Cell. Mol, Biol, 8, 35-42). Increased numbers of eosinophils and IL-2 receptor positive cells were found in the biopsies. No differences in the numbers of total leukocytes, T lymphocytes, elastase-positive neutrophils, macrophages or mast cell subtypes were observed, The number of cells expressing and GM-CSF mRNA significantly increased, In another patient study, the eosinophil phenotype was the same for asthmatic patients and normal individuals. However, eosinophils from asthmatic patients had greater leukotriene C4 producing capacity and migration capacity. There were elevated levels of IL-3, IL-5 and GM-CSF in the circulation of asthmatics but not in normal individuals (Bruijnzeel et al,, 1992 Schweiz. Med. Wochenschr. 122, 298-301).

Efficacy of antibody to IL-5 was assessed in a guinea pig asthma model, The animals were challenged with ovalbumin and assayed for eosinophilia and the responsiveness to the bronchioconstriction substance P. A 30 mg/kg dose of antibody administered ip. blocked ovalbumininduced increased sensitivity to substance P and blocked increases in bronchoalveolar and lung tissue accumulation of eosinophils (Mauser et al., 1993 Am. Rev. Respir. Dis. 148, 1623-7), In a separate study guinea pigs challenged for eight days with ovalbumin were treated with monoclonal antibody to IL-5. Treatment produced a reduction in the ~Bc-lrv I WO 95/23225 PI'(T/11195/00 32 number of eosinophils in bronchoalveolar lavage. No reduction was observed for unchallenged guinea pigs and guinea pigs treated with a control antibody, Antibody treatment completely inhibited the development of hyperreactivity to histamine and arecoline after ovalbumin challenge (van Oosterhout et al., 1993 Am. Rev. Respir. Dis. 147, 548-52) Results obtained from human clinical analysis and animal studies indicate the role of activated T helper cells, cytokines and eosinophils in asthma. The role of IL-5 in eosinophil development and function makes ILa good candidate for target selection. The antibody studies neutralized IL-5 in the circulation thus preventing eosinophilia. Inhibition of the production of IL-5 will achieve the same goal.

Asthma a prominent feature of asthma is the infiltration of eosinophils and deposition of toxic eosinophil proteins major basic protein, eosinophil-derived neurotoxin) in the lung, A number of T-cellderived factors like IL-5 are responsible for the activation and maintainance of eosinophils (Kay, 1991 J. Allergy Clin. Immun, 87, 893). Inhibition of expression in the lungs can decrease the activation of eosinophils and will help alleviate the symptoms of asthma.

Atopy is characterized by the developement of type I hypersensitive reactions associated with exposure to certain environmental antigens, One of the common clinical manifestations of atopy is eosinophilia (accumulation of abnormally high levels of eosinophils in the blood).

Antibodies against IL-5 have been shown to lower the levels of eosinophils in mice (Cook et al,, 1993 in Immunopharmacol, Eosinophils ed, Smith and Cook, pp. 193-216, Academic, London, UK) Parasitic infection-related eosinophilia- infections with parasites like helminths, can lead to severe eosinophilia (Cook et al., 1993 supra). Animal models for eosinophilia suggest that infection of mice, for example, can lead to blood, peritoneal and/or tissue eosinophilia, all of which seem to be lowered to varying degrees by antibodies directed against Pulmonary infiltration eosinophilia- is characterised by accumulation of high levels of eosinophils in pulmonary parenchyma (Gleich, 1990 J. Allergy Clin. Immunol. 85, 422).

IB~dl-l1 I WO 95/23225 I'CT/I B95W00150 33 L-Tryptophan-associated eosinophilia-myalgia syndrome (EMS)- The EMS disease is closely linked to the consumption of Ltryptophan, an essential aminoacid used to treat conditions like insomnia (for review see Varga et al,, 1993 J Invest. Dermatol. 100, 97s). Pathologic and histologic studies have demonstrated high levels of eosinophils and mononuclear inflammatory cells in patients with EMS, It appears that and transforming growth factor play a significant role in the development of EMS (Varga et al,, 1993 supra) by activating eosinophils and other inflammatory cells.

Thus, ribozymes of the present invention that cleave IL-5 mRNA and thereby IL-5 activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits function is described above; available cellular and activity assays are numerous, reproducible, and accurate, Animal models for IL-5 function and for each of the suggested disease targets exist (Cook et al., 1993 supra) and can be used to optimize activity, Example 3: NF-KB Ribozymes that cleave rel A mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. Inflammatory mediators such as lipopolysaccharide (LPS), interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-a) act on cells by inducing transcription of a number of secondary mediators, including other cytokines and adhesion molecules. In many cases, this gene activation is known to be mediated by the transcriptional regulator, NF-KB. One subunit of NF-KB, the re/A gene product (termed RelA or p65) is implicated specifically in the induction of inflammatory responses. Ribozyme therapy, due to its exquisite specificity, is particularly well-suited to target intracellular factors that contribute to disease pathology. Thus, ribozymes that cleave mRNA encoded by rel A or TNF-a may represent novel therapeutics for the treatment of inflammatory and autoimmune disorders, The nuclear DNA-binding activity, NF-KB, was first identified as a factor that binds and activates the immunoglobulin K light chain enhancer in B cells. NF-KB now is known to activate transcription of a variety of other cellular genes cytokines, adhesion proteins, oncogenes and viral I I ~rr~rc I WO 95!23225 P(T/1139 34 proteins) in response to a variety of stimuli phorbol esters, mitogens, cytokines and oxidative stress). In addition, molecular and biochemical characterization of NF-KB has shown that the activity is due to a homodimer or heterodimer of a family of DNA binding subunits. Each subunit bears a stretch of 300 amino acids that is homologous to the oncogene, v-rel. The activity first described as NF-KB is a heterodimer of p49 or p50 with p65, The p49 and p50 subunits of NF-KB (encoded by the nf-KB2 or nf-KB1 genes, respectively) are generated from the precursors NF-KB1 (p105) or NF-KB2 (p10 0 The p65 subunit of NF-KB (now termed Rel A) is encoded by the rel A locus.

The roles of each specific transcription-activating complex now are being elucidated in cells Perkins, et al,, 1992 Proc. Natl Acad. Sci USA 89, 1529-1533). For instance, the heterodimer of NF-KB1 and Rel A (p50/p65) activates transcription of the promoter for the adhesion molecule, VCAM-1, while NF-KB2/RelA heterodimers (p49/p65) actually inhibit transcription Shu, et al., Mol, Cell, Biol, 13, 6283-6289 (1993)), Conversely, heterodimers of NF-KB2/RelA (p49/p65) act with Tat-I to activate transcription of the HIV genome, while NF-KB1/RelA (p50/p65) heterodimers have little effect Liu, N.D, Perkins, R.M. Schmid, G.J, Nabel, J. Virol. 1992 66, 3883-3887). Similarly, blocking rel A gene expression with antisense oligonucleotides specifically blocks embryonic stem cell adhesion; blocking NF-KB1 gene expression with antisense oligonucleotides had no effect on cellular adhesion (Narayanan et al., 1993 Mol, Cell. Biol, 13, 3802-3810), Thus, the promiscuous role initially assigned to NF-KB in transcriptional activation Lenardo, D. Baltimore, 1989 Cell 58, 227-229) represents the sum of the activities of the rel family of DNA-binding proteins. This conclusion is supported by recent transgenic "knock-out" mice of individual members of the rel family. Such "knockouts" show few developmental defects, suggesting that essential transcriptional activation functions can be performed by more than one member of the rel family.

A number of specific inhibitors of NF-KB function in cells exist, including treatment with phosphorothioate antisense oliogonucleotide, treatment with double-stranded NF-KB binding sites, and over expression of the natural inhibitor MAD-3 (an IKB family member). These agents have BbBaL~lr 95/23225 I'c( T/I 195 0 I been used to show that NF-KB is required for induction of a number of molecules involved in inflammation, as described below.

*NF-KB is required for phorbol ester-mediated induction of IL-6 (I.

Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8 (Kunsch and Rosen, 1993 Mol, Cell. Biol. 13, 6137-46).

*NF-KB is required for induction of the adhesion molecules ICAM-1 (Eck, et al., 1993 Mol. Cell. Biol. 13, 6530-6536), VCAM-1 (Shu et al,, supra), and E-selectin (Read, et al., 1994 J. Exp. Med. 179, 503-512) on endothelial cells.

*NF-KB is involved in the induction of the integrin subunit, CD18, and other adhesive properties of leukocytes (Eck et al,, 1993 supra).

The above studies suggest that NF-KB is integrally involved in the induction of cytokines and adhesion molecules by inflammatory mediators, Two recent papers point to another connection between NF-KB and inflammation: glucocorticoids may exert their anti-inflammatory effects by inhibiting NF-KB. The glucocorticoid receptor and p65 both act at NF-KB binding sites in the ICAM-1 promoter (van de Stolpe, et al., 1994 J. Biol.

Chem. 269, 6185-6192). Glucocorticoid receptor inhibits NF-KB-mediated induction of IL-6 (Ray and Prefontaine, 1994 Proc, Natl Acad. Sci USA 91, 752-756). Conversely, overexpression of p65 inhibits glucocorticoid induction of the mouse mammary tumor virus promoter. Finally, protein cross-linking and co-immunoprecipitation experiments demonstrated direct physical interaction between p65 and the glucocorticoid receptor Ribozymes of this invention block to some extent NF-KB expression and can be used to treat disease or diagnose such disease. Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of restenosis, transplant rejection and rheumatoid arthritis. Ribozyme cleavage of relA mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms, The sequence of human and mouse re/A mRNA can be screened for accessible sites using a computer folding algorithm. Potential hammerhead or hairpin ribozyme cleavage sites were identified, These sites are shown in Tables 17, 18 and 21-22. (All sequences are 5' to 3' in the tables.) While mouse and human sequences can be screened and ~sl~R I I \WO 95/23225 195m/() 36 ribozymes thereafter designed, the human targetted sequences are of most utility, The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 19 22, Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity and may be formed of ribonucleotides or other nucleotides or non-nucleotides, Such ribozymes are equivalent to the ribozymes described specifically in the Tables, By engineering ribozyme motifs we have designed several ribozymes directed against re/A mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance, The ability of ribozymes to cleave re/A target sequences in vitro is evaluated.

The ribozymes will be tested for function in vivo by analyzing cytokineinduced VCAM-1, ICAM-1, IL-6 and IL-8 expression levels. Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA and RNA vectors. Cytokine-induced VCAM-1, ICAM-1, IL-6 and IL-8 expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. Rel A mRNA levels will be assessed by Northern analysis, RNAse protection or primer extension analysis or quantitative RT-PCR.

Activity of NF-KB will be monitored by gel-retardation assays. Ribozymes that block the induction of NF-KB activity and/or re/ A mRNA by more than will be identified.

RNA ribozymes and/or genes encoding them will be locally delivered to transplant tissue ex vivo in animal models. Expression of the ribozyme will be monitored by its ability to block ex vivo induction of VCAM-1, ICAM- 1, IL-6 and IL-8 mRNA and protein. The effect of the anti-rel A ribozymes on graft rejection will then be assessed. Similarly, ribozymes will be introduced into joints of mice with collagen-induced arthritis or rabbits with Streptococcal cell wall-induced arthritis. Liposome delivery, cationic lipid delivery, or adeno-associated virus vector delivery can be used. One dose (or a few infrequent doses) of a stable anti-relA ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate inflammatory and immune responses in these diseases, 1r ~L~rYl~ilarP~- r I \VO 95/23225 IK"I 1110/0015i 37 Uses A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders. Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below. In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves rel A mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment, -Rheumatoid arthritis (RA).

Due to the chronic nature of RA, a gene therapy approach is logical.

Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the synovium: high efficiency of gene transfer and expression for several months would be expected Roessler, E.D. Allen, J.M, Wilson, J.W.

Hartman, B. L. Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent, Multiple administrations may be necessary. Retrovirus and adeno-associated virus vectors would lead to permanent gene transfer and expression in the joint.

However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

*Restenosis.

Expression of NF-KB in the vessel wall of pigs causes a narrowing of the luminal space due to excessive deposition of extracellular matrix components. This phenotype is similar to matrix deposition that occurs subsequent to coronary angioplasty. In addition, NF-KB is required for the expression of the oncogene c-myb La Rosa, J.W. Pierce, G.E.

Soneneshein, Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-KB induces smooth muscle proliferation and the expression of excess matrix components: both processes are thought to contribute to reocclusion of vessels after coronary angioplasty.

*Transplantation.

I

23225 P("III'95/00150 38 NF-KB is required for the induction of adhesion molecules (Eck et al,, supra, K. O'Brien, et al., J. Clin. Invest, 92, 945-951 (1993)) that function in immune recognition and inflammatory responses, At least two potential modes of treatment are possible. In the first, transplanted organs are treated ex vivo with ribozymes or ribozyme expression vectors. Transient inhibition of NF the transplanted endothelium may be sufficient to prevent transp ,ociated vasculitis and may significantly modulate graft rejection. In the second, donor B cells are treated ex vivo with ribozymes or ribozyme expression vectors, Recipients would receive the treatment prior to transplant, Treatment of a recipient with B cells that do not express T cell co-stimulatory molecules (such as ICAM-1, VCAM-1, and/or B7 an B7-2) can induce antigen-specific anergy, Tolerance to the donor's histocompatibility antigens could result; potentially, any donor could be used for any transplantation procedure.

*Asthma.

Granulocyte macrophage colony stimulating factor (GM-CSF) is thought to play a major role in recruitment of eosinophils and othe inflammatory cells during the late phase reaction to asthmatic trauma, Again, blocking the local induction of GM-CSF and other inflammatory mediators is likely to reduce the persistent inflammation observed in chronic asthmatics. Aerosol delivery of ribozymes or adenovirus ribozyme expression vectors is a feasible treatment, *Gene Therapy, Immune responses limit the efficacy of many gene transfer techniques. Cells transfocted with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus vectors in terminally differentiated cells is longer in neonatal or immunecompromised animals, Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.

Thus, ribozymes of the present invention that cleave rel A mRNA and thereby NF-KB activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits NF-KB

M

L- -b WO 95123225 P1(I0*5/01() 39 function is described above; available cellular and activity assays are number, reproducible, and accurate, Animal models for NF-KB function (Kitajima, et al., supra) and for each of the suggested disease targets exist and can be used to optimize activity, Example 4: TNF-a Ribozymes that cleave the specific cites in TNF-a mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders.

Tumor necrosis factor-a (TNF-a) is a protein, secreted by activated leukocytes, that is a potent mediator of inflammatory reactions. Injection of TNF-a into experimental animals can simulate the symptoms of systemic and local inflammatory diseases such as septic shock or rheumatoid arthritis.

TNF-a was initially described as a factor secreted by activated macrophages which mediates the destruction of solid tumors in mice (Old, 1985 Science 230, 4225-4231). TNF-a subsequently was found to be identical to cachectin, an agent responsible for the weight loss and wasting syndrome associated with tumors and chronic infections (Beutler, et al,, 1985 Nature 316, 552-554). The cDNA and the genomic locus for TNF-a have been cloned and found to be related to TNF-B (Shakhov et al., 1990 J. Exp, Med, 171, 35-47). Both TNF-a and TNF-B bind to the same receptors and have nearly identical biological activities, The two TNF receptors have been found on most cell types examined (Smith, et al., 1990 Science 248, 1019-1023). TNF-a secretion has been detected from monocytes/macrophages, CD4+ and CD8+ T-cells, B-cells, lymphokine activated killer cells, neutrophils, astrocytes, endothelial cells, smooth muscle cells, as well as various non-hematopoletic tumor cell lines for a review see Turestskaya et al,, 1991 in Tumor Necrosis Factor: Structure, Function, and Mechanism of Action B. B. Aggarwal, J. Vilcek, Eds, Marcel Dekker, Inc., pp. 35-60), TNF-c is regulated transcriptionally and translationally, and requires proteolytic processing at the plasma membrane in order to be secreted (Kriegler et al., 1988 CCll 53, 45-53), Once secreted, the serum half life of TNF-a is approximately 30 minutes.

The tight regulation of TNF-a is important due to the extreme toxicity of this cytokine, Increasing evidence indicates that overproduction of TNF-a

I

WO 95/23225 ICTll B95.100156 during infections can lead to severe systemic toxicity and death (Tracey Cerami, 1992 Am. J. Trop. Med. Hyv. 47, 2-7).

Antisense RNA and Hammerhead ribozymes have been used in an attempt to lower the expression level of TNF-. by targeting specified cleavage sites [Sioud et al,, 1992 J. Mol. Biol. 223; 831; Sioud WO 94/10301; Kisich and co-workers, 1990 abstract (FASEB J. 4, A1860; 1991 slide presentation Leukocyte Biol, sup. 2, 70); December, 1992 poster presentation at Anti-HIV Therapeutics Conference in SanDiego, CA; and "Development of anti-TNF-a ribozymes for the control of TNF-ca gene expression"- Kisich, Doctoral Dissertation, 1993 University of California, Davis] listing various TNFa targeted ribozymes.

Ribozymes of this invention block to some extent TNF-a expression and can be used to treat disease or diagnose such disease, Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of septic shock and rheumatoid arthritis, Ribozyme cleavage of TNF-ca mRNA in these systems may prevent inflammatory cell function and alleviate disease symptoms, The sequence of human and mouse TNF-a mRNA can be screened for accessible sites using a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 23, 25, and 27 28. (All sequences are 5' to 3' in the tables,) While mouse and human sequences can be screened and ribozymes thereafter designed, the human targeted sequences are of most utility. However, mouse targeted ribozymes are useful to test efficacy of action of the ribozyme prior to testing in humans. The nucleotide base position is noted In the Tables as that site to be cleaved by the designated type of ribozyme, (In Table 24, lower case letters indicate positions that are not conserved between the human and the mouse TNF-a sequences,) The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 24, 26 28. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity, For example, stem-loop II sequence of hammerhead ribozymes listed in Tables 24 and 26 can be altered (substitution, deletion, and/or insertion) to contain any

I

WO 95/23225 PCT/IB95/00156 41 sequences provided a minimum of two base-paired stem structure can form, Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 27 and 28 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 24, 26 28 may be formed of ribonucleotides or other nucleotides or nonnucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables or AAV In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves TNF-a RNA is inserted into a plasmid DNA vector or an adenovirus DNA viral vector or AAV or alpha virus or retroviris vectors. Viral vectors have been used to transfer genes to the intact vasculature or to joints of live animals (Willard et al,, 1992 Circulation, 86, 1-473.; Nabel et al., 1990 Science, 249, 1285-1288) and both vectors lead to transient gene expression. The adenovirus vector is delivered as recombinant adenoviral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant adenovirus particles are locally administered to the site of treatment, through the use of an injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

In another preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves TNF-c RNA is inserted into a retrovirus vector for sustained expression of ribozyme(s).

By engineering ribozyme motifs we have designed several ribozymes directed against TNF-a mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave TNF-a target sequences in vitro is evaluated.

The ribozymes will be tested for function in cells by analyzing bacterial lipopolysaccharide (LPS)-induced TNF-a expression levels.

Ribozymes will be delivered to cells by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors. TNF-a expression will be monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis. TNF-a mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension I \VO 95/23225 'PC'T/I IB95/(1()15( 42 analysis or quantitative RT-PCR. Ribozymes that block the induction of TNF-a activity and/or TNF-a mRNA by more than 90% will be identified.

RNA ribozymes and/or genes encoding them will be locally delivered to macrophages by intraperitoneal injection. After a period of ribozyme uptake, the peritoneal macrophages are harvested and induced ex vivo with LPS. The ribozymes that significantly reduce TNF-a secretion are selected. The TNF-a can also be induced after ribozyme treatment with fixed Streptococcus in the peritoneal cavity instead of ex vivo. In this fashion the ability of TNF-a ribozymes to block TNF-a secretion in a localized inflammatory response are evaluated. In addition, we will determine if the ribozymes can block an ongoing inflammatory response by delivering the TNF-a ribozymes after induction by the injection of fixed Streptococcus.

To examine the effect of anti-TNF-a ribozymes on systemic inflammation, the ribozymes are delivered by intravenous injection. The ability of the ribozymes to inhibit TNF-a secretion and lethal shock caused by systemic LPS administration are assessed. Similarly, TNF-a ribozymes can be introduced into the joints of mice with collagen-induced arthritis, Either free delivery, liposome delivery, cationic lipid delivery, adenoassociated virus vector delivery, adenovirus vector delivery, retrovirus vector delivery or plasmid vector delivery in these animal model experiments can be used to supply ribozymes. One dose (or a few infrequent doses) of a stable anti-TNF-a ribozyme or a gene construct that constitutively expresses the ribozyme may abrogate tissue damage in these inflammatory diseases.

Macrophage isolation.

To produce responsive macrophages 1 ml of sterile fluid thioglycollate broth (Difco, Detroit, Ml.) was injected i.p. into 6 week old female C57bl/6NCR mice 3 days before peritoneal lavage. Mice were maintained as specific pathogen free in autoclaved cages in a laminar flow hood and given sterilized water to minimize "spontaneous" activation of macrophages. The resulting peritoneal exudate cells (PEC) were obtained by lavage using Hanks balanced salt solution (HBSS) and were plated at 2.5X10 5 /well in 96 well plates (Costar, Cambridge, MA.) with Eagles minimal essential medium (EMEM) containing 10% heat inactivated fetal esrn I WO 95/23225 'CT/1195/)00156 43 bovine serum. After adhering for 2 hours the wells were washed to remove non-adherent cells. The resulting cultures were 97% macrophages as determined by morphology and staining for non-specific esterase, Transfection of ribozymes into macrophages: The ribozymes were diluted to 2X final concentration, mixed with an equal volume of 11nM lipofectamine (Life Technologies, Gaithersburg, and vortexed. 100 ml of lipid:ribozyme complex was then added directly to the cells, followed immediately by 10 ml fetal bovine serum.

Three hours after ribozyme addition 100 ml of 1 mg/ml bacterial lipopolysaccaride (LPS) was added to each well to stimulate TNF production.

Quantitation of TNF-a in mouse macrophages: Supernatants were sampled at 0, 2, 4, 8, and 24 hours post LPS stimulation and stored at -700C. Quantitation of TNF-a was done by a specific ELISA, ELISA plates were coated with rabbit anti-mouse TNF-a serum at 1:1000 dilution (Genzyme) followed by blocking with milk proteins and incubation with TNF-a containing supernatants, TNF-a was then detected using a murine TNF-a specific hamster monoclonal antibody (Genzyme). The ELISA was developed with goat anti-hamster IgG coupled to alkaline phosphatase.

Assessment of reagent toxicity: Following ribozyme/lipid treatment of macrophages and harvesting of supernatants viability of the cells was assessed by incubation of the cells with 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). This compound is reduced by the mitochondrial dihydrogenases, the activity of which correlates well with cell viability. After 12 hours the absorbance of reduced MTT is measured at 585 nm.

Uses The association between TNF-a and bacterial sepsis, rheumatoid arthritis, and autoimmune disease make TNF-a an attractive target for therapeutic intervention [Tracy Cerami 1992 supra; Williams et al., 1992 Proc. Natl. Acad. Sci. USA 89, 9784-9788; Jacob, 1992 J. Autoimmun. (Supp. 133-143].

111 MaMMM MM WO 95123225 P(T/I i95/001.(5 44 Septic Shock Septic shock is a complication of major surgery, bacterial infection, and polytrauma characterized by high fever, increased cardiac output, reduced blood pressure and a neutrophilic infiltrate into the lungs and other major organs. Current treatment options are limited to antibiotics to reduce the bacterial load and non-steroidal anti-inflammatories to reduce fever. Despite these treatments in the best intensive care settings, mortality from septic shock averages 50%, due primarily to multiple organ failure and disseminated vascular coagulation. Septic shock, with an incidence of 200,000 cases per year in the United States, is the major cause of death in intensive care units. In septic shock syndrome, tissue injury or bacterial products initiate massive immune activation, resulting in the secretion of pro-inflammatory cytokines which are not normally detected in the serum, such as TNF-a, interleukin-11 (IL-11), y-interferon (IFN-y), interleukin-6 (ILand interleukin-8 Other non-cytokine mediators such as leukotriene b4, prostaglandin E2, C3a and C3d also reach high levels (de Boer et al., 1992 Immunopharmacology 24, 135-148).

TNF-a is detected early in the course of septic shock in a large fraction of patients (de Boer et al., 1992 supra). In animal models, injection of TNFa has been shown to induce shock-like symptoms similar to those induced by LPS injection (Beutler et al., 1985 Science 229, 869-871); in contrast, injection of IL-13, IL-6, or IL-8 does not induce shock. Injection of TNF-a also causes an elevation of IL-1B, IL-6, IL-8, PgE 2 acute phase proteins, and TxA 2 in the serum of experimental animals (de Boer et al., 1992 supra). In animal models the lethal effects of LPS can be blocked by preadministration of anti-TNF-a antibodies. The cumulative evidence indicates that TNF-a is a key player in the pathogenesis of septic shock, and therefore a good candidate for therapeutic intervention, Rheumatoid Arthritis Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints leading to bone destruction and loss of joint function. At the cellular level, autoreactive T- lymphocytes and monocytes are typically present, and the synoviocytes often have altered morphology and immunostaining patterns. RA joints have been shown to contain elevated levels of TNF-a, IL-la and IL-1B, IL-6, GM-CSF, and TGF- II~BPII~- Lc. 0 95, 23225 PC'T/1 95IIO 150 (3 (Abney et al., 1991 Imm. Rev. 119, 105-123), some or all of which may contribute to the pathological course of the disease, Cells cultured from RA joints spontaneously secrete all of the proinflammatory cytokines detected in vivo. Addition of antisera against TNF-a to these cultures has been shown to reduce IL-la/B production by these cells to undetectable levels (Abney et al,, 1991 Supra), Thus, TNF-a may directly induce the production of other cytokines in the RA joint, Addition of the anti-inflammatory cytokine, TGF-B, has no effect on cytokine secretion by RA cultures. Immunocytochemical studies of human RA surgical specimens clearly demonstrate the production of TNF-a, IL-la/3, and IL-6 from macrophages near the cartilage/pannus junction when the pannus in invading and overgrowing the cartilage (Chu et al,, 1992 Br. J.

Rheumatology 31, 653-661). GM-CSF was shown to be produced mainly by vascular endothelium in these samples. Both TNF-a ana TGF- have been shown to be fibroblast growth factors, and may contribute to the accumulation of scar tissue in the RA joint. TNF-a has also been shown to increase osteoclast activity and bone resorbtion, and may have a role in the bone erosion commonly found in the RA joint (Cooper et al., 1992 Clin.

Exp. Immunol. 89, 244-250).

Elimination of TNF-a from the rheumatic joint would be predicted to reduce overall inflammation by reducing induction of MHC class II, IL-la/B, 11-6, and GM-CSF, and reducing T-cell activation. Osteoclast activity might also fall, reducing the rate of bone erosion at the joint. Finally, elimination of TNF-a would be expected to reduce accumulation of scar tissue within the joint by removal of a fibroblast growth factor.

Treatment with an anti-TNF-a antibody reduces joint swelling and the histological severity of collagen-induced arthritis in mice (Williams et al., 1992 Proc. Natl. Acad. Sci. USA 89, 9784-9788). In addition, a study of RA patients who have received i.v, infusions of anti-TNF-a monoclonal antibody reports a reduction in the number and severity of inflamed joints after treatment. The benefit of monoclonal antibody treatment in the long term may be limited by the expense and immunogenicity of the antibody.

Psoriasis Psoriasis is an inflammatory disorder of the skin characterized by keratinocyte hyperproliferation and immune cell infiltrate (Kupper, 1990 J.

I

1ILgle I WO 95/23225 PCTIB95/(l150 46 Clin. Invest. 86, 1783-1789). It is a fairly common condition, affecting of the population. The disorder ranges in severity from mild, with small flaky patches of skin, to severe, involving inflammation of the entire epidermis. The cellular infiltrate of psoriasis includes T-lymphocytes, neutrophils, macrophages, and dermal dendrocytes. The majority of Tlymphocytes are activated CD4+ cells of the TH-1 phenotype, although some CD8+ and CD4-/CD8- are also present. B lymphocytes are typically not found in abundance in psoriatic plaques.

Numerous hypotheses have been offered as to the proximal cause of psoriasis including auto-antibodies and auto-reactive T-cells, overproduction of growth factors, and genetic predisposition. Although there is evidence to support the involvement of each of these factors in psoriasis, they are neither mutually exclusive nor are any of them necessary and sufficient for the pathogenesis of psoriasis (Reeves, 1991 Semin. Dermatol. 10, 217).

The role of cytokines in the pathogenesis of psoriasis has been investigated, Among those cytokines found to be abnormally expressed were TGF-x IL-la, IL-13, IL-1ra, IL-6, IL-8, IFN-y, and TNF-a In addition to abnormal cytokine production, elevated expression of ICAM-1, ELAM-1, and VCAM has been observed (Reeves, 1991 supra). This cytokine profile is similar to that of normal wound healing, with the notable exception that cytokine levels subside upon healing. Keratinocytes themselves have recently been shown to be capable of secreting EGF, TGF-a, IL-6, and TNF-a, which could increase proliferation in an autocrine fashion (Oxholm et 1991 APMIS 99, 58-64).

Nickoloff et al., 1993 (J Dermatol Sci. 6, 127-33) have proposed the following model for the initiation and maintenance of the psoriatic plaque: Tissue damage induces the wound healing response in the skin.

Keratinocytes secrete IL-la, IL-1B, IL-6, IL-8, TNF-a, These factors activate the endothelium of dermal capillaries, recruiting PMNs, macrophages, and T-cells into the wound site.

Dermal dendrocytes near the dermal/epidermal junction remain activated when they should return to a quiescent state, and subsequently secrete cytokines including TNF-a, IL-6, and IL-8. Cytokine expression, in gl~PII Il WO 95/23225 PC"171I395/00(156 47 turn, maintains the activated state of the endothelium, allowing extravasation of additional immunocytes, and the activated state of the keratinocytes which secrete TGF-a and IL-8. Keratinocyte IL-8 recruits immunocytes from the dermis into the epidermis. During passage through the dermis, T-cells encounter the activated dermal dendrocytes which efficiently activate the TH-1 phenotype, The activated T-cells continue to migrate into the epidermis, where they are stimulated by keratinocyteexpressed ICAM-1 and MHC class II. IFN-y secreted by the T-cells synergizes with the TNF-a from dermal dendrocytes to increase keratinocyte proliferation and the levels of TGF-o, IL-8, and IL-6 production.

IFN-y also feeds back to the dermal dendrocyte, maintaining the activated phenotype and the inflammatory cycle.

Elevated serum titres of IL-6 increases synthesis of acute phase proteins including complement factors by the liver, and antibody production by plasma cells, Increased complement and antibody levels increases the probability of autoimmune reactions.

Maintenance of the psoriatic plaque requires continued expression of all of these processes, but attractive points of therapeutic intervention are TNF-a expression by the dermal dendrocyte to maintain activated endothelium and keratinocytes, and IFN-y expression by T-cells to maintain activated dermal dendrocytes.

There are 3 million patients in the United States afflicted with psoriasis. The available treatments for psoriasis are corticosteroids. The most widely prescribed are TEMOVATE (clobetasol propionate), LIDEX (fluocinonide), DIPROLENE (betamethasone propionate), PSORCON (diflorasone diacetate) and TRIAMCINOLONE formulated for topical application, The mechanism of action of corticosteroids is multifactorial, This is a palliative therapy because the underlying cause of the disease remains, and upon discontinuation of the treatment the disease returns.

Discontinuation of treatment is often prompted by the appearance of adverse effects such as atrophy, telangiectasias and purpura, Corticosteroids are not recommended for prolonged treatments or when treatment of large and/or inflamed areas is required. Alternative treatments include retinoids, such as etretinate, which has been approved for treatment of severe, refractory psoriasis. Alternative retinoid-based treatments are in advanced clinical trials. Retinoids act by converting r BBae~r I W\O 95/23225 1C1/Ii/Illl90015 48 keratinocytes to a differentiated state and restoration of normal skin development. Immunosuppressive drugs such as cyclosporine are also in the advanced stages of clinical trials. Due to the nonspecific mechanism of action of corticosteroids, retinoids and immunosuppressives, these treatments exhibit severe side effects and should not be used for extended periods of time unless the condition is life-threatening or disabling. There is a need for a less toxic, effective therapeutic agent in psoriatic patients.

HIV and AIDS The human immunodeficiency virus (HIV) causes several fundamental changes in the human immune system from the time of infection until the development of full-blown acquired immunodeficiency syndrome (AIDS). These changes include a shift in the ratio of CD4+ to CD8+ T-cells, sustained elevation of IL-4 levels, episodic elevation of TNFa and TNF-1 levels, hypergammaglobulinemia, and lymphoma/leukemia (Rosenberg Fauci, 1990 Immun. Today 11, 176; Weiss 1993 Science 260, 1273). Many patients experience a unique tumor, Kaposi's sarcoma and/or unusual opportunistic infections Pneumocystis carinii, cytomegalovirus, herpesviruses, hepatitis viruses, papilloma viruses, and tuberculosis). The immunological dysfunction of individuals with AIDS suggests that some of the pathology may be due to cytokine dysregulation.

Levels of serum TNF-a and IL-6 are often found to be elevated in AIDS patients (Weiss, 1993 supra). In tissue culture, HIV infection of monocytes isolated from healthy individuals stimulates secretion of both TNF-a and IL-6. This response has been reproduced using purified gp120, the viral coat protein responsible for binding to CD-4 (Buonaguro et al., 1992 J. Virol. 66, 7159). It has also been demonstrated that the viral gene regulator, Tat, can directly induce TNF transcription. The ability of HIV to directly stimulate secretion of TNF-a and IL-6 may be an adaptive mechanism of the virus. TNF-a has been shown to upregulate transcription of the LTR of HIV, increasing the number of HIV-specific transcripts in infected cells. IL-6 enhances HIV production, but at a post-transcriptional level, apparently increasing the efficiency with which HIV transcripts are translated into protein. Thus, stimulation of TNF-a secretion by the HIV virus maj promote infection of neighboring CD4+ cells both by enhancing virus production from latently infected cells and by driving replication of the virus in newly infected cells.

~1 0 9523225 P('T/195/00150 49 The role of TNF-a in HIV replication has been well established in tissue culture models of infection (Sher et al,, 1992 Immun. Rev. 127, 183), suggesting that the mutual induction of HIV replication and TNF-a replication may create positive feedback in vivo. However, evidence for the presence of such positive feedback in infected patients is not abundant.

TNF-a levels are found to be elevated in some, but not all patients tested, Children with AIDS who were given zidovudine had reduced levels of TNFc compared to those not given zidovudine (Cremoni et al., 1993 AIDS 7, 128), This correlation lends support to the hypothesis that reduced viral replication is physiologically linked to TNF-a levels, Furthermore, recently it has been shown that the polyclonal B cell activation associated with HIV infection is due to membrane-bound TNF-a. Thus, levels of secreted TNF-a may not accurately reflect the contribution of this cytokine to AIDS pathogenesis, Chronic elevation of TNF-a has been shown to shown to result in cachexia (Tracey et al., 1992 Am. J. Trop. Med. Hyg. 47, increased autoimmune disease (Jacob, 1992 supra), lethargy, and immune suppression in animal models (Aderka et al., 1992 Isr. J. Med. Sci. 28, 126- 130), The cachexia associated with AIDS may be associated with chronically elevated TNF-a frequently observed in AIDS patients.

Similarly, TNF-a can stimulate the proliferation of spindle cells isolated from Kaposi's sarcoma lesions of AIDS patients (Barillari et al., 1992 J Immunol 149, 3727), A therapeutic agent that inhibits cytokine gene expression, inhibits adhesion molecule expression, and mimics the anti-inflammatory effects of glucocorticoids (without inducing steroid-responsive genes) is ideal for the treatment of inflammatory and autoimmune disorders, Disease targets for such a drug are numerous. Target indications and the delivery options each entails are summarized below, In all cases, because of the potential immunosuppressive properties of a ribozyme that cleaves the specified sites in TNF-a mRNA, uses are limited to local delivery, acute indications, or ex vivo treatment.

*Septic shock.

I

1 6 \VO 95/23225 I'(T71195/((I0015(0 Exogenous delivery of ribozymes to macrophages can be achieved by intraperitoneal or intravenous injections. Ribozymes will be delivered by incorporation into liposomes or by complexing with cationic lipids.

*Rheumatoid arthritis (RA), Due to the chronic nature of RA, a gene therapy approach is logical.

Delivery of a ribozyme to inflamed joints is mediated by adenovirus, retrovirus, or adeno-associated virus vectors. For instance, the appropriate adenovirus vector can be administered by direct injection into the synovium: high efficiency of gene transfer and expression for several months would be expected Roessler, ED. Allen, J.M. Wilson, J.W, Hartman, B, L, Davidson, J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the course of the disease could be reversed by the transient, local administration of an anti-inflammatory agent. Multiple administrations may be necessary. Retrovirus and adeno-associated virus vectors would lead to permanent gene transfer and expression in the joint, However, permanent expression of a potent anti-inflammatory agent may lead to local immune deficiency.

*Psoriasis The psoriatic plaque is a particularly good candidate for ribozyme or vector delivery. The stratum corneum of the plaque is thinned, providing access to the proliferating keratinocytes. T-cells and dermal dendrocytes can be efficiently targeted by trans-epidermal diffusion Organ culture systems for biopsy specimens of psoriatic and normal skin are described in current literature (Nickoloff et al,, 1993 Supra).

Primary human keratinocytes are easily obtained and will be grown into epidermal sheets in tissue culture. In addition to these tissue culture models, the flaky skin mouse develops psoriatic skin in response to UV light. This model would allow demonstration of animal efficacy for ribozyme treatments of psoriasis.

*Gene Therapy.

Immune responses limit the efficacy of many gene transfer techniques. Cells transfected with retrovirus vectors have short lifetimes in immune competent individuals. The length of expression of adenovirus ill l~l~AR~3"1 ~I \VO 95/23225 I/1 ii95/011015( 51 vectors in terminally differentiated cells is longer in neonatal or immunecompromised animals. Insertion of a small ribozyme expression cassette that modulates inflammatory and immune responses into existing adenovirus or retrovirus constructs will greatly enhance their potential.

Thus, ribozymes of the present invention that cleave TNF-a mRNA and thereby TNF-a activity have many potential therapeutic uses, and there are reasonable modes of delivering the ribozymes in a number of the possible indications. Development of an effective ribozyme that inhibits TNF-a function is described above; available cellular and activity assays are number, reproducible, and accurate, Animal models for TNF-a function and for each of the suggested disease targets exist and can be used to optimize activity.

Example 5: p210 bc rab l Chronic myelogenous leukemia exhibits a characteristic disease course, presenting initially as a chronic granulocytic hyperplasia, and invariably evolving into an acute leukemia which is caused by the clonal expansion of a cell with a less differentiated phenotype the blast crisis stage of the disease), CML is an unstable disease which ultimately progresses tu a terminal stage which ,esembles acute leukemia, This lethal disease affects approximately 16,000 patients a year, Chemotherapeutic agents such as hydroxyurea or busulfan can reduce the leukemic burden but do not impact the life expectancy of the patient (e.

approximately 4 years). Consequently, CML patients are candidates for bone marrow transplantation (BMT) therapy. However, for those patients which survive BMT, disease recurrence remains a major obstacle (Apperley et al,, 1988 Br. J. Haematol. 69, 239), The Philadelphia (Ph) chromosome which results from the translocation of the abl oncogene from chromosome 9 to the bcr gene on chromosome 22 is found in greater than 95% of CML patients and in 25% of all cases of acute lymphoblastic leukemia Fourth International Workshop on Chromosomes in Leukemia 1982, Cancer Genet. Cvtogenet. 11, 316], In virtually all Ph-positive CMLs and approximately 50% of the Ph-positive ALLs, the leukemic cells express bcrabl fusion mRNAs in which exon 2 (b2-a2 junction) or exon 3 (b3-a2 junction) from the major breakpoint cluster region of the bcr gene is spliced

I

I--I 9 2.322 IP( '7 52 to exon 2 of the abl gene, Heisterkamp et al., 1985 Nature 315, 758; Shtivelman et al,, 1987, Blood 69, 971), In the remaining cases of Phpositive ALL, the first exon of the bcr gene is spliced to exon 2 of the abl gene (Hooberman et al,, 1989 Proc. Nat. Acad. Sci. USA 86, 4259; Heisterkamp et al., 1988 Nucleic Acids Res. 16, 10069), The b3-a2 and b2-a2 fusion mRNAs encode 210 kd bcr-abl fusion proteins which exhibit oncogenic activity (Daley et al,, 1990 Science 247, 824; Heisterkamp et al., 1990 Nature 344, 251), The importance of the bcrabl fusion protein (p210bcr-ab l in the evolution and maintenance of the leukemic phenotype in human disease has been demonstrated using antisense oligonucleotide inhibition of p 2 1 0 bcr-abl expression. These inhibitory molecules have been shown to inhibit the in vitro proliferation of leukemic cells in bone marrow from CML patients. Szczylik et al., 1991 Science 253, 562).

Reddy, U.S. Patent 5,246,921 (hereby incorporated by reference herein) describes use of ribozymes as therapeutic agents for leukemias, such as chronic myelogenous leukemia (CML) by targeting the specific junction region of bcr-abl fusion transcripts, It indicates causing cleavage by a ribozyme at or near the breakpoint of such a hybrid chromosome, specifically it includes cleavage at the sealoence GUX, where X is A, U or G. The one example presented is to cuiave the sequence 5' AGC AG AGUU (cleavage site) CAA AAGCCCU-3'.

Scanlon WO 91/18625, WO 91/18624, and WO 91/18913 and Snyder et al., W093/03141 and W094/13793 describe a ribozyme effective to cleave oncogenic variants of H-ras RNA. This ribozyme is said to inhibit H-ras expression in response to external stimuli.

The invention features use of ribozymes to inhibit the development or expression of a transformed phenotype in man and other animals by modulating expression of a gene that contributes to the expression of CML.

Cleavage of targeted mRNAs expressed in pre-neoplastic and transformed cells elicits inhibition of the transformed state.

The invention can be used to treat cancer or pre-neoplastic conditions. Two preferred administration protocols can be used, either in vivo administration to reduce the tumor burden, or ex vivo treatment to L WO 95/23225 PI'(Tl l B500) 53 eradicate transformed cells from tissues such as bone marrow prior to reirnplantation, This inventior features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associated with development or maintenance of CML, The mRNA targets are present in the 425 nucleotides surrounding the fusion sites of the bcr and abl sequences in the b2-a2 and b3-a2 recombinant mRNAs, Other sequences in the 5' portion of the bcrmRNA or the 3' portion of the abl mRNA may also be targeted for ribozyme cleavage.

Cleavage at any of these sites in the fusion mRNA molecules will result in inhibition of translation of the fusion protein in treated cells, The invention provides a class of chemical cleaving agents which exhibit a high degree of specificity for the mRNA causative of CML. Such enzymatic RNA molecules can be delivered exogenously or endogenously to afflicted cells. In the preferred hammerhead motif the small size (less than 40 nucleotides, preferably between 32 and 36 nucleotides in length) of the molecule allows the cost of treatment to be reduced.

The smallest ribozyme delivered for any type of treatment reported to date (by Rossi et al, 1992 supra) is an in vitro transcript having a length of 142 nucleotides, Synthesis of ribozymes greater than 100 nucleotides in length is very difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. Delivery of ribozymes by expression vectors is primarily feasible using only ex vivo treatments, This limits the utility of this approach. In this invention, an alternative approach uses smaller ribozyme motifs and exogenous delivery. The simple structure of these molecules also increases the ability of the ribozyme to invade targeted regions of the mRNA structure, Thus, unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-ribozyme flanking sequences to interfere with correct folding of the ribozyme structure, as well as complementary binding of the ribozyme to the mRNA target.

The enzymatic RNA molecules of this invention can be used to treat human CML or precancerous conditions. Affected animals can be treated at the time of cancer detection or in a prophylactic manner. This timing of treatment will reduce the number of affected cells and disable cellular II_ C~ 95/23225 PC"l'-111S'lill50 54 replication. This is possible because the ribozymes are designed to disable those structures required for successful cellular proliferation.

Ribozymes of this invention block to some extent p210bcr'ab l expression and can be used to treat disease or diagnose such disease.

Ribozymes will be delivered to cells in culture and to tissues in animal models of CML. Ribozyme cleavage of bcr/abl mRNA in these systems may prevent or alleviate disease symptoms or conditions.

The sequence of human bcr/abl mRNA can be screened for accessible sites using a computer folding algorithm. Regions of the mRNA that did not form secondary folding structures and that contain potential hammerhead or hairpin ribozyme cleavage sites can be identified. These sites are shown in Table 29 (All sequences are 5' to 3' in the tables), The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

The sequences of the chemically synthesized ribozymes most useful in this study are shown in Table 30. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Table 30 (5'-GGCCGAAAGGCC-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence provided, a minimum of two base-paired stem structure can form, The sequences listed in Tables 30 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables, By engineering ribozyme motifs we have designed several ribozymes directed against bcr-abl mRNA sequences, These have been synthesized with modifications that improve their nuclease resistance as described above, These ribozymes cleave bcr-abl target sequences in vitro.

The ribozymes are tested for function in vivo by exogenous delivery to cells expressing bcr-abl, Ribozymes are delivered by incorporation into liposomes, by complexing with cationic lipids, by microinjection, or by expression from DNA vectors, Expression of bcr-abl is monitored by ELISA, by indirect immunofluoresence, and/or by FACS analysis, Levels of WO 95/23225 PITII 95!00150 bcr-abl mRNA are assessed by Northern analysis, RNase protection, by primer extension analysis or by quantitative RT-PCR techniques.

Ribozymes that block the induction of p210bc r a b l protein and mRNA by more than 20% are identified.

Example 6: RSV This invention relates to the use of ribozymes as inhibitors of respiratory syncytial virus (RSV) production, and in particular, the inhibition of RSV replication.

RSV is a member of the virus family paramyxoviridae and is classified under the genus Pneumovirus (for a review see Mclntosh and Chanock, 1990 in Virology ed. B.N. Fields, pp. 1045, Raven Press Ltd, NY). The infectious virus particle is composed of a nucleocapsid enclosed within an envelope. The nucleocapsid is composed of a linear negative singlestranded non-segmented RNA associated with repeating subunits of capsid proteins to form a compact structure and thereby protect the RNA from nuclease degradation. The entire nucleocapsid is enclosed by the envelope. The size of the virus particle ranges from 150 300 nm in diameter. The complete life cycle of RSV takes place in the cytoplasm of infected cells and the nucleocapsid never reaches the nuclear compartment (Hall, 1990 in Principles and Practice of Infectious Diseases ed. Mandell et al., Churchill Livingstone, NY).

The RSV genome encodes ten viral proteins essential for viral production. RSV protein products include two structural glycoproteins (G and F) found in the envelope spikes, two matrix proteins [M and M2 (22K)] found in the inner membrane, three proteins localized in the nucleocapsid P and one protein that is present on the surface of the infected cell and two nonstructural proteins [NS1 (1C) and NS2 found only in the infected cell. The mRNAs for the 10 RSV proteins have similar and 3' ends. UV-inactivation studies suggest that a single promoter is used with multiple transcription initiation sites (Barik et al., 1992 J. Virol. 66, 6813), The order of transcription corresponding to the protein assignment on the genomic RNA is 1C, 1B, N, P, M, SH, G, F, 22K and L genes (Huang et al., 1985 Virus Res. 2, 157) and transcript abundance corresponds to the order of gene assignment (for example the 1C and 1B mRNAs are much more abundant than the L mRNA. Synthesis of viral message begins

I

~BRrPe WVO 95/23225 P(T/IB95/0(15(.

56 immediately after RSV infection of cells and reaches a maximum at 14 hours post-infection (Mclntosh and Chanock, supra), There are two antigenic subgroups of RSV, A and B, which can circulate simultaneously in the community in varying proportions in different years (Mclntosh and Chanock, supra). Subgroup A usually predominates.

Within the two subgroups there are numerous strains. By the limited sequence analysis available It seems that homology at the nucleotide level is more complete within than between subgroups, although sequence divergence has been noted within subgroups as well. Antigenic determinates result primarily from both surface glycoproteins, F and G. For F, at least half of the neutralization epitopes have been stably maintained over a period of 30 years. For G however, A and B subgroups may be related antigenically by as little as a few percent. On the nucleotide level, however, the majority of the divergence in the coding region of G is found in the sequence for the extracellular domain (Johnson et al,, 1987, Proc.

NatL Acad. Sci. USA 84, 5625).

Respiratory Syncytial Virus (RSV) is the major cause of lower respiratory tract illness during infancy and childhood (Hall, supra) and as such is associated with an estimated 90,000 hospitalizations and 4500 deaths in the United States alone (Update: respiratory syncytial virus activity United States, 1993, Mmwr Morb Mortal Wkly Rep, 42, 971).

Infection with RSV generally outranks all other microbial agents leading to both pneumonia and bronchitis. While primarily affecting children under two years of age, immunity is not complete and reinfection of older children and adults, especially hospital care givers (Mclntosh and Chanock, supra), is not uncommon. Immunocompromised patients are severely affected and RSV infection is a major complication for patients undergoing bone marrow transplantation Uneventful RSV respiratory disease resembles a common cold and recovery is in 7 to 12 days, Initial symptoms (rhinorrhea, nasal congestion, slight fever, etc.) are followed in 1 to 3 days by lower respiratory tract signs of infection that include a cough and wheezing. In severe cases, these mild symptoms quickly progress to tachypnea, cyanosis, and listlessness and hospitalization is required. In infants with underlying cardiac or respiratory disease, the progression of symptoms is especially rapid and can lead to respiratory failure by the second or third day of illness. With _I WO 95123225 PCT/II95/0I156 57 modern intensive care however, overall mortality is usually less than 5% of hospitalized patients (Mclntosh and Chanock, supra).

At present, neither an efficient vaccine nor a specific antiviral agent is available. An immune response to the viral surface glycoproteins can provide resistance to RSV in a number of experimental animals, and a subunit vaccine has been shown to be effective for up to 6 months in children previously hospitalized with an RSV infection (Tristam et al., 1993, J. Infect. Dis. 167, 191). An attenuated bovine RSV vaccine has also been shown to be effective in calves for a similar length of time (Kubota et al,, 1992 J. Vet, Med. Sci, 54, 957). Previously however, a formalin-inactivated RSV vaccine was implicated in greater frequency of severe disease in subsequent natural infections with RSV (Connors et al., 1992 J. Virol. 66, 7444).

The current treatment for RSV infection requiring hospitalization is the use of aerosolized ribavirin, a guanosine analog [Antiviral Agents and Viral Diseases of Man, 3rd edition, 1990. (eds. G.J. Galasso, R.J, Whitley, and T.C. Merigan) Raven Press Ltd., Ribavirin therapy is associated with a decrease in the severity of the symptoms, improved arterial oxygen and a decrease in the amount of viral shedding at the end of the treatment period. It is not certain, however, whether ribavirin therapy actually shortens the patients' hospital stay or diminishes the need for supportive therapies (Mclntosh and Chanock, supra). The benefits of ribavirin therapy are especialy clear for high risk infants, those with the most serious symptoms or for patients with underlying bronchopulmonary or cardiac disease, Inhibition of the viral polymerase complex is supported as the main mechanism for inhibition of RSV by ribavirin, since viral but not cellular polypeptide synthesis is inhibited by ribavirin in RSV-infected cells (Antiviral Agents and Viral Diseases of Man, 3rd edition, 1990. (eds, G,J.

Galasso, R.J. Whitley, and T.C, Merigan) Raven Press Ltd., NY], Since ribavirin is at least partially effective against RSV infection when delivered by aerosolization, it can be assumed that the target cells are at or near the epithelial surface, In this regard, RSV antigen had not spread any deeper than the superficial layers of the respiratory epithelium in autopsy studies of fatal pneumonia (Mclntosh and Chanock, supra).

Jennings et al,, WO 94/13688 indicates that targets for specific types of ribozymes include respiratory syncytical virus.

I~i~RPls~sa W\O 95!23225 PCT I B95(ci 58 The invention features novel enzymatic RNA molecules, or ribozymes, and methods for their use for inhibiting production of respiratory syncytial virus (RSV). Such ribozymes can be used in a method for treatment of diseases caused by these related viruses in man and other animals. The invention also features cleavage of the genomic RNA and mRNA of these viruses by use of ribozymes. In particular, the ribozyme molecules described are targeted to the NS1 NS2 (1B) and N viral genes.

These genes are known in the art (for a review see Mclntosh and Chanock, 1990 supra Ribozymes that cleave the specified sites in RSV mRNAs represent a novel therapeutic approach to respiratory disorders. Applicant indicates that ribozymes are able to inhibit the activity of RSV and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave these sites in RSV mRNAs encoding 1C, 1B and N proteins may be readily designed and are within the invention.

Also, those of ordinary skill in the art, will find that it is clear from the examples described that ribozymes cleaving other mRNAs encoded by RSV M, SH, G, F, 22K and L) and the genomic RNA may be readily designed and are within the invention.

In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables 31, 33, 35, 37 and 38.

Examples of such ribozymes are shown in Tables 32, 34, 36-38. Examples of such ribozymes consist essentially of sequences defined in these Tables. By "consists essentially of" is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage, Ribozymes of this invention block to some extent RSV production and can be used to treat disease or diagnose such disease, Ribozymes will be delivered to cells in culture and to cells or tissues in animal models of respiratory disorders, Ribozyme cleavage of RSV encoded mRNAs or the genomic RNA in these systems may alleviate disease symptoms.

I

95/23225 I)C"r B95/o00156 59 While all ten RSV encoded proteins (1C, 1B, N, P, M, SH, 22K, F, G, and L) are essential for viral life cycle and are all potential targets for ribozyme cleavage, certain proteins (mRNAs) are more favorable for ribozyme targeting than the others. For example RSV encoded proteins 1C, 1B, SH and 22K are not found in other members of the family paramyxoviridae and appear to be unique to RSV, In contrast the ectodomain of the G protein and the signal sequence of the F protein show significant sequence divergence at the nucleotide level among various RSV sub-groups (Johnson et al., 1987 supra), RSV proteins 1C, 1B and N are highly conserved among various subtypes at both the nucleotide and amino acid levels. Also, 1C, 1B and N are the most abundant of all RSV proteins.

The sequence of human RSV mRNAs encoding 1C, 1B and N proteins are screened for accessible sites using a computer folding algorithm, Hammerhead or hairpin ribozyme cleavage sites were identified. These sites are shown in Tables 31, 33, 34, 37 and 38 (All sequences are 5' to 3' in the tables.) The nucleotide base position is noted in the Tables as that site to be cleaved by the designated type of ribozyme.

Ribozymes of the hammerhead or hairpin motif are designed to anneal to various sites in the mRNA message. The binding arms are complementary to the target site sequences described above, The ribozymes are chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., 1987 J, Am, Chem. Soc., 109, 7845-7854 and in Scaringe et al., 1990 Nucleic Acids Res., 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The average stepwise coupling yields were Inactive ribozymes were synthesized by substituting a U for G5 and a U for A14 (numbering from Hertel et al., 1992 Nucleic Acids Res,, 3252). Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840). Hairpin ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51). All ribozymes are modified extensively to enhance stability by modification with nuclease resistant i WO 95/23225 P(cT/1B95/(0015(> groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes are purified by gel electrophoresis using general methods or are purified by high pressure liquid chromatography and are resuspended in water.

The sequences of the chemically synthesized ribozymes useful in this study are shown in Tables 32, 34, 36, 37 and 38. Those in the art will recognize that these sequences are representative only of many more such sequences where the enzymatic portion of the ribozyme (all but the binding arms) is altered to affect activity. For example, stem-loop II sequence of hammerhead ribozymes listed in Tables 32 and 34(5'-GGCCGAAAGGCCcan be altered (substitution, deletion, and/or insertion) to contain any sequences provided a minimum of two base-paired stem structure can form. Similarly, stem-loop IV sequence of hairpin ribozymes listed in Tables 37 and 38 (5'-CACGUUGUG-3') can be altered (substitution, deletion, and/or insertion) to contain any sequence, provided a minimum of two base-paired stem structure can form. The sequences listed in Tables 32, 34, 36, 37 and 38 may be formed of ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes are equivalent to the ribozymes described specifically in the Tables.

By engineering ribozyme motifs we have designed several ribozymes directed against RSV encoded mRNA sequences. These ribozymes are synthesized with modifications that improve their nuclease resistance. The ability of ribozymes to cleave target sequences in vitro is evaluated.

Numerous common cell lines can be infected with RSV for experimental purposes. These include HeLa, Vero and several primary epithelial cell lines. A cotton rat animal model of experimental human RSV infection is also available, and the bovine RSV is quite homologous to the human viruses, Rapid clinical diagnosis is through the use of kits designed for the immunofluorescence staining of RSV-infected cells or an ELISA assay, both of which are adaptable for experimental study. RSV encoded mRNA levels will be assessed by Northern analysis, RNAse protection, primer extension analysis or quantitative RT-PCR. Ribozymes that block the induction of RSV activity and/or 1C, 1B and N protein encoding mRNAs by more than 90% will be identified.

I

I~Y- WVO 95/23225 PCT/ 1 B95/00156 61 Optimizing Ribozyme Activity Ribozyme activity can be optimized as described by Draper et al,, PCT W093/23569, The details will not be repeated here, but include altering the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see Eckstein et al., International Publication No.

WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci, 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al,, International Publication No. WO 91/03162, as well as Jennings et al., WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules. All these publications are hereby incorporated by reference herein.), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

Sullivan, et al., PCT W094/02595, incorporated by reference herein, describes the general methods for delivery of enzymatic RNA molecules Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. The RNA/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.

Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Sullivan, et al., supra and Draper, et al,, supra which have been incorporated by reference herein.

Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given r SlllY~I*16 I~ll~ W\O 95/23225 P('T/Il95/00) 62 pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.

Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Enzvmol,, 217, 47-66; Zhou et al,, 1990 Mol. Cell. Biol., 10, 4529- 37). Several investigators have demonstrated that ribozymes expressed from such promoters can function in mammalian cells Kashani-Sabet et al., 1992 Antisense Res. Dev,, 2, 3-15; Ojwang et al., 1992 Proc. Natl.

Acad. Sci. U S A, 89, 10802-6; Chen et al., 1992 Nucleic Acids Res., 4581-9; Yu et al., 1993 Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al., 1992 EMBO J. 11,4411-8; Lisziewicz et al., 1993 Proc. Natl. Acad.

Sci. U. S. 90, 8000-4). The above ribozyme transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral, or alpha virus vectors).

In a preferred embodiment of the invention, a transcription unit expressing a ribozyme that cleaves target RNA is inserted into a plasmid DNA vector, a retrovirus DNA viral vector, an adenovirus DNA viral vector or an adeno-associated virus vector or alpha virus vector. These and other vectors have been used to transfer genes to live animals (for a review see Friedman, 1989 Science 244, 1275-1281; Roemer and Friedman, 1992 Eur. J. Biochem. 208, 211-225) and leads to transient or stable gene expression. The vectors are delivered as recombinant viral particles. DNA may be delivered alone or complexed with vehicles (as described for RNA above). The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, through the use of a catheter, stent or infusion pump.

Diagnostic uses Ribozymes of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By \\X0 95/23225 l(T/I B95(100 63 using multiple ribozymes described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease, In this manner, other genetic targets may be defined as important mediators of the disease, These experiments will lead to better treatment of the disease progression by affording the possibility of combinational therapies multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules).

Other in vitro uses of ribozymes of this invention are well known in the art, and include detection of the presence of mRNA associated with ICAM-1, relA, TNF-a, p210, bcr-abl or RSV related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type or mutant forms of the target RNA are used for the assay. The first ribozyme is used to identify wild-type RNA present in the sample and the second ribozyme will be used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme efficiencies in the reactions and the absence of cleavage of the "nontargeted" RNA species. The cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wildtype and mutant RNAs in the sample population. Thus each analysis will require two ribozymes, two substrates and one unknown sample which will be combined into six reactions, The presence of cleavage products will be determined using an RNAse protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype ICAM-1, rel A, TNF, p2o10 b c r-abl or RSV) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels will be adequate and will I_ WO 95/23225 P(T/ 1B95/()19 156 64 decrease the cost of the initial diagnosis. Higher mutant form to wild-type ratios will be correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

II. Chemical Synthesis Of Ribozymes There follows the chemical synthesis, deprotection, and purification of RNA, enzymatic RNA or modified RNA molecules in greater than milligram quantities with high biological activity. Applicant has determined that the synthesis of enzymatically active RNA in high yield and quantity is dependent upon certain critical steps used during its preparation.

Specifically, it is important that the RNA phosphoramidites are coupled efficiently in terms of both yield and time, that correct exocyclic amino protecting groups be used, that the appropriate conditions for the removal of the exocyclic amino protecting groups and the alkylsilyl protczting groups on the 2'-hydroxyl are used, and that the correct work-up and purification procedure of the resulting ribozyme be used.

To obtain a correct synthesis in terms of yield and biological activity of a large RNA molecule about 30 to 40 nucleotide bases), the protection of the amino functions of the bases requires either amide or substituted amide protecting groups, which must be, on the one hand, stable enough to survive the conditions of synthesis, and on the other hand, removable at the end of the synthesis. These requirements are met by the amide protecting groups shown in Figure 8, in particular, benzoyl for adenosine, isobutyryl or benzoyl for cytidine, and isobutyryl for guanosine, which may be removed at the end of the synthesis by incubating the RNA in NH3/EtOH (ethanolic ammonia) for 20 h at 65 In the case of the phenoxyacetyl type protecting groups shown in Figure 8 on guanosine and adenosine and acetyl protecting groups on cytidine, an incubation in ethanolic ammonia for 4 h at 65 "C iu used to obtain complete removal of these protecting groups. Removal of the alkylsilyl 2'-hydroxyl protecting groups can be accomplished using a tetrahydrofuran solution of TBAF at room temperature for 8-24 h.

The most quantitative procedure for recovering the fully deprotected RNA molecule is by either ethanol precipitation, or an anion exchange cartridge desalting, as described in Scaringe et al. Nucleic Acids Res.

1990, 18, 5433-5341. The purification of the long RNA sequences may be

I

11~1~ WO 95/23225 IC(1Tll 95/0015( accomplished by a two-step chromatographic procedure in which the molecule is first purified on a reverse phase column with either the trityl group at the 5' position on or off, This purification is accomplished using an acetonitrile gradient with triethylammonium or bicarbonate salts as the aqueous phase, In the case of the trityl on purification, the trityl group may be removed by the addition of an acid and drying of the partially purified RNA molecule. The final purification is carried out on an anion exchange column, using alkali metal perchlorate salt gradients to elute the fully purified RNA molecule as the appropriate metal salts, e.g. Na Li etc. A final de-salting step on a small reverse-phase cartridge completes the purification procedure. Applicant has found that such a procedure not only fails to adversely affect activity of a ribozyme, but may improve its activity to cleave target RNA molecules.

Applicant has also determined that significant (see Tables 39-41) improvements in the yield of desired full length product (FLP) can be obtained by: 1. Using 5-S-alkyltetrazole at a delivered or effective concentration of 0.25-0.5 M or 0.15-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. (By delivered is meant that the actual amount of chemical in the reaction mix is known. This is possible for large scale synthesis since the reaction vessel is of size sufficient to allow such manipulations. The term effective means that available amount of chemical actually provided to the reaction mixture that is able to react with the other reagents present in the mixture. Those skilled in the art will recognize the meaning of these terms from the examples provided herein.) The time for this step is shortened from 10-15 m, vide supra, to 5-10 m.

Alkyl, as used herein, refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons, More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted, When substitu'ed the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO 2 or N(CH 3 2 amino, or SH.

The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to g r ~y B"arramu- ~r~lu~I~ '5!23225 IT21I B<05/(0) 66 7 carbons, more preferably 1 to 4 carbons, The alkenyl group may be substituted or unsubstituted, When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO 2 halogen, N(CH 3 2 amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.

Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO 2 or

N(CH

3 2 amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated 7 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms, Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an wnere R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen.

2. Using 5-S-alkyltetrazole at an effective, or final, concentration of 0.1-0.35 M for the activation of the RNA (or analogue) amidite during the coupling step. The time for this step is shortened from 10-15 m, vide supra, to 5-10 m.

3. Using alkylamine (MA, where alkyl is preferably methyl, ethyl, propyl or butyl) or NH40H/alkylamine (AMA, with the same preferred alkyl groups as noted for MA) 65 °C for 10-15 m to remove the exocyclic \WO 95/23225 PCT'I B 95!( 67 amino protecting groups (vs 4-20 h 55-65 °C using NH 4 0H/EtOH or

NH

3 /EtOH, vide supra), Other alkylamines, e.g. ethylamine, propylamine, butylamine etc, may also be used, 4. Using anhydrous triethylamine*hydrogen fluoride (aHF*TEA) 65 °C for 0,5-1.5 h to remove the 2'-hydroxyl alkylsilyl protecting group (vs 8 24 h using TBAF, vide supra or TEA*3HF )r 24 h (Gasparutto et al.

Nucleic Acids Res. 1992, 20, 5159-5166), Other alkylamine*HF complexes may also be used, e.g. trimethylamine or diisopropylethylamine.

The use of anion-exchange resins to purify and/or analyze the fully deprotected RNA. These resins include, but are not limited to, quartenary or tertiary amino derivatized stationary phases such as silica or polystyrene. Specific examples include Dionex-NA100®, Mono-Q®, Poros-

Q®.

Thus, the invention features an improved method for the coupling of RNA phosphoramidites; for the removal of amide or substituted amide protecting groups; and for the removal of 2'-hydroxyl alkylsilyl protecting groups. Such methods enhance the production of RNA or analogs of the type described above with substituted 2'-groups), and allow efficient synthesis of large amounts of such RNA. Such RNA may also have enzymatic activity and be purified without loss of that activity. While specific examples are given herein, those in the art will recognize that equivalent chemical reactions can be performed with the alternative chemicals noted above, which can be optimized and selected by routine experimentation.

In another aspect, the invention features an improved method for the purification or analysis of RNA or enzymatic RNA molecules 28-70 nucleotides in length) by passing said RNA or enzymatic RNA molecule over an HPLC, reverse phase and/or an anion exchange chromatography column, The method of purification improves the catalytic activity of enzymatic RNAs over the gel purification method (see Figure Draper et al., PCT WO93/23569, incorporated by reference herein, disclosed reverse phase HPLC purification, The purification of long RNA molecules may be accomplished using anion exchange chromatography, particularly in conjunction with alkali perchlorate salts. This system may be used to purify very long RNA molecules, In particular, it is advantageous to ~r~aL~ I I WO 95/23225 IcT1 1195100156 68 use a Dionex NucleoPak 100© or a Pharmacia Mono Q® anion exchange column for the purification of RNA by the anion exchange method. This anion exchange purification may be used following a reverse-phase purification or prior to reverse phase purification. This method results in the formation of a sodium salt of the ribozyme during the chromatography.

Replacement of the sodium alkali earth salt by other metal salts, e.g., lithium, magnesium or calcium perchlorate, yields the corresponding salt of the RNA molecule during the purification.

In the case of the 2-step purification procedure, in which the first step is a reverse phase purification followed by an anion exchange step, the reverse phase purification is best accomplished using polymeric, e.g.

polystyrene based, reverse-phase media, using either a 5'-trityl-on or trityl-off method. Either molecule may be recovered using this reversephase method, and then, once detritylated, the two fractions may be pooled and then submitted to an anion exchange purification step as described above.

The method includes passing the enzymatically active RNA molecule over a reverse phase HPLC column; the enzymatically active RNA molecule is produced in a synthetic chemical method and not by an enzymatic process; and the enzymatic RNA molecule is partially blocked, and the partially blocked enzymatically active RNA molecule is passed over a reverse phase HPLC column to separate it from other RNA molecules.

In more preferred embodiments, the enzymatically active RNA molecule, after passage over the reverse phase HPLC column, is deprotected and passed over a second reverse phase HPLC column (which may be the same as the reverse phase HPLC column), to remove the enzymatic RNA molecule from other components. In addition, the column is a silica or organic polymer-based C4, C8 or C18 column having a porosity of at least 125 A, preferably 300 A, and a particle size of at least 2 rpm, preferably 5 .im.

Activation The synthesis of RNA molecules may be accomplished chemically or enzymatically. In the case of chemical synthesis the use of tetrazole as an activator of RNA phosphoramidites is known (Usman et al. J. Am. Chem.

~II~UIIIY WO 95/23225 P'cTI 95/00156 69 Soc. 1987, 109, 7845-7854). In this, and subsequent reports, a 0.5 M solution of tetrazole is allowed to react with the RNA phosphoramidite and couple with the polymer bound 5'-hydroxyl group for 10 m, Applicant has determined that using 0.25-0,5 M solutions of 5-S-alkyltetrazoles for only min gives equivalent or better results, The following exemplifies the procedure.

Example 7: Synthesis of RNA and Ribozymes Using as Activating Agent The method of synthesis used follows the general procedure for RNA synthesis as described in Usman et al,, 1987supra and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the and phosphoramidites at the 3'-end, The major difference used was the activating agent, 5-S-ethyl or -methyltetrazole 0.25 M concentration for 5 min.

All small scale syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 pmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 m coupling step for methylated RNA. A 6.5-fold excess (162.5 [pL of 0.1 M 32.5 p.mol) of phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 .tL of 0.25 M 100 gpmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by .olorimetric quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems.

All large scale syntheses were conducted on a modified (eight amidite port capacity) 390Z (ABI) synthesizer using a 25 pmol scale protocol with a 5-15 min coupling step for alkylsilyl protected RNA and 7,5 m coupling step for 2'-O-methylated RNA. A six-fold excess (1.5 mL of 0.1 M 150 .Lmol) of phosphoramidite and a forty-five-fold excess of S-ethyl tetrazole (4.5 mL of rr g I WO 95/23225 C'I1 1195/()0156 0,25 M 1125 pmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 390Z, determined by colorimetric quantitation of the trityl fractions, was 95.0-96.7%.

Oligonucleotide synthesis reagents for the 390Z: Detritylation solution was 2% DCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25-0,5 M in acetonitrile) was made up from the solid obtained from Applied Biosystems.

Deprotection The first step of the deprotection of RNA molecules may be accomplished by removal of the exocyclic amino protecting groups with either NH 4 0H/EtOH:3/1 (Usman et al. J, Am, Chem. Soc. 1987, 109, 7845- 7854) or NH 3 /EtOH (Scaringe et al. Nucleic Acids Res. 1990, 18, 5433- 5341) for -20 h 55-65 OC, Applicant has determined that the use of methylamine or NH 4 0H/methylamine for 10-15 min 55-65 °C gives equivalent or better results. The following exemplifies the procedure.

Example 8: RNA and Ribozyme Deprotection of Exocyclic Amino Protecting Groups Using Methylamine (MA) or NH4OH/Methylamine (AMA) The polymer-bound oligonucleotide, either trityl-on or off, was suspended in a solution of methylamine (MA) or (AMA) 55-65 °C for 5-15 min to remove the exocyclic amino protecting groups. The polymer-bound oligoribonucleotide was transferred from the synthesis column to a 4 mL glass screw top vial. NH 4 0H and aqueous methylamine were pre-mixed in equal volumes, 4 mL of the resulting reagent was added to the vial, equilibrated for 5 m at RT and then heated at or 65 °C for 5-15 min, After cooling to -20 the supernatant was removed from the polymer support. The support was washed with 1,0 mL of EtOH:MeCN:H 2 0/3:1:1, vortexed and the supernatant was then added to the first supernatant, The combined supernatants, containing the oligoribonucleotide, were dried to a white powder. The same procedure was followed for the aqueous methylamine reagent.

Table 40 is a summary of the results obtained using the improvements outlined in this application for base deprotection.

r~lL~YI~ WO 95123225 P("CT/I 195/00156 71 The second step of the deprotection of RNA molecules may be accomplished by removal of the 2'-hydroxyl alkylsilyl protecting group using TBAF for 8-24 h (Usman et al, J, Am. Chem, Soc. 1987, 109, 7845- 7854). Applicant has determined that the use of anhydrous TEA*HF in Nmethylpyrrolidine (NMP) for 0.5-1.5 h 55-65 °C gives equivalent or better results. The following exemplifies this procedure.

Example 9: RNA and Ribozyme Deprotection of 2'-Hvdroxyl Alkylsilyl Protecting Groups Using Anhydrous TEA*HF To remove the alkylsilyl protecting groups, the ammonia-deprotected oligoribonucleotide was resuspended in 250 pL of 1,4 M anhydrous HF solution (1.5 mL N-methylpyrrolidine, 750 iIL TEA and 1,0 mL TEA-3HF) and heated to 65 °C for 1.5 h. 9 mL of 50 mM TEAB was added to quench the reaction. The resulting solution was loaded onto a Qiagen 500® anion exchange cartridge (Qiagen Inc.) prewashed with 10 mL of 50 mM TEAB.

After washing the cartridge with 10 mL of 50 mM TEAB, the RNA was eluted with 10 mL of 2 M TEAB and dried down to a white powder.

Table 41 is a summary of the results obtained using the improvements outlined in this application for alkylsilyl deprotection, Example 10: HPLC Purification, Anion Exchange column For a small scale synthesis, the crude material was diluted to 5 mL with diethylpyrocarbonate treated water, The sample was injected onto either a Pharmacia Mono Q® 16/10 or Dionex NucleoPac® column with 100% buffer A (10 mM NaCIO 4 A gradient from 180-210 mM NaC0O 4 at a rate of 0.85 mM/void volume for a Pharmacia Mono Q® anion-exchange column or 100-150 mM NaC0O 4 at a rate of 1.7 mM/void volume for a Dionex NucleoPac® anion-exchange column was used to elute the RNA.

Fractions were analyzed by a HP-1090 HPLC with a Dionex NucleoPac® column. Fractions containing full length product at >80% by peak area were pooled.

For a trityl-off large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose® Fast Flow column. The column was thoroughly washed with 10 mM sodium perchlorate buffer. The oligonucleotide was eluted from the column with WVO 95/23225 P 1195 (50 151, 72 300 mM sodium perchlorate. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material in the synthesis.

The eluent was diluted four fold in sterile H 2 0 to lower the salt concentration and applied to a Pharmacia Mono Q® 16/10 column. A gradient from 10-185 mM sodium perchlorate was run over 4 column volumes to elute shorter sequences, the full length product was then eluted in a gradient from 185-214 mM sodium perchlorate in 30 column volumes, The fractions of interest were analyzed on a HP-1090 HPLC with a Dionex NucleoPac@ column. Fractions containing over 85% full length material were pooled. The pool was applied to a Pharmacia RPC® column for desalting, For a trityl-on large scale synthesis, the crude material was desalted by applying the solution that resulted from quenching of the desilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose® Fast Flow column. The column was thoroughly washed with 20 mM NH 4 C0 3

CH

3 CN buffer, The oligonucleotide was eluted from the column with 1.5 M

NH

4 C0 3 H/10% acetonitrile. The eluent was quantitated and an analytical HPLC was run to determine the percent full length material present in the synthesis. The oligonucleotide was then applied to a Pharmacia Resource RPC column. A gradient from 20-55% B (20 mM NH4C0 3 H/25% CH 3

CN,

buffer A 20 mM NH 4 C0 3 H/10% CH 3 CN) was run over 35 column volumes. The fractions of interest were analyzed on a HP-1090 HPLC with a Dionex NucleoPac column. Fractions containing over 60% full length material were pooled, The pooled fractions were then submitted to manual detritylation with 80% acetic acid, dried down immediately, resuspended in sterile H 2 0, dried down and resuspended in H 2 0 again. This material was analyzed on a HP 1090-HPLC with a Dionex NucleoPac column. The material was purified by anion exchange chromatography as in the trityl-off scheme (vide supra).

Example 11 Ribozyme Activity Assay Purified 5'-end labeled RNA substrates (15-25-mers) and purified end labeled ribozymes (-36-mers) were both heated to 95 quenched on ice and equilibrated at 37 separately, Ribozyme stock solutions were 1 200 nM, 40 nM or 8 nM and the final substrate RNA concentrations were 1 nM. Total reaction volumes were 50 The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCl 2 Reactions were

I

II_

\VO 95/23225 PCT/I 1B95/00(150 73 initiated by mixing substrate and ribozyme solutions at t 0, Aliquots of pL were removed at time points of 1, 5, 15, 30, 60 and 120 m, Each aliquot was quenched in formamide loading buffer and loader onto a denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).

Example 12: One pot deprotection of RNA Applicant has shown that aqueous methyl amine is an efficient reagent to deprotect bases in an RNA molecule. However, in a time consuming step (2-24 hrs), the RNA sample needs to be dried completely prior to the deprotection of the sugar 2'-hydroxyl groups. Additionally, deprotection of RNA synthesized on a large scale 100 pmol) becomes challenging since the volume of solid support used is quite large.

In an attempt to minimize the time required for deprotection and to simplify the process of deprotection of RNA synthesized on a large scale, applicant describes a one pot deprotection protocol (Fig. 12). According to this protocol, anhydrous methylamine is used in place of aqueous methyl amine. Base deprotection is carried out at 65 °C for 15 min and the reaction is allowed to cool for 10 min. Deprotection of 2'-hydroxyl groups is then carried out in the same container for 90 min in a TEA*3HF reagent, The reaction is quenched with 16 mM TEAB solution.

Referring to Fig. 13, hammerhead ribozyme targeted to site B is synthesized using RNA phosphoramadite chemistry and deprotected using either a two pot or a one pot protocol. Profiles of these ribozymes on an HPLC column are compared. The figure shows that RNAs deprotected by either the one pot or the two pot protocols yield similar full-length product profiles. Applicant has shown that using a one pot deprotection protocol, time required for RNA deprotection can be reduced considerably without compromising the quality or the yield of full length RNA.

Referring to Fil. 14, hammerhead ribozymes targeted to site B (from Fig, 13) are tested for their ability to cleave RNA. As shown in the figure 14, ribozymes that are deprotected using one pot protocol have catalytic activity comparable to ribozymes that are deprotected using a two pot protocol.

I

U WO 95/23225 PCT/1B95/00156 74 Example 12a Improved protocol for the synthesis of phosphorothioate containing RNA and ribozymes using 5-S-Alkyltetrazoles as Activating Agent The two sulfurizing reagents that have been used to synthesize ribophosphorothioates are tetraethylthiuram disulfide (TETD; Vu and Hirschbein, 1991 Tetrahedron Letter 31, 3005), and 3H-1,2-benzodithiol-3one 1,1-dioxide (Beaucage reagent; Vu and Hirschbein, 1991 supra).

TETD requires long sulfurization times (600 seconds for DNA and 3600 seconds for RNA). It has recently been shown that for sulfurization of DNA oligonucleotides, Beaucage reagent is more efficient than TETD (Wyrzykiewicz and Ravikumar, 1994 Bioorganic Med. Chem. 4, 1519).

Beaucage reagent has also been used to synthesize phosphorothioate oligonucleotides containing 2'-deoxy-2'-fluoro modifications wherein the wait time is 10 min (Kawasaki et al., 1992 J. Med. Chem).

The method of synthesis used follows the procedure for RNA synthesis as described herein and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The sulfurization step for RNA described in the literature is a 8 second delivery and 10 min wait steps (Beaucage and lyer, 1991 Tetrahedron 49, 6123). These conditions produced about sulfurization as measured by HPLC analysis (Morvan et al., 1990 Tetrahedron Letter 31, 7149). This 5% contaminating oxidation could arise from the presence of oxygen dissolved in solvents and/or slow release of traces of iodine adsorbed on the inner surface of delivery lines during previous synthesis.

A major improvement is the use of an activating agent, ethyltetrazole or 5-S-methyltetrazole at a concentration of 0.25 M for 5 min.

Additionally, for those linkages which are phosporothioate, the iodine solution is replaced with a 0.05 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage reagent) in acetonitrile, The delivery time for the sulfurization step is reduced to 5 seconds and the wait time is reduced to 300 seconds.

RNA synthesis is conducted on a 394 (ABI) synthesizer using a modified 2.5 jmol scale protocol with a reduced 5 min coupling step for alkylsilyl protected RNA and 2.5 min coupling step for 2'-O-methylated RNA. A 6.5-fold excess (162.5 p.L of 0.1 M 32.5 mol) of phosphoramidite RECTIFIED SHEET (RULE 91)

ISA/EP

I

_I \WO 95/,23225 P(T/1 9')5/(10 and a 40-fold excess of S--ethyl tetrazole (400 p.L of 0.25 M 100 pmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle.

Average coupling yields on the 394 synthesizer, determined by colorimetric quantitation of the trityl fractions, was 97.5-99%. Other oligonucleotide synthesis reagents for the 394 synthesizer: detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from Applied Biosystems. Sulfurizing reagent was obtained from Glen Research.

Average sulfurization efficiency (ASE) is determined using the formula: ASE (PS/Total) 1 /n 1 where, PS integrated 31P NMR values of the P=S diester Total integration value of all peaks n length of oligo Referring to tables 42 and 43, effects of varying the delivery and the wait time for sulfurization with Beaucage's reagent is described. These data suggest that 5 second wait time and 300 second delivery time is the condition under which ASE is maximum.

Using the above conditions a 36 hammerhead ribozyme is synthesized which is targeted to site C. The ribozyme is synthesized to contain phosphorothioate linkages at four oositions towards the 5' end.

RNA cleavage activity of this ribozyme is shown in Fig, 16. Activity of the phosphorothioate ribozyme is comparable to the activity of a ribozyme lacking any phosphorothioate linkages.

Example 13: Protocol for the synthesis of 2'-N-phtalimido-nucleoside phosphoramidite The 2'-amino group of a 2'-deoxy-2'-amino nucleoside is normally protected with N-(9-flourenylmethoxycarbonyl) (Fmoc; Imazawa and Eckstein, 1979 supra, Pieken et al., 1991 Science 253, 314). This protecting group is not stable in CH3CN solution or even in dry form during

-I

~UIIII~m~l~- 81- WO 95/23225 PCT/1B95/00(15( 76 prolonged storage at -20 oC. These problems need to be overcome in order to achieve large scale synthesis of RNA, Applicant describes the use of alternative protecting groups for the 2'amino group of 2'-deoxy-2'-amino nucleoside. Referring to Figure 17, phosphoramidite 17 was synthesized starting from 2'-deoxy-2'aminonucleoside (12) using transient protection with Markevich reagent (Markiewicz J. Chem, Res. 1979, S, 24). An intermediate 13 was obtained in 50% yield, however subsequent introduction of N-phtaloyl (Pht) group by Nefken's method (Nefkens, 1960 Nature 185, 306), desilylation dimethoxytrytilation (16) and phosphitylation led to phosphoramidite 17.

Since overall yield of this multi-step procedure was low applicant investigated some alternative approaches, concentrating on selective introduction of N-phtaloyl group without acylation of 5' and 3' hydroxyls.

When 2'-deoxy-2'-amino-nucleoside was reacted with 1.05 equivalents of Nefkens reagent in DMF overnight with subsequent treatment with Et3N (1 hour) only 10-15% of N and 5'(3')-bis-phtaloyl derivatives were formed with the major component being N-Pht-derivative The N,O-bis by-products could be selectively and quantitively converted to N-Pht derivative 15 by treatment of crude reaction mixture with cat. KCN/MeOH.

A convenient "one-pot" procedure for the synthesis of key intermediate 16 involves selective N-phthaloylation with subsequent dimethoxytrytilation by DMTCI/Et3N and resulting in the preparation of DMT derivative 16 in 85% overall yield as follows. Standard phosphytilation of 16 produced phosphoramidite 17 in 87% yield. One gram of 2'-amino nucleoside, for example 2'-amino uridine (US Biochemicals® part 77140) was co-evaporated twice from dry dimethyl formamide (Dmf) and dried in vacuo overnight. 50 mis of Aldrich sure-seal Dmf was added to the dry 2'-amino uridine via syringe and the mixture was stirred for 10 minutes to produce a clear solution. 1.0 grams (1,05 eq.) of Ncarbethoxyphthalimide (Nefken's reagent, 98% Jannsen Chimica) was added and the solution was stirred overnight. Thin layer chromatography (TLC) showed 90% conversion to a faster moving products (10% ETOH in CHCI3) and 57 1L of TEA (0.1 eq.) was added to effect closure of the phthalimide ring. After 1 hour an additional 855 4l (1.5 eq.) of TEA was added followed by the addition of 1.53 grams (1.1 eq.) of DMT-CI -~YIPn~BII- -I WVO 95/23225 PC"TIIB95/00150 77 (Lancaster Synthesis@, The reaction mixture was left to stir overnight and quenched with ETOH after TLC showed greater than desired product. Dmf was removed under vacuum and the mixture was washed with sodium bicarbonate solution aq., 500 mis) and extracted with ethyl acetate (2x 200 mis). A 25mm x 300mm flash column (75 grams Merck flash silica) was used for purification. Compound eluted at 80 to ethyl acetate in hexanes (yield: 80% purity: >95% by 1

HNMR).

Phosphoramidites were then prepared using standard protocols described above.

With phosphoramidite 17 in hand applicant synthesized several ribozymes with 2'-deoxy-2'-amino modifications. Analysis of the synthesis demonstrated coupling efficiency in 97-98% range. RNA cleavage activity of ribozymes containing 2'-deoxy-2'-amino-U modifications at U4 and/or U7 positions (see Figure wherein the 2'-amino positions were either protected with Fmoc or Pht, was identical. Additionally, complete deprotection of 2'-deoxy-2'-amino-Uridine was confirmed by basecomposition analysis. The coupling efficiency of phosphoramidite 17 was not effected over prolonged storage (1-2 months) at low temperatures.

Protecting 2' Position with a SEM Group There follows a method using the 2'-(trimethylsilyl)ethoxymethyl protecting group (SEM) in the synthesis of oligoribonucleotides, and in particular those enzymatic molecules described above. For the synthesis of RNA it is important that the 2'-hydroxyl protecting group be stable throughout the various steps of the synthesis and base deprotection. At the same time, this group should also be readily removed when desired. To that end the t-butyldimethylsilyl group has been efficacious (Usman,N.; Ogilvie,K,K.; Jiang,M.-Y,; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and Scaringe,S.A.; Franklyn,C.; Usman,N, Nucl, Acids Res.

1990, 18, 5433-5441). However, long exposure times to tetra-nbutylammonium fluoride (TBAF) are generally required to fully remove this protecting group from the 2'-hydroxyl. In addition, the bulky alkyl substituents can prove to be a hindrance to coupling thereby necessitating longer coupling times. Finally, it has been shown that the TBDMS group is base labile and is partially deprotected during treatment with ethanolic ammonia (Scaringe,SA.; Franklyn,C.; Usman,N. Nucl. Acids Res. 1990,

II

811191~1W~"Ul~rrs~lp13 II I I- WO 95/23225 I'(T/1195/O0150 78 18, 5433-5441 and Stawinski,J.; Stromberg,R.; Thelin,M.; Westman,E.

Nucleic Acids Res. 1988, 16, 9285-9298).

The (trimethylsilyl)ethoxymethyl ether (SEM) seems a suitable substitute. This protecting group is stable to base and all but the harshest acidic conditions. Therefore it is stable under the conditions required for oligonucleotide synthesis. It can be readily introduced and the oxygen carbon bond makes it unable to migrate. Finally, the SEM group can be removed with BF 3 *OEt 2 very quickly.

There follows a method for synthesis of RNA by protecting the 2'position of a nucleotide during RNA synthesis with a (trimethylsilyl)ethoxymethyl (SEM) group. The method can involve use of standard RNA synthesis conditions as discussed below, or any other equivalent steps. Those in the art are familiar with such steps. The nucleotide used can be any normal nucleotide or may be substituted in various positions by methods well known in the art, as described by Eckstein et al., International Publication No. WO 92/07065, Perrault et al., Nature 1990, 344, 565-568, Pieken et al., Science 1991, 253, 314-317, Usman,N.; Cedergren,R.J. Trends in Biochem. Sci. 1992, 17, 334-339, Usman et al., PCT W093/15187, and Sproat,B. European Patent Application 92110298.4.

This invention also features a method for covalently linking a SEM group to the 2'-position of a nucleotide, The method involves contacting a nucleoside with an SEM-containing molecule under SEM bonding conditions. In a preferred embodiment, the conditions are dibutyltin oxide, tetrabutylammonium fluoride and SEM-CI. Those in the art, however, will recognize that other equivalent conditions can also be used.

In another aspect, the invention features a method for removal of an SEM group from a nucleoside molecule or an oligonucleotide. The method involves contacting the molecule or oligonucleotide with boron trifluoride etherate (BF 3 .OEt 2 under SEM removing conditions, in acetonitrile.

Referring to Figure 18, there is shown the method for solid phase synthesis of RNA. A 2',5'-protected nucleotide is contacted with a solid phase bound nucleotide under RNA synthesis conditions to form a dinucleotide, The protecting group at the 2'-position in prior art st I 'r 2.22 I l105 llll( 79 methods can be a silyl ether, as shown in the Figure. In the mothod of the present invention, an SEM group is used in place of the silyl ether.

Otherwise RNA synthesis can be performed by standard methodology.

Referring to Figure 9, there is shown the synthesis of protected nucleosides and phosphoramadites. Briefly, a nucleoside is protected at the or 3'-position by contacting with a derivative of SEM under appropriate conditions. Specifically, those conditions include contacting the nucleoside with dibutyltin oxide and SEM chloride. The 2 regioisomers are separated by chromatography and the 2'protected moiety is converted into a phosphoramidite by standard procedure. The 3'-protected nucleoside is converted into a succinate derivative suitable for derivatization of a solid support.

Referring to Figure 20, a prior art method for deprotection of RNA using silyl ethers is shown, This contrasts with the method shown in Figure 21 in which deprotection of RNA containing an SEM group is performed, In step 1, the base protecting groups and cyanoethyl groups are removed by standard procedure. The SEM group is then removed as shown in the Figure. The details of the synthesis of phosphoramidites and SEM protected nucleosides and their use in synthesis of oligonucleotides and subsequent deprotection of Example 14: Synthesis of 2'-O-((trimethylsilvl)ethoxymethvl)-5'-O- Dimethoxvtrityl Uridine (2) Referring to Figure 19, 5'-O-dimethoxytrityl uridine 1 (1.0 g, 1.83 mmol) in CH 3 CN (18 mL) was added dibutyltin oxide (1,0 g, 4.03 mmol) and TBAF (1 M, 2.38 mL, 2.38 mmol). The mixture was stirred for 2 h at RT (about 20-25'C) at which time (trimethylsilyl)ethoxymethyl chloride (SEM- Cl) (487 pL, 2.75 mmol) was added, The reaction mixture was stirred overnight and then filtered and evaporated, Flash chromatography hexanes in ethyl acetate) yielded 347 mg of 2'-hydroxyl protected nucleoside 2 and 314 mg of 3'-hydroxyl protected nucleoside 3.

Example 15: Synthesis of 2'-O-((trimethylsilyl)ethoxymethyl) Uridine (4) Nucleoside 2 was detritylated following standard methods, as shown in Figure 19.

N 0 95 23225 N't 1105 jExatple 16:Sy5ntheis of 2'-O tri mcltQ- jij hq~y~pptt vlj- 3At Nucleoside 4 was acetylated following standard methods, as shown in Figure 19, Example 17: Synthesis of 5'3'-O-Acety Uridine 6 Referring to Figure 19, the fully protected uridine 5 (32 mg, 0.07 mmol) was dissolved in CH 3 CN (700 pL) and BF 3 *OEt 2 (17.5 tL, 0.14 mmol) was added. The reaction was stirred 15 m and MeOH was added to quench the reaction. Flash chromatography MeOH in CH 2 C1 2 gave 20 mg (88%1) of SEM deprotected nucleoside 6.

Examole 18: Synthesis of 2'-O-((trimethylsilyl)ethoxymethy-3'-O- Dimethoxvtritvl Uridine (2) Nucleoside 3 was succinylated and coupled tc the suppol following standard procedures, as shown in Figure 19.

Example 19: Synthesis of 2'-O-((trimethylsilvl)ethoxymethyl)-5'-O- Dimethoxytrityl Uridine 3'-(2-Cvanoethyl N,N-diisopropylphosphoramidite) U8) Nucleoslde 3 was phosphitylated following standard methods, as shown in Figure 19, Example 20: Synthesis of RNA Using 2'-O-SEM Protection Referring to Figure 18, the method of synthesis used follows the general procedure for RNA synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M.-Y.; Cedergren,R.J. J. Am. Chem, Soc. 1987, 109, 7845-7854 and in Scaringe,S.A,; Franklyn,C.; Usman,N, Nucl, Acids Res.

1990, 18, 5433-5441, The phosphoramidite 8 was coupled following standard RNA methods to provide a 10-mer of uridylic acid, Syntheses were conducted on a 394 (ABI) synthesizer using a modified 2.5 imol scale protocol with a 10 m coupling step. A thirteen-fold excess (325 IL of 0,1 M 32,5 ltmol) of phosphoramidite and a 80-fold excess of tetrazole (400 ItL of 0.5 M 200 gmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394, determined by colorimetric quantitation of the trityl fractions, were 98-99%.

Other oligonucleotide synthesis reagents for the 394: Detritylation solution was 2% TCA in methylene chloride; capping was performed with 16% N-

I

_IL, I 0 95/23225 1 11395 Ii(115i, 81 Methyl imidazole in THF and 10%o acetic anhydride/10o 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9 0 water in THF.

Fisher Synthesis Grade acetonitrile was used directly from the reagent bottle.

Referring to Figure 21, the homopolymor was base deprotected with

NH

3 /EtOH at 65 The solution was decanted and the support was washed twice with a solution of 1:1:1 H 2 0:CH3CN:MeOH. The combined solutions were dried down and then diluted with CH 3 CN (1 mL). BF 3 *OEt 2 I.L, 30 I.mol) was added to the solution and aliquots were removed at ten time points, The results indicate that after 30 min deprotection is complete, as shown in Figure 22.

IIl. Vectors Expressing Ribozymes There follows a method for expression of a ribozyme in a bacterial or eucaryotic cell, and for production of large amounts of such a ribozyme. In general, the invention features a method for preparing multi-copy cassettes encoding a defined ribozyme structure for production of a ribozyme at a decreased cost. A vector is produced which encodes a plurality of ribozymes which are cleaved at their 3' and 5' ends from an RNA transcript producted from the vector by only one other ribozyme. The system is useful for scaling up production of a ribozyme, which may be either modified or unmodified, in situ or in vitro. Such vector systems can be used to express a desired ribozyme in a specific cell, or can be used in an in vitro system to allow productiuon of large amounts of a desired riboqyne, The vectors of this invention allow a higher yield synthesis of a ribozyme in the form of an RNA transcript which is cleaved in situ or in vitro before or after transcript Isolation, Thus, this invention is distinct from the prior art in that a single ribozyme is used to process the 3' and 5' ends of each therapeutic, transacting or desired ribozyme instead of processing only one end, or only one ribozyme, This allows smaller vectors to be derived with multiple transacting ribozymes released by only one other ribozyme from the mRNA transcript. Applicant has also provided methods by which the activity of such ribozymes is increased compared to those in the art, by designing ribozyme-encoding vectors and the corresponding transcript such that C I WO 95123225 IC '1 111' 1 ill it folding of the mRNA does not interfere with processing by the releasing ribozyme, The stability of the ribozyme produced in this method can be enhanced by provision of sequences at the termini of the ribozymes as described by Draper et al,, PCT WO 93/23509, hereby incorporated by reference herein, The method of this invention is advantageous since it provides high yield synthesis of ribozymes by use of low cost transcription-based protocols, compared to existing chemical ribozyme synthesis, and can use isolation techniques currently used to purify chemically synthesized oligonucleotides, Thus, the method allows synthesis of ribozymes in high yield at low cost for analytical, diagnostic, or therapeutic applications.

The method is also useful for synthesis of ribozymes in vitro for ribozyme structural studies, enzymatic studies, target RNA accessibility studies, transcription inhibition studies and nuclease protection studies, much is described by Draper et al,, PCT WO 93/23509 hereby incorporated by reference herein.

The method can also be used to produce ribozymes in situ either to increase the intracellular concentration of a desired therapeutic ribozyme, or to produce a concatameric transcript for subsequent in vitro isolation of unit length ribozyme, The desired ribozyme can be used to inhibit gene expression in molecular genetic analyses or in infectious cell systems, and to test the efficacy of a therapeutic molecule or treat afflicted cells, Thus, in general, the invention features a vector which includes a bacterial, viral or eucaryotic promoter within a plasmid, cosmid, phagmid, virus, viro" virusoid or phage vector. Other vectors are equally suitable and include double-stranded, or partially double-stranded DNA, formed by an amplification method such as the polymerase chain reaction, or doublestranded, partially double-stranded or single-stranded RNA, formed by sitedirected homologous recombination into viral or viroid RNA genomes.

Such vectors need not be circular. Transcriptionally linked to the promoter region is a first ribozyme-encoding region, and nucleotide sequences encoding a ribozyme cleavage sequence which is placed on either side of a region encoding a therapeutic or otherwise desired second ribozyme.

I

a II II 0) 23225 i' I 1l1'5 11115(1 83 Suitable restriction endonuclease sites can be provided to ease construction of this vector in DNA vectors or in requisite DNA vectors of an RNA expression system. The desired second ribozyme may be any desired type of ribozyme, such as a hammerhead, hairpin hepatitis delta virus (HDV) or other catalytic center, and can include group I and group II introns, as discussed above, The first ribozyme is chosen to cleave the encoded cleavage sequence, and may also be any desired ribozyme, for example, a Tetrahymena derived ribozyme, which may, for example, include an imbedded restriction endonuclease site in the center of a selfrecognition sequence to aid in vector construction. This endonuclease site is useful for construction of the vector, and subsequent analysis of the vector, When the promoter of such a vector is activated an RNA transcript is produced which includes the first and second ribozyme sequences. The first ribozyme sequence is able to act, under appropriate conditions, to cause cleavage at the cleavage sites to release the second ribozyme sequences. These second ribozyme sequences can then act at their target RNA sites, or can be isolated for later use or analysis, Thus, in one aspect the invention features a vector which includes a first nucleic acid sequence (encoding a first ribozyme having intramolecular cleaving activity), and a second nucleic acid sequence (encoding a second ribozyme having intermolecular cleaving enzymatic activity) flanked by nucleic acid sequences encoding RNA which is cleaved by the first ribozyme to release the second ribozyme from the RNA transcript encoded by the vector, The second ribozyme may be flanked by the first ribozyme either on the 5' side or 3' side. If desired, the first ribozyme may be encoded on a separate vector and may have intermolecular cleaving activity.

As discussed above, the first ribozyme can be chosen to be any selfcleaving ribozyme, and the second ribozyme may be chosen to be any desired ribozyme. The flanking sequences are chosen to include sequences recognized by the first ribozyme. When the vector is caused to express RNA from these nucleic acid sequences, that RNA has the ability under appropriate conditions to cleave each of the flanking regions and thereby release one or more copies of the second ribozyme. If desired, several different second ribozymes can be produced by the same vector, or LL~Lbr I 0) 9523225 N' 1 llt 1 95 (111 IS 84 several different vectors can be placed in the same vessol or coll to produce different ribozymes, In preferred embodiments, the vector includes a plurality of the nucleic acid sequences encoding the second ribozyme, each flanked by nucleic acid sequences recognized by the first ribozyme, Most preferably, such a plurality includes at least six to nine or even between 60 100 nucleic acid sequences. In other preferred embodiments, the vector includes a promoter which regulates expression of the nucleic acid encoding the ribozymes from the vector; and the vector is chosen from a plasmid, cosmid, phagmid, virus, viroid or phage. In a most preferred embodiment, the plurality of nucleic acid sequences are identical and are arranged in sequential order such that each has an identical end nearest to the promoter, If desired, a poly(A) sequence adjacent to the sequence encoding the first or second ribozyme may be provided to increase stability of the RNA produced by the vector; and a restriction endonuclease site adjacent to the nucleic acid encoding the first ribozyme is provided to allow insertion of nucleic acid encoding the second ribozyme during construction of the vector.

In a second aspect, the invention features a method for formation of a ribozyme expression vector by providing a vector including nucleic acid encoding a first ribozyme, as discussed above, and providing a singlestranded DNA encoding a second ribozyme, as discussed above. The single-stranded DNA is then allowed to anneal to form a partial duplex DNA which can be filled in by a treatment with an appropriate enzyme, such as a DNA polymerase in the presence of dNTPs, to form a duplex DNA which can then be ligated to the vector, Large vectors resulting from this method can then be selected to insure that a high copy number of the single-stranded DNA encoding the second ribozyme is incorporated into the vector, In a further aspect, the invention features a method for production of ribozymes by providing a vector as described above, expressing RNA from that vector, and allowing cleavage by the first ribozyme to release the second ribozyme.

In preferred embodiments, three different ribozyme motifs are used as cis-cleaving ribozymes. The hammerhead, hairpin, and hepatitis delta

I

~LI

i123225 PCK I 'H1 135111ll1( virus (HDV) ribozyme motifs consist of small, well-defined sequences that rapidly self-cleave in vitro (Symons, 1992 Ann Rv. Bipochem. 61, 641).

While structural and functional differences exist among the three ribozyme motifs, they self-process efficiently in vivo. All three ribozyme motifs selfprocess to 87-95% completion in the absence of 3' flanking sequences. In vitro, the self-processing constructs described in this invention are significantly more active than those reported by Taira et al,, 1990 su and Altschuler et al,, 1992 Gene. 122, 85. The present invention enables the use of cis-cleaving ribozymes to efficiently truncate RNA molecules at specific sites in vivo by ensuring lack of secondary structure which prevents processing.

Isolation of Therapeutic Ribozyme The preferred method of isolating therapeutic ribozyme is by a chromatographic technique, The HPLC purification methods and reverse HPLC purification methods described by Draper et al., PCT WO 93/23509, hereby incorporated by reference herein, can be used. Alternatively, the attachment of complementary oligonucleotides to cellulose or other chromatography columns allows isolation of the therapeutic second ribozyme, for example, by hybridization to the region between the flanking arms and the enzymatic RNA, This hybridization will select against the short flanking sequences without the desired enzymatic RNA, and against the releasing first ribozyme. The hybridization can be accomplished in the presence of a chaotropic agent to prevent nuclease degradation, The oligonucleotides on the matrix can be modified to minimize nuclease activity, for example, by provision of 2'-O-methyl RNA oligonucleotides, Such modifications of the oligonucleotide attached to the column matrix will allow the multiple use of the column with minimal oligo degradation. Many such modifications are known in the art, but a chemically stable nonreducible modification is preferred, For example, phosphorothioate modifications can also be used, The expressed ribozyme RNA can be isolated from bacterial or eucaryotic cells by routine procedures such as lysis followed by guanidine isothiocyanate isolation.

The current known self-cleaving site of Tetrahymena can be used in an alternative vector of this invention, If desired, the full-length

E

r I \VO )5/;2322?s PC I 1 1')5 (11I(5 86 Tetrahymena sequence may be used, or a shorter sequence may be used It is preferred that, in order to decrease the superfluous sequences in the self-cleaving site at the 5' cleavage end, the hairpin normally present in the Tetrahymena ribozyme should contain the therapeutic second ribozyme 3' sequence and its complement. That is, the first releasing ribozymeencoding DNA is provided in two portions, separated by DNA encoding the desired second ribozyme. For example, if the therapeutic second ribozyme recognition sequence is CGGACGA/CGAGGA, then CGAGGA is provided in the self-cleaving site loop such that it is in a stem structure recognized by the Tetrahymena ribozyme. The loop of the stem may include a restriction endonuclease site into which the desired second ribozyme-encoding DNA is placed, If desired, the vector may be used in a therapeutic protocol by use of the systems described by Lechner, PCT WO 92/13070, hereby incorporated by reference herein, to allow a timed expression of the therapeutic second ribozyme, as well as an appropriate shut off of cell or gene function. Thus, the vector will include a promoter which appropriately expresses enzymatically active RNA only in the presence of an RNA or another molecule which indicates the presence of an undesired organism or state. Such enzymatically active RNA will then kill or harm the cell in which it exists, as described by Lechner, id., or act to cause reduced expression of a desired protein product.

A number of suitable RNA vectors may also be used in this invention.

The vectors include plant viroids, plant viruses which contain single or double-stranded RNA genomes and animal viruses which contain RNA genomes, such as the picornaviruses, myxoviruses, paramyxoviruses, hepatitis A virus, reovirus and retroviruses, In many instances cited, use of these viral vectors also results in tissue specific delivery of the ribozymes.

Example 21: Design of self-processing cassettes In a preferred embodiment, applicant compared the in vitro and in vive cis-cleaving activity of three different ribozyme motifs-the hammerhead, the hairpin and the hepatitis delta virus ribozyme-in order to assess their potential to process the ends of transcripts in vivo. To make a direct comparison among the three, however, it is important to design the ribozyme-containing transcripts to be as similar as possible. To this end,

M

~r~a 1 I \VO 95,':23225 P( I15') I I 87 all the ribozyme cassettes contained the same trans-acting hammerhead ribozyme followed immediately by one of the three cis-acting ribozymes (Figure 23-25), For simplicity, applicant refers to each cassette by an abbreviation that indicates the downstream cis-cleaving ribozyme only.

Thus HH refers to the cis-cleaving cassette containing a hammerhead ribozyme, while HP and HDV refer to the cassettes containing hairpin and hepatitis delta virus cis-cleaving ribozymes, respectively. The general design of the ribozyme cassettes, as well as specific differences among the cassettes, are outlined below, A sequence predicted to form a stable stem-loop structure is included at the 5' end of all the transcripts, The hairpin stem contains the T7 RNA polymerase initiation sequence (Milligan Uhlenbeck, 1989 Methods Enzymol. 180, 51) and its complement, separated be a stable tetra-loop (Antao et al,, 1991 Nucleic Acids Res, 19, 5901), By incorporating the T7 initiation sequence into a stem-loop structure, applicant hoped to avoid nonproductive base pairing interactions with either the trans-acting ribozyme or with the cis-acting ribozyme, The presence of a hairpin at the end of a transcript may also contribute to the stability of the transcript in vivo. These are non-limiting examples, Those in the art will recognize that other embodiments can be readily generated using a variety of promoters, initiator sequences and stem-loop structure combinations generally known in the art, The trans-acting ribozyme used in this study is targeted to a site B (5'.,,CUGGAGUQCGACCUUC*.3'), The 5' binding arm of the ribozyme, GAAGGUC-3', and the core of the ribozyme, CUGAUGAGGCCGAAAGGCOGAA-3', remain constant in all cases. In addition, all transcripts also contain a single nucleotide between the stem-loop and the first nucleotide of the ribozyme. The linker nucleotide was required to obtain the same activity in vitro that was measured with an identical ribozyme lacking the 5' hairpin, Because the three cis-cleaving ribozymes have different requirements at the site of cleavage, slight differences were unavoidable at the 3' end of the processed transcript. The junction between the trans- and cis-acting ribozyme is, however, designed so that there is minimal extraneous sequence left at the 3' end of the transcleaving ribozyme once cis-cleavage occurs. The only differences between the constructs lie in the 3' binding arm of the ribozyme, where Cr /23 225 P( I 1115 il ?ii, 88 either 6 or 7 nucleotides, complementary to the target sequence are present and where, after processing, two to five extra nucleotides remain, The cis-cleaving hammerhead ribozyme used in the HH cassette is based on the design of Grosshans and Cech, 1991 gura. As shown in Figure 23, the 3' binding arm of the trans-acting ribozyme is included in the required base-pairing interactions of the cis-cleaving ribozyme to form stem I, Two extra nucleotides, UC, were included at the end of the 3' binding arm to form the self-processing hammerhead ribozyme site (Ruffner et al., 1990 supra) which remain on the 3' end of the trans-acting ribozyme following self-processing.

The hairpin ribozyme portion of the HP self-processing construct is based on the minimal wild-type sequence (Hampel Tritz, 1989 supra). A tetra-loop at the end of helix 1 side of the cleavage site) serves to link the two portions and thus allows a minimal five nucleotides to remain at the end of the released trans-acting ribozyme following self-processing, Two variants of HP were designed: HP(GU) and HP(GC). The HP(GU) was constructed with a GU wobble base pair in helix 2 (A52G substitution; Figure 24). This slight destabilization of helix 2 was intended to improve self-processing activity by promoting product release and preventing the reverse reaction (Berzal-Herranz et al,, 1992 Genes Dev. 6, 129; Chowrira et al,, 1993 Biochemistry 32, 1088). The HP(GC) cassette was constructed as a control for strong base-pairing interactions in helix 2 (U77C and A52G substitution; Figure 24). Another modification to discourage the reverse ligation reaction of the hairpin ribozyme was to shorten helix 1 (Figure 24) by one base pair relative to the wild-type sequence (Chowrira Burke, 1991 Biochemistry 30, 8518), The HDV ribozyme self-processes efficiently when the nucleotide 5' to the cleavage site is a pyrimidine, and somewhat less so when adenosine is in that position, No other sequence requirements have been identified upstream of the cleavage site, however, we have observed some decrease in activity when a stem-loop structure was present within 2 nt of the cleavage site, The HDV self-processing construct (Fig 25) was designed to generate the trans-acting hammerhead ribozyme with only two additional nucleotides at its 3' end after self-processing. The HDV sequence used here is based on the anti-genomic sequence (Perrota Been, 1992 supra) 95,23225 PC i 11195'00 89 but includes the modifications of Been et al., 1992 (Biochemistry 31, 11843) in which cis-cleavage activity of the ribozyme was improved by the substitution of a shortened helix 4 for a wild-type stem-loop (Figure To prepare DNA inserts that encode self-processing ribozyme cassettes, partially overlapping top- and bottom-strand oligonucleotides (60-90 nucleotides) were designed to include sequences for the T7 promoter, the trans-acting ribozyme. the cis-cleaving ribozyme and appropriate restriction sites for use in cloning (see Fia. 26). The singlestrand portions of annealed oligonucleotides were converted to doublestrands using Sequenase® Biochemicals). Insert DNA was ligated into EcoR1/Hindlll-digested pucl8 and transformed into E. coil strain using standard protocols (Maniatis et al., 1982 in Molecular Cloning Cold Spring Harbor Press). The identity of positive clones was confirmed by sequencing small-scale plasmid preparations.

Larger scale preparations of plasmid DNA for use as in vitro transcription templates and in transactions were prepared using the protocol and columns from QIAGEN Inc. (Studio City, CA) except that an additional ethanol precipitation was included as the final step.

Example 22: RNA Processing in vitro Transcription reactions containing linear plasmid templates were carried out essentially as described (Milligan Uhlenbeck, 1989 Supra; Chowrira Burke, 1991 Supra), In order to prepare 5' end-labeled transcripts, standard transcription reactions were carried out in the presence of 10-20 gCi [y- 3 2 P]GTP, 200 .tM each NTP and 0.5 to 1 jg of linearized plasmid template. The concentration of MgCI2 was maintained at 10 mM above the total nucleotide concentration, To compare the ability of the different ribozyme cassettes to selfprocess in vitro, each construct was transcribed and allowed to undergo self-processing under identical conditions at 370C, For these comparisons, equal amounts of linearized DNA templates bearing the various ribozyme cassettes were transcribed in the presence of [y- 3 2 P]GTP to generate end-labeled transcripts. In this manner only the full-length, unprocessed transcripts and the released trans-ribozymes are visualized by autoradiography. In all reactions, Mg 2 was included at 10 mM above the nucleotide concentration so that cleavage by all the ribozyme cassettes F)r~ 0 95/23225 S( '1 1 )5 0i0156 would be supported, Transcription templates were linearized at several positions by digestion with different restriction enzymes so that selfprocessing in the presence of increasing lengths of downstream sequence could be compared (see Fig 26). The resulting transcripts have either non-ribozyme nucleotides at the 3' end (Hindlll-digested template), 220 nucleotides (Ndel digested templates) or 454 nucleotides of downstream sequence (Rcal digested template).

As shown in Figure 27, all four ribozyme cassettes are capable of selfprocessing and yield RNA products of expected sizes. Two nucleotides essential for hammerhead ribozyme activity (Ruffner et al,, 1990 supra) have been changed in the HH(mutant) core sequence (see Figure 23) and so this transcript is unable to undergo self-processing (Fig. 27), This is evidenced by the lack of a released 5' RNA in the HH(mutant), although the full-length RNAs are present Comparison of the amounts of released trans-ribozyme (Fig. 27) indicate that there are differences in the ability of these ribozymes to self-process in vitro, especially with respect to the presence of downstream sequence, For the two HP constructs, it is clear that HP(GC) is more efficient than the HP(GU) ribozyme, both in the presence and in the absence of extra downstream sequence, In addition, the activity of HP(GU) falls off more dramatically when downstream sequence is present. The stronger G:C base pair likely contributes to the HP(GC) construct's ability to fold correctly (and/or more quickly) into the productive structure, even when as much as 216 extra nucleotides are present downstream. The HH ribozyme construct is also quite efficient at self-processing, and slightly better than the HP(GU) construct even when downstream sequence is present.

Of the three ribozyme motifs, the presence of extra downstream sequence seems to most affect the efficiency of HDV. When no extra sequence is present downstream, HDV is quite efficient and self-processes to approximately the same level as the HH and HP(GC) cassettes.

However, when extra downstream sequence is present, the self-processing activity seems to decrease almost as dramatically as is seen with the (suboptimal) HP(GU) cassette.

M

\O 23225 P( I 1fl l156 91 Example 23: Kinetics of self-processing reaction Hindlll-digested template (250 ng) was used in a standard transcription reaction mixture containing: 50 mM Tris.HCI pH 8,3; 1 mM ATP, GTP and UTP; 50 pM CTP; 40 pCi [x- 3 2 P]CTP; 12 mM MgCl2; 10 mM DTT, The transcription/self-processing reaction was initiated by the addition of T7 RNA polymerase (15 U/pl). Aliquots of 5 .l were taken at regular time intervals and the reaction was stopped by adding an equal volume of 2x formamide loading buffer (95% formamide, 15 mM EDTA, dyes) and freezing on dry ice. The samples were resolved on a polyacrylamide sequencing gel and results were quantitated by Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). Ribozyme selfcleavage rates were determined from non-linear, least-squares fits (KaleidaGraph, Synergy Software,Reeding, PA) of the data to the equation: 1 k (Fraction Uncleaved Transcript) (1-e k where t represents time and k represents the unimolecular rate constant for cleavage (Long Uhlenbeck, 1994 Proc. Natl. Acad. Sci. USA 91, 6977), Linear templates were prepared by digesting the plasmids with Hindlll so that transcripts will contain only four to five vector-derived nucleotides at the 3' end (see Figure 23-25). By comparison of the unimolecular rate constant determined for each construct, it is clear that HH is the most efficient at self-processing (Table 44). The HH transcript self-processes 2fold faster than HDV and 3-fold faster than HP(GC) transcripts. Although the HP(GU) RNA undergoes self-processing, it is at least 6-fold slower than the HP(GC) construct. This is consistent with previous observations that the stability of helix 2 is essential for self-processing and trans-cleavage activity of the hairpin ribozyme (Hampel et al., 1990 supra; Chowrira Burke, 1991 supra), The rate of HH self-cleavage during transcription measured here (1.2 min 1 is similar to the rate measured by Long and Uhlenbeck 1994 supra using a HH that has a different stem I and stem III, Self-processing rates during transcription for HP and HDV have not been previously reported, However, self-processing of the HDV ribozyme-as measured here during transcription-is significantly slower than when tested after isolation from a denaturing gel (Been et al., 1992 supra). This decrease likely reflects the difference in protocol as well as the presence of flanking sequence in the HDV construct used here.

I

II W\O 9523225 1 ,I 1 I ll15(1 92 Example 24: Effect of downstream sequences on trans-cleavag. in vitro Transcripts containing the trans ribozyme with or without 3' flanking sequences were assayed for their ability to cleave their target in trans. To this end, transcripts from three templates were resolved on a preparative gel and bands corresponding both to processed trans-acting ribozymes from the HH transcription reaction, and to full-length HH(mutant) and AHDV transcripts were isolated. In all three transcripts the trans-acting ribozyme portion is identical-with the exception of sequences at their 3' ends, The HH trans-acting ribozyme contains only an additional UC at its 3' end, while Hr.(mutant) and AHDV have 52 and 37 nucleotides, respectively, at their 3' ends, A 622 nucleotide, internally-labeled target RNA was incubated, under ribozyme excess conditions, along with the three ribozyme transcripts in a standard reaction buffer, To make internally-labeled substrate RNA for trans-ribozyme cleavage reactions, a 622 nt region (containing hammerhead site P) was synthesized by PCR using primers that place the T7 RNA promoter upstream of the amplified sequence. Target RNA was transcribed in a standard transcription buffer in the presence of 3 2 P]CTP (Chowrira Burke, 1991 supra). Thq reaction mixture was treated with 15 units of ribonuclease-free DNasel, extracted with phenol followed chloroform:isoamyl alcohol precipitated with isopropanol and washed with 70% ethanol, The dried pellet was resuspended in 20 pI DEPC-treated water and stored at -200C.

Unlabeled ribozyme (1pM) and internally labeled 622 nt substrate RNA (<10 nM) were denatured and renatured separately in a standard cleavage buffer (containing 50 mM Tris-HCI pH 7.5 and 10 mM MgCI2) by heating to 900C for 2 min. and slow cooling to 37°C for 10 min, The reaction was initiated by mixing the ribozyme and substrate mixtures and incubating at 37°C, Aliquots of 5 pl were taken at regular time intervals, quenched by adding an equal volume of 2X formamide gel loading buffer and frozen on dry ice. The samples were resolved on 5%0 polyacrylamide sequencing gel and results were quantitatively analyzed by radioanalytic imaging of gels with a Phosphorlmager® (Molecular Dynamics, Sunnyvale,

CA),

The HH trans-acting ribozyme cleaves the target RNA approximately faster than the AHDV transcript and greater than 20-fold faster than V- PIIIII~IP---- ')5'23225 PC'I /1WB 5!I 93 the HH(mutant) transcript (Figure 28). The additional nucleotides at the end of HH(mutant) form 7 base-pairs with the 3' target-binding arm of the trans-acting ribozyme (Figure 23). This interaction must be disrupted (at a cost of 6 kcal/mole) to make the trans-acting ribozyme available for binding the target sequence. In contrast, the additional nucleotides at the end of AHDV were not designed to form any strong, alternative base-pairing with the trans-ribozyme, Nevertheless, the AHDV sequences are predicted to form multiple structures involving the 3' target-binding arm of the trans ribozyme that have stabilities ranging from 1-2 kcal/mole. Thus, the observed reductions in activity for the AHDV and HH(mutant) constructs are consistent with the predicted folded structures, and it reinforces the view that the flanking sequences can decrease the catalytic efficiency of a ribozyme through nonproductive interactions with either the ribozyme or the substrate or both.

Example 25: RNA self-processing in vivo Since three of the constructs (HH, HDV and HP(GC)) self-process efficiently in solution, the affect of the mammalian cellular milieu on ribozyme self-processing was next explored by applicant. A transient expression system was employed to investigate ribozyme activity in vivo. A mouse cell line (OST7-1) that constitutively expresses T7 RNA polymerase in the cytoplasm was chosen for this study (Elroy-Stein and Moss, 1990 Proc. Natl. Acad, Sci. USA 87, 6743). In these cells plasmids containing a ribozyme cassette downstream of the T7 promoter will be transcribed efficiently in the cytoplasm (Elroy-Stein Moss, 1990 supra).

Monolayers of a mouse L9 fibroblast cell line (OST7-1; Elroy-Stein and Moss, 1990 supra) were grown in 6-well plates with 5x10 5 cells/well.

Cells were transfected with circular plasmids (5 tg/well) using the calcium phosphate-DNA precipitation method (Maniatis et al,, 1982 supra). Cells were lysed (4 hours post-transfection) by the addition of standard lysis buffer (200 pl/well) containing 4M guanadinium isothiocyanate, 25 mM sodium citrate (pH 0.5% sarkosyl (Chomczynski and Sacchi, 1987 Anal. Biochem. 162, 156), and 50 mM EDTA pH 8.0, The lysate was extracted once with water-saturated phenol followed by one extraction with chloroform:isoamyl alcohol Total cellular RNA was precipitated with an equal volume of isopropanol, The RNA pellet was resuspended in 0.2

C"&

I I \VO 9)5/23225 P( I I il I)5 I 94 M ammonium acetate and reprecipitated with ethan 1o pellet was then washed with 70% ethanol and resuspended in DEPC-teated water, Purified cellular RNA (3 .tg/reaction) was first denatured in the presence of a 5' end-labeled DNA primer (100 pmol) by heating to 90'C for 2 min, in the absence of Mg 2 and then snap-cooling on ice for at least min, This protocol allows for efficient annealing of the primer to its complementary RNA sequence. The primer was extended using Superscript II reverse transcriptase (8 U/pl; BRL) in a buffer containing mM TrisHCI pH 8.3; 10 mM DTT; 75 mM KCI; 1 mM MgCl2; 1 mM each dNTP, The extension reaction was carried out at 42°C for 10 min, The reaction was terminated by adding an equal volume of 2x formamide gel loading buffer and freezing on crushed dry ice, The samples were resolved on a 10% polyacrylamide sequencing gel. The primer sequences are as follows: HH primer, 5'-CTCCAGTTTCGAGCTTT-3'; HDV primer, AAGTAGCCCAGGTCGGACC-3'; HP primer, ACCAGGTAATATACCACAAC-3', As shown in Figure 29, specific bands corresponding to full-length precursor RNA and 3' cleavage products were detected from cells transfected with the self-processing cassettes, All three constructs, in addition to being transcriptionally active, appear to self-process efficiently in the cytoplasm of OST7-1 cells, In particular, the HH and HP(GC) constructs self-process to greater than 95%, The overall extent of selfprocessing in OST7-1 cells appears to be strikingly similar to the extent of self-processing in vitro (Figure 29 "In Vitro +MgCI2" vs, "Cellular"), Consistent with the in vitro self-processing results, the HP(GU) cassette self-processed to approximately 50% in OST7-1 cells, As expected, transfection with plasmids containing the HH(mutant) cassette yielded a primer-extension product corresponding to the full-length RNA with no detectable cleavage products (Figure 29), The latter result strongly suggests that the primer extension band corresponding to the 3' cleavage product is not an artifact of reverse transcription, Applicant was concerned with the possibility that RNA self-processing might occur during cell lysis, RNA isolation and /or the primer extension assay. Two precautions were taken to exclude this possibility, First, 50 mM EDTA was included in the lysis buffer, EDTA is a strong chelator of divalent

-I

O ')5123225 IC l' lI1195:()(1 1( metal ions such as Mg2+ and Ca 2 that are necessary for ribozymo activity. Divalent metal ions are therefore unavailable to self-processing RNAs following cell lysis. A second precaution involved using primers in the primer-extension assay that were designed to hybridize to essential regions of the processing ribozyme. Binding of these primers should prevent the 3' cis-acting ribozymes from folding into the conformation essential for catalytic activity, Two experiments were carried out to further eliminate the possibility that self-processing is occurring either during RNA preparations or during the primer extension analysis. The first experiment involves primer extension analysis on full-length precursor RNAs that were added to nontransfected OST7-1 lysates after cell lysis, Thus, only if self-processing is occurring at some point after lysis would cleavage products be detected.

Full-length precursor RNAs were prepared by transcribing under conditions of low Mg 2 (5 mM) and high NTP concentration (total 12 mM) in an attempt to eliminate the free Mg 2 required for the self-processing reaction (Michel et al. 1992 Genes Dev, 6, 1373). The full-length precursor RNAs were gel-purified, and a known amount was added to lysates of nontransfected OST7-1 cells. RNA was purified from these lysates and incubated for 1 hr in DEPC-treated water at 370 C prior to the standard primer extension analysis (Figure 29, in vitro "-MgCI2" control). The predominant RNA detected in all cases corresponds to the primer extension product of full-length precursor RNAs, If, instead, the purified RNA containing the full-length precursor is incubated in 10 mM MgCl2 prior to the primer extension analysis, most or all of the RNA detected by primer extension analysis undergoes cleavage (Figure 29, in vitro "+MgCl2" control). These results indicate that the standard RNA isolation and primer extension protocols used here do not provide a favorable environment for RNA self-processing, even though the RNA in question is inherently able to undergo self-cleavage.

In a second experiment to demonstrate lack of self-processing during work up, internally-labeled precursor RNAs were prepared and added to non-transfected OST7-1 lysates as in the previous control, The internallylabeled precursor RNAs were carried through the RNA purification and primer extension reactions (in the presence of unlabeled primers) and analyzed to determine the extent of self-processing. By this analysis, the Illb \VO tJS23225 PCT/Illl 51if)15 96 vast majority of the added full-length RNA remained intact during the entire process of RNA isolation and primer extension, These two control experiments validate the protocols used and support applicant's conclusion that the self-processing reactions catalyzed by HH, HDV and HP(GC) cassettes are occurring in the cytoplasm of OST7-1 cells.

Sequences in figures 23 through 25 are meant to be non-limiting examples, Those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art.

In addition, those in the art will recognize that Applicant provides guidance through the above examples as to how to best design vectors of this invention so that secondary structure of the mRNA allows efficient cleavage by releasing ribozymes. Thus, the specific constructs are not limiting in this invention. Such constructs can be readily tested as described above for such secondary structure, either by computer folding algorithms or empirically. Such constructs will then allow at least completion of release of ribozymes, which can be readily determined as described above or by methods known in the art, That is, any such secondary structure in the RNA does not reduce release of the ribozymes by more than IV. Ribozymes Expressed by RNA Polymerase III Applicant has determined that the level of production of a foreign RNA, using a RNA polymerase III (pol III) based system, can be significantly enhanced by ensuring that the RNA is produced with the 5' terminus and a 3' region of the RNA molecule base-paired together to form a stable intramolecular stem structure, This stem structure is formed by hydrogen bond interactions (either Watson-Crick or non-Watson-Crick) between nucleotides in the 3' region (at least 8 bases) and complementary nucleotides in the 5' terminus of the same RNA molecule.

Although the example provided below involves a type 2 pol III gene unit, a number of other pol III promoter systems can also be used, for example, tRNA (Hall et al., 1982 Cell 29, 5S RNA (Nielson et al., 1993, Nucleic Acids Res. 21, 3631-3636), adenovirus VA RNA (Fowlkes and Shenk, 1980 Cell 22, 405-413), U6 snRNA (Gupta and Reddy, 1990

I

LLU_ 0 95,23225 1l5 l(O 97 Nucleic Acids Res, 19, 2073-2075), vault RNA (Kickoefer et al., 1993 J.

Biol. Chem. 268, 7868-7873), telomerase RNA (Romero and Blackburn, 1991 Cell 67, 343-353), and others, The construct described in this invention is able to accumulate RNA to a significantly higher level than other constructs, even those in which and 3' ends are involved in hairpin loops, Using such a construct the level of expression of a foreign RNA can be increased to between 20,000 and 50,000 copies per cell, This makes such constructs, and the vectors encoding such constructs, excellent for use in decoy, therapeutic editing and antisense protocols as well as for ribozyme formation. In addition, the molecules can be used as agonist or antagonist RNAs (affinity RNAs), Generally, applicant believes that the intramolecular base-paired interaction between the 5' terminus and the 3' region of the RNA should be in a double-stranded structure in order to achieve enhanced RNA accumulation.

Thus, in one preferred embodiment the invention features a pol III promoter system a type 2 system) used to synthesize a chimeric RNA molecule which includes tRNA sequences and a desired RNA a tRNA-based molecule), The following exemplifies this invention with a type 2 pol III promoter and a tRNA gene. Specifically to illustrate the broad invention, the RNA molecule in the following example has an A box and a B box of the type 2 pol III promoter system and has a 5' terminus or region able to base-pair with at least 8 bases of a complementary 3' end or region of the same RNA molecule, This is meant to be a specific example, Those in the art will recognize that this is but one example, and other embodiments can be readily generated using other pol III promoter systems and techniques generally known in the art.

By "terminus" is meant the terminal bases of an RNA molecule, ending in a 3' hydroxyl or 5' phosphate or 5' cap moiety, By "region" is meant a stretch of bases 5' or 3' from the terminus that are involved in base-paired interactions. It need not be adjacent to the end of the RNA. Applicant has determined that base pairing of at least one end of the RNA molecule with a region not more than about 50 bases, and preferably only 20 bases, from 9P-1 II \WO 95123225 C I '(51 I)Oll 1 Sc 98 the other end of the molecule provides a useful molecule able to be expressed at high levels, By region" is meant a stretch of bases 3' from the terminus that are involved in intramolecular bas-paired interaction with complementary nucleotides in the 5' terminus of the same molecule. The 3' region can be designed to include the 3' terminus. The 3' region therefore is 0 nucleotides from the 3' terminus, For example, in the S35 construct described in the present invention (Fig, 40) the 3' region is one nucleotide from the 3' terminus. In another example, the 3' region is 43 nt from 3' terminus, These examples are not meant to be limiting. Those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art, Generally, it is preferred to have the 3' region within 100 bases of the 3' terminus.

By "tRNA molecule" is meant a type 2 pol III driven RNA molecule that is generally derived from any recognized tRNA gene. Those in the art will recognize that DNA encoding such molecules is readily available and can be modified as desired to alter one or more bases within the DNA encoding the RNA molecule and/or the promoter system. Generally, but not always, such molecules include an A box and a B box that consist of sequences which are well known in the art (and examples of which can be found throughout the literature). These A and B boxes have a certain consensus sequence which is essential for a optimal pol III transcription.

By "chimeric tRNA molecule" is meant a RNA molecule that includes a pol III promoter (type 2) region. A chimeric tRNA molecule, for example, might contain an intramolecular base-paired structure between the 3' region and complementary 5' terminus of the molecule, and includes a foreign RNA sequence at any location within the molecule which does not affect the activity of the type 2 pol III promoter boxes. Thus, such a foreign RNA may be provided at the 3' end of the B box, or may be provided in between the A and the B box, with the B box moved to an appropriate location either within the foreign RNA or another location such that it is effective to provide pol Ill transcription, In one example, the RNA molecule may include a hammerhead ribozyme with the B box of a type 2 pol III promoter provided in stem II of the ribozyme. In a second example, the B box may be provided in stem IV region of a hairpin ribozyme. A specific example of such RNA molecules is provided below. Those in the art will

I

\O (;112325 S( "i IB9)5/(i( recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art, By "desired RNA" molecule is meant any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include antisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA.

By "antisense RNA" is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et al., 1991 Annu. Rev.

Biochem, 60, 631-652). By "enzymatic RNA" is meant an RNA molecule with enzymatic activity (Cech, 1988 J.American. Med. Assoc. 260, 3030- 3035), Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA, Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

By "decoy RNA" is meant an RNA molecule that mimics the natural binding domain for a ligand, The decoy RNA therefore competes with natural binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy" and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al,, 1990 Cell 63, 601-608). This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art, By "therapeutic editing RNA" is meant an antisense RNA that can bind to its cellular target (RNA or DNA) and mediate the modification of a specific base.

By "agonist RNA" is meant an RNA molecule that can bind to protein receptors with high affinity and cause the stimulation of specific cellular pathways.

~ss~ I \VO )5/23225 P(1"IIl 95 (l 15,( 100 By "antagonist RNA" is meant an RNA molecule that can bind to cellular proteins and prevent it from performing its normal biological function (for example, see Tsai et al,, 1992 Proc, Nail, Acad, Sci, USA 89, 8864-8868).

In other aspects, the invention includes vectors encoding RNA molecules as described above, cells including such vectors, methods for producing the desired RNA, and use of the vectors and cells to produce this

RNA,

Thus, the invention features a transcribed non-naturally occuring RNA molecule which includes a desired therapeutic RNA portion and an intramolecular stem formed by base-pairing interactions between a 3' region and complementary nucleotides at the 5' terminus in the RNA, The stem preferably includes at least 8 base pairs, but may have more, for example, 15 or 16 base pairs.

In preferred embodiments, the 5' terminus of the chimeric tRNA includes a portion of the precursor molecule of the primary tRNA molecule, of which 8 nucleotides are involved in base-pairing interaction with the 3' region; the chimeric tRNA contains A and B boxes; natural sequences 3' of the B box are deleted, which prevents endogenous RNA processing; the desired RNA molecule is at the 3' end of the B box; the desired RNA molecule is between the A and the B box; the desired RNA molecule includes the B box; the desired RNA molecule is selected from the group consisting of antisense RNA, decoy RNA, therapeutic editing RNA, enzymatic RNA, agonist RNA and antagonist RNA; the molecule has an intramolecular stem resulting from a base-paired interaction between the terminus of the RNA and a complementary 3' region within the same RNA, and includes at least 8 bases; ?;nd the 5' terminus is able to base pair with at least 15 bases of the 3' region.

In most preferred embodiments, the molecule is transcribed by a RNA polymerase III based promoter system, a type 2 pol III promoter system; the molecule is a chimeric tRNA, and may have the A and B boxes of a type 2 pol III promoter separated by between 0 and 300 bases; DNA vector encoding the RNA molecule of claim 51.

II' WO 95/23225 P( J 11W95I)|51 101 In other related aspects, the invention features an RNA or DNA vector encoding the above RNA molecule, with the portions of the vector encoding the RNA functioning as a RNA pol III promoter; or a cell containing the vector or a method to provide a desired RNA molecule in a cell, by introducing the molecule into a cell with an RNA molecule as described above. The cells can be derived from animals, plants or human beings.

In order for RNA-based gene therapy approaches to be effective, sufficient amounts of the therapeutic RNA must accumulate in the appropriate intracellular compartment of the treated cells. Accumulation is a function of both promoter strength of the antiviral gene, and the intracellular stability of the antiviral RNA. Both RNA polymerase II (pol II) and RNA polymerase III (pol III) based expression systems have been used to produce therapeutic RNAs in cells (Sarver Rossi, 1993 AIDS Res, Human Retroviruses 9, 483-487; Yu et al,, 1993 P.N,A.S.(USA) 90, 6340- 6344). However, pol III based expression cassettes are theoretically more attractive for use in expressing antiviral RNAs for the following reasons.

Pol II produces messenger RNAs located exclusively in the cytoplasm, whereas pol III produces functional RNAs found in both the nucleus and the cytoplasm. Pol II promoters tend to be more tissue restricted, whereas pol III genes encode tRNAs and other functional RNAs necessary for basic "housekeeping" functions in all cell types. Therefore, pol III promoters are likely to be expressed in all tissue types, Finally, pol III transcripts from a given gene accumulate to much greater levels in cells relative to pol II genes, Intracellular accumulation of therapeutic RNAs is also dependent on the method of gene transfer used. For example, the retroviral vectors presently used to accomplish stable gene transfer, integrate randomly into the genome of target cells. This random integration leads to varied expression of the transferred gene in individual cells comprising the bulk treated cell population. Therefore, for maximum effectiveness, the transferred gene must have the capacity to express therapeutic amounts of the antiviral RNA in the entire treated cell population, regardless of the integration site.

1. 95, 23225 f l 1195 M(I A6 102 Pol III System The following is just one non-limiting example of the invention. A pol III based genetic element derived from a human tRNAi m et gene and termed A3-5 (Fig. 33; Adeniyi-Jones et al., 1984 supra), has been adapted to express antiviral RNAs (Sullenger et al,, 1990 Mol. Cell. Biol. 10, 6512- 6523), This element was inserted into the DC retroviral vector (Sullenger et al., 1990 Mol. Cell, Biol. 10, 6512-6523) to accomplish stable gene transfer, and used to express antisense RNAs against moloney murine leukemia virus and anti-HIV decoy RNAs (Sullenger et al,, 1990 Mol. Cell, Biol. 10, 6512-6523; Sullenger et al,, 1990 Cell 63, 601-608; Sullenger et al., 1991 J, Virol. 65, 6811-6816; Lee et al,, 1992 The New Biologist 4, 66- 74), Clonal lines are expanded from individual cells present in the bulk population, and therefore express similar amounts of the therapeutic RNA in all cells. Development of a vector system that generates therapeutic levels of therapeutic RNA in all treated cells would represent a significant advancement in RNA based gene therapy modalities.

Applicant examined hammerhead (HHI) ribozyme (RNA with enzymatic activity) expression in human T cell lines using the A3-5 vector system (These constructs are termed "A3-5/HHI"; Fig. 34). On average, ribozymes were found to accumulate to less than 100 copies per cell in the bulk T cell populations. In an attempt to improve expression levels of the chimera, the applicant made a series of modified A3-5 gene units containing enhanced promoter elements to increase transcription rates, and inserted structural elements to improve the intracellular stability of the ribozyme transcripts (Fig. One of these modified gene units, termed gave rise to more than a 100-fold increase in ribozyme accumulation in bulk T cell populations relative to the original A3-5/HHI vector system.

Ribozyme accumulation in individual clonal lines from the pooled T cell populations ranged from 10 to greater than 100 fold more than those achieved with the original A3-5/HHI version of this vector, The S35 gene unit may be used to express other therapeutic RNAs including, but not limited to, ribozymes, antisense, decoy, therapeutic editing, agonist and antagonist RNAs, Application of the S35 gene unit would not be limited to antiviral therapies, but also to other diseases, such as cancer, in which therapeutic RNAs may be effective. The S35 gene unit may be used in the context of other vector systems besides retroviral L~IOTI~ I \VO 95/23225 l'C I I1.1( 103 vectors, including but not limited to, other stable gene transfer systems such as adeno-associated virus (AAV; Carter, 1992 Curr, Opin. Genet. Dev.

3, 74), as well as transient vector systems such as plasmid delivery and adenoviral vectors (Berkner, 1988 BioTechniques 6, 616-629).

As described below, the S35 vector encodes a truncated version of a tRNA wherein the 3' region of the RNA is base-paired to complementary nucleotides at the 5' terminus, which includes the 5' precursor portion that is normally processed off during tRNA maturation. Without being bound by any theory, Applicant believes this feature is important in the level of expression observed. Thus, those in the art can now design equivalent RNA molecules with such high expression levels. Below are provided examples of the methodology by which such vectors and tRNA molecules can be made, Vectors The use of a truncated human tRNAimet gene, termed A3-5 (Fig. 33; Adeniyi-Jones et al., 1984 supra), to drive expression of antisense RNAs, and subsequently decoy RNAs (Sullenger et al,, 1990 supra) has recently been reported. Because tRNA genes utilize internal pol III promoters, the antisense and decoy RNA sequences were expressed as chimeras containing tRNAimet sequences. The truncated tRNA genes were placed into the U3 region of the 3' moloney murine leukemia virus vector LTR (Sullenger et al,, 1990 supra).

Base-Paired Structures Since the A3-5 vector combination has been successfully used to express inhibitory levels of both antisense and decoy RNAs, applicant cloned ribozyme-encoding sequences (termed as "A3-5/HHI") into this vector to explore its utility for expressing therapeutic ribozymes. However, low ribozyme accumulation in human T cell lines stably transduced with this vector was observed (Fig. 35). To try and improve accumulation of the ribozyme, applicant incorporated various RNA structural elements (Fig. 34) into one of the ribozyme chimeras Two strategies were used to try and protect the termini of the chimeric transcripts from exonucleolytic degredation. One strategy involved the incorporation of stem-loop structures into the termini of the transcript. Two

I

\O 9'5/23225 I 'I 195/111 104 such constructs were cloned, S3 which contains a stem-loop structure at the 3' end, and S5 which contains stem-loop structures at both ends of the transcript (Figure 34). The second strategy involved modification of the 3' terminal sequences such that the 5' terminus and the 3' end sequences can form a stable base-paired stem. Two such constructs were made: in which the 3' end was altered to hybridize to the 5' leader and acceptor stem of the tRNAimet domain, and S35Plus which was identical to S35 but included more extensive structure formation within the non-ribozyme portion of the A3-5 chimeras (Figure 34), These stem-loop structures are also intended to sequester non-ribozyme sequences in structures that will prevent them from interfering with the catalytic activity of the ribozyme.

These constructs were cloned, producer cell lines were generated, and stably-transduced human MT2 (Harada et al., 1985 supra) and CEM (Nara Fischinger, 1988 supra) cell lines were established (Curr. Protocols Mol, Bio/. 1992, ed. Ausubel et al., Wiley Sons, NY). The RNA sequences and structure of S35 and S35 Plus are provided in Figures 40-47.

Referring to Figure 48, there is provided a general structure for a chimeric RNA molecule of this invention, Each N independently represents none or a number of bases which may or may not be base paired. The A and B boxes are optional and can be any known A or B box, or a consensus sequence as exemplified in the figure, The desired nucleic acid to be expressed can be any location in the molecule, but preferably is on those places shown adjacent to or between the A and B boxes (designated by arrows), Figure 49 shows one example of such a structure in which a desired RNA is provided 3' of the intramolecular stem, A specific example of such a construct is provided in Figures 50 and 51, Example 26: Cloning of A3-5-Ribozyme Chimera Oligonucleotides encoding the S35 insert that overlap by at least nucleotides were designed GATCCACTCTGCTGTTCTGTTTTGA 3' and 5' CGCGTCAAAAACAGAACAGCAGAGTG The oligonucleotides M each) were denatured by boiling for 5 min in a buffer containing mM TrisHCI, pH8.0, The oligonucleotides were allowed to anneal by snap cooling on ice for 10-15 min.

The annealed oligonucleotide mixture was converted into a doublestranded molecule using Sequenase® enzyme (US Biochemicals) in a

M

\VO 95123225 P"I/I I195/1l I i5( 105 buffer containing 40 mM Tris.HCI, pH7.5, 20 mM MgCI2, 50 mM NaCI, mM each of the four deoxyribonucleotide triphosphates, 10 mM DTT. The reaction was allowed to proceed at 370C for 30 min. The reaction was stopped by heating to 70°C for 15 min.

The double stranded DNA was digested with appropriate restriction endonucleases (BamHI and Mlul) to generate ends that were suitable for cloning into the A3-5 vector, The double-stranded insert DNA was ligated to the A3-5 vector DNA by incubating at room temperature (about 20°C) for 60 min in a buffer containing 66 mM Tris.HCI, pH 7.6, 6.6 mM MgCI2, 10 mM DTT, 0.066 piM ATP and 0.1U/il T4 DNA Ligase (US Biochemicals).

Competent E, coli bacterial strain was transformed with the recombinant vector DNA by mixing the cells and DNA on ice for 60 min.

The mixture was heat-shocked by heating to 37°C for 1 min. The reaction mixture was diluted with LB media and the cells were allowed to recover for min at 37°C, The cells were plated on LB agar plates and incubated at 37 0 C for 18 h.

Plasmid DNA was isolated from an overnight culture of recombinant clones using standard protocols (Ausubel et al,, Cu, Protocols Mol.

Biology 1990, Wiley Sons, NY).

The identity of the clones were determined by sequencing the plasmid DNA using the Sequenase DNA sequencing kit (US Biochemicals).

The resulting recombinant A3-5 vector contains the S35 sequence.

The HHI encoding DNA was cloned into this A3-5-S35 containing vector using Sacll and BamHI restriction sites, Example 27: Northern analysis RNA from the transduced MT2 cells were extracted and the presence of A3-5/ribozyme chimeric transcripts were assayed by Northern analysis (Curr, Protocols Mol. Biol. 1992, ed. Ausubel et al., Wiley Sons, NY).

Northern analysis of RNA extracted from MT2 transductants showed that chimeras of appropriate sizes were expressed (Fig. 35.36).

In addition, these results demonstrated the relative differences in accumulation among the different constructs (Figure 35.36). The pattern of IIPL ~lk I 0 '5?23225 P( 1110("H05(> 106 expression seen from the A3-5/HHI ribozyme chimera was similar to 12 other ribozymes cloned into the A3-5 vector (not shown). In MT-2 cell line, ribozyme chimeras accumulated, on average, to less than 100 copies per cell.

Addition of a stem-loop onto the 3' end of A3-5/HHI did not lead to increased A3-5 levels (S3 in Fig. 35.36). The S5 construct containing both and 3' stem-loop structures also did not lead to increased ribozyme levels (Fig. 35.36).

Interestingly, the S35 construct expression in MT2 cells was about 100-fold more abundant relative to the original A3-5/HHI vector transcripts (Fig. 35,36). This may be due to increased stability of the S35 transcript.

Example 28: Cleavage activity To assay whether ribozymes transcribed in the transduced cells contained cleavage activity, total RNA extracted from the transduced MT2 T cells were incubated with a labeled substrate containing the HHI cleavage site (Figure 37). Ribozyme activity in all but the S35 constructs, was too low to detect, However, ribozyme activity was detectable in transduced T cell RNA. Comparison of the activity observed in the transduced MT2 RNA with that seen with MT2 RNA in which varying amounts of in vitro transcribed S5 ribozyme chimeras, indicated that beteen 1-3 nM of S35 ribozyme was present in S35-transduced MT2 RINA, This level of activity corresponds to an intracellular concentration of 5,000-15,000 ribozyme molecules per cell, Example 29: Clonal variation Variation in the ribozyme expression levels among cells making up the bulk population was determined by generating several clonal cell lines from the bulk S35 transduced CEM line (Curr. Protocols Mol, Biol, 1992, ed, Ausubel et al,, Wiley Sons, NY) and the ribozyme expression and activity levels in the individual clones were measured (Figure 38 and 39).

All the individual clones were found to express active ribozyme. The ribozyme activity detected from each clone correlated well with the relative amounts of ribozyme observed by Northern analysis, Steady state ribozyme levels among the clones ranged from approximately 1,000 molecules per cell in clone G to 11,000 molecules per cell in clone H (Fig.

19* I~Y~BWieAL~QII~ n~ aaa~~ 9 23225 I11i')? 107 38). The mean accumulation among the clones, calculated by averaging the ribozyme levels of the clones, exactly equaled the level measured in the parent bulk population. This suggests that the individual clones are representative of the variation present in the bulk population, The fact that all 14 clones were found to express ribozyme indicate that the percentage of cells in the bulk population expressing ribozymA is also very high. In addition, the lowest level of expression in the clones was still more than 10-fold that seen in bulk cells transduced with the original vector. Therefore, the S35 gene unit should be much more effective in a gene therapy setting in which bulk cells are removed, transduced and then reintroduced back into a patient.

Example 30: Stability Finally, the bulk S35-transduced line, resistant to G418, was propogated for a period of 3 months (in the absence of G418) to determine if ribozyme expression was stable over extended periods of time. This situation mimicks that found in the clinic in which bulk cells are transduced and then reintroduced into the patient and allowed to propogate. There was a modest 30% reduction of ribozyme expression after 3 months. This difference probably arose from cells with varying amount of ribozyme expression and exhibiting different growth rates in the culture becoming slightly more prevalent in the culture, However, ribozyme expression is apparently stable for at least this period of time.

Example 31: Design and construction of TRZ-tRNA Chimera A transcription unit, termed TRZ, is designed that contains the motif (Figure 52). A desired RNA ribozyme) can be inserted into the indicated region of TRZ tRNA chimera. This construct might provide additional stability to the desired RNA. TRZ-A and TRZ-B are non-limiting examples of the TRZ-tRNA chimera.

Referring to Fig. 53-54, a hammerhead ribozyme targeted to site I (HHITRZ-A; Fig. 53) and a hairpin ribozyme (HPITRZ-A; Fig. 54), also targeted to site I, is cloned individually into the indicated region of TRZ tRNA chimera. The resulting ribozyme trancripts retain full RNA cleavage activity (see for example Fig, 55). Applicant has shown that efficient a~ 0 95 23225 Pi'('lliil 108 expression of these TRZ tRNA chimera can be achieved in mammalian cells.

Besides ribozymes, desired RNAs like antisense, therapeutic editing RNAs, decoys, can be readily inserted into the indicated region of TRZtRNA chimera to achieve therapeutic levels of RNA expression in mammalian cells.

Sequences listed in Figures 40-47 and 50 54 are meant to be nonlimiting examples. Those skilled in the art will recognize that variants (mutations, insertions and deletions) of the above examples can be readily generated using techniques known in the art, are within the scope of the present invention.

Example 32: Ribozvme expression in T cell lines Ribozyme expression in T cell lines stably-transduced with either a retroviral-based or an Adeno-associated virus (AAV)-based ribozyme expression vector (Figure 56), The human T cell lines MT2 and CEM were transduced with either retroviral or AAV vectors encoding a neomycin slelctable marker and a ribozyme (S35/HHI) expressed from pol III meti tRNA-driven promoter. Cells stably-transduced with the vectors were selectivelyt expanded medium containing the neomycin antibiotic derivative, G418 (0,7 mg/ml), Ribozyme expression in the stable cell lines was then alalyzed by Northern analysis. The probe used to detect ribozyme transcripts also cross-hybridized with human met i tRNA sequences, Refering to Figure 56, S35/HHI RNA accumulates to significant levels in MT2 and CEM cells when transduced with either the retrovirus or the AAV vector.

These are meant to be non-limiting examples, those skilled in the art will recognize that other vectors such as adenovirus vector (Figure 57), plasmid DNA vector, alpha virus vectors and the other derivatives there of, can be readily generated to deliver the desired RNA, using techniques known in the art and are within the scope of this invention, Additionally, the transcription units can be expressed individually or in multiples using pol II and/or pol III promoters, References cited herein, as well as Draper WO 93/23569, 94/02495, 94/06331, Sullenger WO 93/12657, Thompson WO 93/04573, and Sullivan

I

I__II_

\VO 95/23225 PC'T/1195/100156 109 WO 94/04609, and 93/11253 describe methods for use of vectors decribed herein, and are incorporated by reference herein, In particular these vectors are useful for administration of antisense and decoy RNA molecules, Example 33: Ligated Ribozymes are catalytically active The ability of ribozymes generated by ligation methods, described in Draper et al,, PCT WO 93/23569, to cleave target RNA was tested on either matched substrate RNA (Fig. 58) or long (622 nt) RNA (Fig. 59, 60 and 61), Matched substrate RNAs were chemically synthesized using solidphase RNA synthesis chemistry (Scaringe et al., 1990 Nucleic Acids Res.

18, 5433-5441). Substrate RNA was 5' end-labeled using [y.

3 2 pj ATP and polynucleotide kinase (Curr. Protocols Mol, Biol. 1992, ed. Ausubel et al,, Wiley Sons, NY). Ribozyme reactions were carried out under ribozyme excess conditions (kcat/KM; Herschlag and Cech, 1990 Biochemistry 29, 10159-10171). Briefly, ribozyme and substrate RNA were denatured and renatured separately by heating to 900C and snap cooling on ice for 10 min in a buffer containing 50 mM Tris, HCI pH 7,5 and 10 mM MgCl2, Cleavage reaction was initiated by mixing the ribozyme with the substrate at 37°C. Aliquots of 5 pl were taken at regular intervals of time and the reaction was stopped by mixing with equal volume of formamide gel loading buffer (Curr. Protocols Mol, Biol, 1992, ed, Ausubel et al,, Wiley Sons, NY). The samples were resolved on 20 polyacrylamide-urea gel, Refering to Fi, 58, -AG refers to the free energy of binding calculated for base-paired interactions between the ribozyme and the substrate RNA (Turner and Sugimoto, 1988 Supra). RPI A is a HH ribozyme with 6/6 binding arms. This ribozyme was synthesized chemically either as a one piece ribozyme or was synthesized in two fragments followed by ligation to generate a one piece ribozyme. The kcat/KM values for the two ribozymes were comparable, A template containing T7 RNA polymerase promoter upstream of 622 nt long target sequence, was PCR amplified from a DNA clone, The target RNA (containing HH ribozyme cleavage sites B, C and D) was transcribed from this PCR amplified template using T7 RNA polymerase. The transcript was internally labeled during transcription by including [a- 3 2 P] CTP as one of the four ribonucleotide triphosphates. The transcription mixture was \VO 95/23225 PC'TI 1B95/0(15 110 treated with DNase-1, following transcription at 37°C for 2 hours, to digest away the DNA template used in the transcription. RNA was precipitated with Isopropanol and the pellet was washed two times with 70% ethanol to get rid of salt and nucleotides used in the transcription reaction. RNA is resuspended in DEPC-treated water and stored at 4°C. Ribozyme cleavage reactions were carried out under ribozyme excess (kcat/KM) conditions [Herschlag and Cech 1990 supral. Briefly, 1000 nM ribozyme and 10 nM internally labeled target RNA were denatured separately by heating to 90°C for 2 min in the presence of 50 mM Tris.HCI, pH 7.5 and 13 mM MgCl2, The RNAs were renatured by cooling to 37°C for 10-20 min, Cleavage reaction was initiated by mixing the ribozyme and target RNA at 370C. Aliquots of 5 il were taken at regular intervals of time and the reaction was quenched by adding equal volume of stop buffer. The samples were resolved on a sequencing gel.

Example 34: Hammerhead ribozymes with 2 base-paired stem II are catalytically active To decrease the cost of chemical synthesis of RNA, applicant was interested in determining whether the length of stem II region of a typical hammerhead ribozyme (2 4 bp stem II) can be shortened without decreasing the catalytic efficiency of the HH ribozyme, The length of stem II was systematically shortened by one base-pair at a time. HH ribozymes with three and two base-paired stem II were chemically synthesized using solid-phase RNA phosphoramidite chemistry (Scaringe et al,, 1990 supra), Matched and long substrate RNAs were synthesized and ribozyme assays were carried out as described in example 33. Referring to figures 62, 63 and 64, data shows that shortening stem II of a hammerhead ribozyme does not significantly alter the catalytic efficiency, It is applicant's opinion that hammerhead ribozymes with 2 2 base-paired stem II region are catalytically active.

Example 35: Synthesis of catalytically active hairpin ribozymes RNA molecules were chemically synthesized having the nucleotide base sequence shown in fia 65 for both the 5' and 3' fragments. The 3' fragments are phosphorylated and ligated to the 5' fragment essentially as described in example 37. As is evident from the Figure 65, the 3' and fragments can hybridize together at helix 4 and are covalently linked via I I WO 95/23225 PCTIB9500150 111 GAAA sequence. When this structure hybridizes to a substrate, a ribozymesubstrate complex structure is formed, While helix 4 is shown as 3 base pairs it may be formed with only 1 or 2 base pairs, nM mixtures of ligated ribozymes were incubated with 1-5 nM end-labeled matched substrates (chemically synthesized by solid-phase synthesis using RNA phosphoramidite chemistry) for different times in mM Tris/HCI pH 7.5, 10 mM MgCI2 and shown to cleave the substrate efficiently (Fig.66), The target and the ribozyme sequences shown in Fig. 62 and 65 are meant to be non-limiting examples. Those in the art will recognize that other embodiments can be readily generated using other sequences and techniques generally known in the art.

V, Constructs of Hairpin Ribozymes There follows an improved trans-cleaving hairpin ribozyme in which a new helix a sequence able to form a double-stranded region with another single-stranded nucleic acid) is provided in the ribozyme to basepair with a 5' region of a separate substrate nucleic acid. This helix is provided at the 3' end of the ribozyme after helix 3 as shown in Figure 3, In addition, at least two extra bases may be provided in helix 2 and a portion of the substrate corresponding to helix 2 may be either directly linked to the portion able to hydrogen bond to the 3' end of the hairpin or may have a linker of atleast one base, By trans-cleaving is meant that the ribozyme is able to act in trans to cleave another RNA molecule which is not covalently linked to the ribozyme itself. Thus, the ribozyme is not able to act on itself in an intramolecular cleavage reaction.

By "base-pair" is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other nontraditional types (for example Hoogsteen type) of interactions.

The increase in length of helix 2 of a hairpin ribozyme (with or without helix 5) has several advantages, These include improved stability of the ribozyme-target complex in vivo In addition, an increase in the recognition sequence of the hairpin ribozyme improves the specificity of the ribozyme. This also makes possible the targeting of potential hairpin RECTIFIED SHEET (RULE W)

ISA/EP

IIIIR~CIIIP \WO 95/23225 PCT/I 1B95100156 112 ribozyme sites that would otherwise bp inaccessible due to neighboring secondary structure.

The increase in length of helix 2 of a hairpin ribozyme (with or without helix 5) enhances trans-ligation reaction catalyzed by the ribozyme. Transligation reactions catalyzed by the regular hairpin ribozyme (4 bp helix 2) is very inefficient (Komatsu et al., 1993 Nucleic Acids Res. 21, 185). This is attributed to weak base-pairing interactions between substrate RNAs and the ribozyme. By increasing the length of helix 2 (with or without helix the rate of ligation (in vitro and in vivo) can be enhanced several fold.

Results of experiments suggest that the length of H2 can be 6 bp without significantly reducing the activity of the hairpin ribozyme. The H2 arm length variation does not appear to be sequence dependent, HP ribozymes with 6 bp H2 have been designed against five different target RNAs and all five ribozymes efficiently cleaved their cognate target RNA.

Additionally, two of these ribozymes were able to successfully inhibit gene expression TNF-a) in mammalian cells, Results of these experiments are shown below, HP ribozymes with 7 and 8 bp H2 are also capable of cleaving target RNA in a sequence-specific manner, however, the rate of the cleavage reaction is lower than those catalyzed by HP ribozymes with 6 bp H2.

Example 36: 4 and 6 base pair H2 Referring to Figures 67-72, HP ribozymes were synthesized as described above and tested for activity. Surprisingly, those with 6 base pairs in H2 were still as active as those with 4 base pairs.

VI. Chemical Modification Oligonucleotides with 5'-C-alkyl Group The introduction of an alkyl group at the 5'-position of a nucleoside or nucleotide sugar introduces an additional center of chirality into the sugar moiety. Referring to Fig. 75, the general structures of belonging to the D-allose, 2, and L-talose, 3, sugar families are shown.

The family names are derived from the known sugars D-allose and L-talose

(R

1

CH

3 in 2 and 3 in Figure 75). Useful specific D-allose and L-talose IBY~SII~II I \VO 95/23225 P(T/11 B95100150 113 nucleotide derivatives are shown in Figure 76, 29-32 and Figure 77, 58- 61 respectively, This invention relates to the use of 5'-C-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA, and also as antisense oligonucleotides. As the term is used in this application, enzymatic nucleic acids are catalytic nucleic molecules that contain alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript, Also within the invention are 5'-C-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides.

Such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 5'-C-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair forming region then the enhanced stability that it provides to the molecule is advantageous, In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of less consequence. Thus, for example, if a 5'-C-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has 10-fold higher stability in vivo then it has utility in the present invention, The same analysis is true for antisense oligonucleotides containing such modifications. The invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis.

Thus, in one aspect, the invention features 5'-C-alkylnucleosides, that is a nucleotide base having at the 5'-position on the sugar molecule an alkyl moiety. In a related aspect, the invention also features alkylnucleotides, and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably slPaR~II W!O 95/23225 '("ITI151I00 156 114 includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above. In preferred embodiments, the sugar of the nucleoside or nucleotide is in an optically pure form, as the talose or allose sugar.

Examples of various alkyl groups useful in this invention are shown in Figure 75, where each R 1 group is any alkyl. These examples are not limiting in the invention, Specifically, an "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 or N(CH 3 2 amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy,

NO

2 halogen, N(CH 3 2 amino, or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO 2 or N(CH 3 2 amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated 7 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted, The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring II~P~QIAI 95,23225 I( 15,1B1 )5100(150 115 atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an where R is either alkyl, aryl, alkylaryl or hydrogen.

In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 5'-C-alkylnucleotides; e.g.

enzymatic nucleic acids having a 5'-C-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 5'-position an alkyl group, In other related aspects, the invention features triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

The 5'-C-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them, While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised.

In another aspect, the invention features a method for conversion of a protected allo sugar to a protected talo sugar. In the method, the protected allo sugar is contacted with triphenyl phosphine, diethylazodicarboxylate, and p-nitrobenzoic acid under inversion causing conditions to provide the protected talo sugar, While one example of such conditions is provided below, those in the art will recognize other such conditions. Applicant has found that such conversion allows for ready synthesis of all types of nucleotide bases as exemplified in the figures.

While this invention is applicable to all oligonucleotides, applicant has found that the modified molecules of this invention are particulary useful for enzymatic RNA molecules. Thus, below is provided examples of such 9523225 PI'"I1 1l)115001150( 116 molecules, Those in the art will recognize that equivalent procedures can be used to make other molecules without such enzymatic activity.

Specifically, Figure 1 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided. This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims, Referring to Figure 1, the preferred sequence of a hammerhead ribozyme in a to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 5'-C-alkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described, Substitutions of any nucleotide with any of the modified nucleotides shown in Figure 75 are possible.

The following are non-limiting examples showing the synthesis of nucleic acids using 5'-C-alkyl-substituted phosphoramidites and the syntheses of the amidites.

Example 37: Synthesis of Hammerhead Ribozvmes Containing nucleotides Other Modified Nucleotides The method of synthesis would follow the procedure for normal RNA synthesis as described in Usman,N,; Ogilvie,K,K.; Jiang,M.-Y.; Cedergren,RJ. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433- 5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 26-29 and 56-59), These 5'-C-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 intron catalytic nucleic acids, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure, Example 38: Methyl-2,3-O-lsopropylidine-6-Deox -[-D-allofuranoside (4) A suspension of L-rhamnose (100 g, 0.55 mol), CuS0 4 (120 g) and conc. H 2 S0 4 (4.0 mL) in 1.0 L of dry acetone was mixed for 24 h at RT, then filtered. Conc. NH 4 0H (5 mL) was added to the filtrate and the newly formed precipitate was filtered. The residue was concentrated in vacuo, coevaporated with pyridine (2 x 300 mL), dissolved in pyridine (500 mL) and cooled to 0 A solution of p-toluenesufonylchloride (107 g 0.56 WVO 95/23225 P1(" 1I Iv)5$'(ll 117 mmol) in dry DCE (500 mL) was added dropwise over 0,5 h. The reaction mixture was left for 16 h at RT, The reaction was quenched by adding icewater (0,5 L) and, after mixing for 0,5 h, was extracted with chloroform (0,75 The organic layer was washed with H 2 0 (2 x 500 mL), 10% H 2

SO

4 (2 x 300 mL), water (2 x 300 mL), sat, NaHCO 3 (2 x 300 mL), brine (2 x 300 mL), dried over MgSO 4 and evaporated to dryness. The residue 115 g) was dissolved in dry MeOH (1 L) and treated with NaOMe (23,2 g, 0,42 mmol) in MeOH, The reaction mixture was left for 16 h at 20 OC, neutralized with dry CO2 and evaporated to dryness. The residue was suspended in chloroform (750 mL), filtered concentrated to 100 mL and purified by flash chromatography in CHC1 3 to yield 45 g of compound 4, Example 39: Methyl-2,3-O-lsopropylidine-5-O-t-Butyldiphenylsilyl-6- Deoxy-D-D-Allofuranoside To solution of methylfuranoside 4 (12.5 g 62.2 mmol) and AgN03 (21.25 g, 125.0 mmol) in dry DMF (300 mL) t-butyldiphenylsilyl chloride (22.2 g 81 mmol) was added dropwise under Ar over 0.5 h. The reaction mixture was stirred for 4 h at RT, diluted with CHC1 3 (200 mL), filtered and evaporated to dryness (below 40 °C using a high vacuum oil pump), The residue was dissolved in CH 2 C1 2 (300 mL) washed with sat, NaHC03 (2 x 50 mL), brine (2 x 50 mL), dried over MgS04 and evaporated to dryness.

The residue was purified by flash chromatography in CH 2

CI

2 to yield 20.0 g of compound Examole 40: Methyl-5-O-t-Butyldiphenylsilyl-6-Deoxy-p-D-Allofuranoside Methylfuranoside 5 (13.5 g, 30.6 mmol) was dissolved in 2:1:1 200 mL) and stirred at 24 °C for m. The reaction mixture was cooled to -10 neutralized with conc,

NH

4 0H (140 mL) and extracted with CH 2 Cl 2 (500 mL), The organic layer was separated, washed with sat, NaHCO 3 (2 x 75 mL), brine (2 x 75 mL), dried over MgS0 4 and evaporated to dryness. The product 6 was purified by flash chromatography using a 0-10% MeOH gradient in CH 2 C1 2 Yield g LIL ~C I WO 95/23225 SP( l/ lI1 5 (15(1 118 Example 41: Methyl-2,3-di-O-Benzoyl-5-O-t-Butvldiphenvlsilvl-6-Deoxy-i- D-Allofuranoside Methylfuranoside 6 (7.0 g, 17.5 mmol) was coevaporated with pyridine (2 x 100 mL) and dissolved in pyridine (100 mL), Benzoyl chloride (5.4 g, 38.5 mmol) was added and the reaction mixture was left at RT for 16 h. Dry EtOH (50 mL) was added and the reaction mixture was evaporated to dryness after 0.5 h. The residue was dissolved in CH2CI2 (300 mL), washed with sat. NaHCO 3 (2 x 75 mL), brine (2 x 75 mL) dried over MgSO 4 and evaporated to dryness. The product was purified by flash chromatography in CH 2 C12 to yield 9,5 g of compound 7.

Example 42: 1-O-Acetyl-2,3-di-O-benzoyl-5-O-t-Butvldiphenvlsilyl-6- Deoxy-p-o-Allofuranose Dibenzoate 7 (4.7 g, 7.7 mmol) was dissolved in a mixture of AcOH (10.0 mL), Ac20 (20.0 mL) and EtOAc (30 mL) and the reaction mixture was cooled 0 OC. 98% H 2

SO

4 (0.15 mL) was then added, The reaction mixture was kept at 0 °C for 16 h, and then poured into a cold 1:1 mixture of sat.

NaHCO 3 and EtOAc (150 mL). After 0.5 h of vigorous stirring the organic phase was separated, washed with brine (2 x 75 mL), dried over MgSO 4 evaporated to dryness and coevaporated with toluene (2 x 50 mL). The product was purified by flash chromatography using a gradient of MeOH in CH2CI 2 Yield: 4.0 g (82% as a mixture of a and p isomers), Example 43: 1-(2',3'-di-O-Benzoyl-5'-O-t-Butvldiphenylsilvl-6'-Deoxy-p-D- Allofuranosyl)uracil Uracil (1.44 g, 11.5 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour, The reaction mixture was cooled to RT, evaporated to dryness and coevaporated with dry toluene (2 x 50 mL), To the residue was added a solution of acetates 8 (6.36 g, 10.0 mmol) in dry CH3CN (100 mL), followed by CFsSOsSiMe 3 (2.8 g, 12.6 mmol). The reaction mixture was kept at 24 °C for 16 h, concentrated to 1/3 of its original volume, diluted with 100 mL of CH 2

CI

2 and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness. The product 9 was purified by flash chromatography using a gradient of 0-5% MeOH in CH 2 C1 2 Yield: 5.7 g

-I

0 1)5/23225 IiI H9S!)1() i 119 Example 44: N-Benzoyl-l-(2',3'-Di-O-Benzoyl-5'-O-t-Butvldiphenylsilvl-6'- Deoxy-3-D-Allofuranosyl)Cytosine

N

4 -benzoylcytosine (1.84 g, 8,56 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (3 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a solution of of acetates 8 (3.6 g, 5,6 mmol) in dry CH 3 CN (100 mL), followed by CF 3

SO

3 SiMe 3 (4.76 g, 21.4 mmol). The reaction mixture was boiled under reflux for 5 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH 2 Cl 2 (100 mL) and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness.

Purification by flash chromatography using a gradient of 0-5% MeOH in

CH

2

CI

2 yielded 1.8 g of compound Example 45: /N--Benzoyl-9-(2',3'-di-O-Benzoyl-5'-O-t-Butyldiphenylsill-6' Deoxy-p-D-Allofuranosyl)adenine (11).

N

6 -benzoyladenine (2.86 g, 11.86 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (7 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL), To the residue was added a solution of of acetates 8 (3.6 g, 5.6 mmol) in dry CH 3 CN (100 mL) followed by CF 3

SO

3 SiMe 3 (6.59 g, 29.7 mmol). The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH 2

CI

2 (100 mL) and extracted with sat. NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgSO 4 and evaporated to dryness. The product 11 was purified by flash chromatography using a gradient of MeOH in CH 2 012. Yield: 2.7 g Example 46: N 2 -lsobutyryl-9-(2',3'-di-O-Benzoyl-5'-O-t-Butvldiphenylsilv- 6'-Deoxy-0-D-Allofuranosyl)auanine (12).

N

2 -lsobutyrylguanine (1.47 g 11.2 mmol) was suspended in mixture of hexamethyldisilazane (100 mL) and pyridine (50 mL) and boiled under reflux until complete dissolution (6 h) occurred, and then for an additional hour. The reaction mixture was cooled to RT evaporated to dryness and coevaporated with dry toluene (2 x 50 mL). To the residue was added a

I

\VO 95123225 "171 Ill95/00 120 solution of of acetates 8 (3,4 g, 5.3 mmol) in dry CH 3 CN (100 mL) followed by CF 3

SO

3 SiMe 3 (6.22 g, 28.0 mmol), The reaction mixture was boiled under reflux for 8 h, cooled to RT, concentrated to 1/3 of its original volume, diluted with CH 2 Cl 2 (100 mL) and extracted with sat, NaHCO 3 (2 x 50 mL), brine (2 x 50 mL) dried over MgS0 4 and evaporated to dryness. The product 12 was purified by flash chromatography using a gradient of 0-2% MeOH in CH2CI2, Yield: 2,1g Example 47: AiN -Benzoyl-9-(2',3'-di-O-benzoyl-6'-Deoxy- -D-Allofuranosyl)adenine Nucleoside 11 (1,65 g, 2.0 mmol) was dissolved in THF (50 mL) and a 1 M solution of TBAF in THF (4 mL) was added. The reaction mixture was kept at RT for 4 h, evaporated to dryness and the product purified by flash chromatography using a gradient of 0-5% MeOH in CH2CI 2 to yield 1.0 g of compound Example 48: /v5-Benzoyl-9-(2',3'-di-O-Benzoyl-5'-O-Dimethoxytrityl-6'- Deoxy--D-Allofuranosyl)-adenine (19).

Nucleoside 15 (0,55 g, 0.92 mmol) was dissolved it; dry CH 2 C1 2 mL). AgNO 3 (0,34 g, 2.0 mmol), dimethoxytrityl chloride (0.68 g, 2.0 mmol) and sym-collidine (0,48 g) were added under Ar, The reaction mixture was stirred for 2h, diluted with CH 2

CI

2 (100 mL), filtered, evaporated to dryness and coevaporated with toluene (2 x 50 mL). Purification by flash chromatography using a gradient of 0-5% MeOH in CH2C1 2 yielded 0.8 g of compound 19.

Example 49: N/-Benzoyl-9-(-5'-O-Dimethoxytrityl-6'-Deoxy--D.Allofuranosyl)adenine (23).

Nucleoside 19 (1,8 g, 2 mmol) was dissolved in dioxane (50 mL), cooled to 0 OC and 2 M NaOH (50 mL) was added. The reaction mixture was kept at 0 °C for 45 m, neutralized with Dowex 50 (Pyr+ form), filtered and the resin was washed with MeOH (2 x 50 mL). The filtrate was then evaporated to dry;,ss, Purification by flash chromatography using a gradient of 0-10% MeOH in CH 2

CI

2 yielded 1.1 g of 23.

0 1) 11 11.3 2 2 5 \~O952322 C 1 1111195100115 121 Example 50: AN -Benzoyl-9-(-5'-O-Dimethoxvtrityl-2'-O-t-butyldimethylsilvl- 6'-Deoxy- 3-D-Allofuranosyl')adenine (27).

Nucleoside 23 (1.2 g, 1.8 mmol) was dissolved in dry THF (50 mL), Pyridine (0.50 g, 8 mmol) and AgNO 3 (0.4 g, 2,3 mmol) were added. After the AgNQ 3 dissolved (1.5 t-butyldimethylsilyl chloride (0.35 g ,2,3 mmol) was a:rli the reaction mixture was stirred at RT for 16 h. The reaction mixtu, diluted with CH 2

CI

2 (100 mL), filtered into sat, NaHCO 3 (50 mL), extracted, the organic layer washed with brine (2 x mL), dried over MgSO 4 and evaporated to dryness. The product 27 was 1 0 purified by flash chromatography using a hexanes:EtOAc 7:3 gradient.

Yield: 0.7 g Example 51: N,5-Benzoyl-g-(-5'-O-Dimethoxytrityl-2'-O--t-butyldimethvlsiI

I.

6'-Deoxy-f3-D-Allofuranosyl)adenine-3'-(2-OyanoethyI N, N-diisopropylk phosphoramidite) (3j1L) 1 5 Standard phosphitylatlon of 27 according to Scaringe,S.A.; Franklyn,O.; Usman,N, Nucleic Acids Res. 1990, 18, 5433-5441 yielded phosphoramidite 31 in 73% yield, Example 52: Methyl-5-0-v-Nitrobenzoyl-2,3--sopropylidine-6-deoxy-f-L- Tallofuranoside Methylfuranoside 4 (3.1 g 14.2 mmol) was dissolved in dry dioxane (200 mL), p-nitrobenzoic acid (10.0 g, 60 mmol) and triphenylphosphine (15.74 g, 60.0 mmol) were added followed by DEAD (10,45 g, 60,0 mmol), The reaction mixture was left at RT for 16 h, EtCH (5 ml-) was added, and after 0.5 h the reaction mixture was evaporated to dryness, The residue was dissolved in 0H 2 01 2 (300 ml-) washed with sat. NaHCO 3 (2 x 75 mL), brine (2 x 75 ml-) dried over MgSO4 and evaporated to dryness, Purification by flash chromatography using a hexanes:EtOAc 9:1 gradient yielded 4.1 g of compound 33. Subsequent dlebenzoylation (NaOMe/MeOH) and silylation (see preparation of 5) led to Ltalofuranoside 34 which was converted to phosphoramidites 58-61 using the same methodology as described above for the preparation of the phosphoramidites of the D-allo-Isomers 29-32, The alkyl substituted nucleotidles of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage VWO 95 23225 I'CT/l1195'(O156 122 or antisense situations. Such oligonucieotides can be formed enzymatically using triphosphate forms by standard procedure, Administration of such oligonucleotides is by standard procedure. See Sullivan et al,, PCT WO 94/ 02595.

The ribozymes and the target RNA containing site O were synthesized, deprotected and purified as described above. RNA cleavage assay was carried our at 370C in the presence of 10 mM MgCl2 as described above, Applicant has substituted 5'-C-Me-L-talo nucleotides at positions A6, A9, A9 G10, C11.1 and C11.1 G10, as shown in Figure 78 (HH-O1 to HH-O 1,2,4 and 5 showed almost wild type activity (Figure 79), However, HH-03 demonstrated low catalytic activity. Ribozymes HH-01, 2, 3, 4 and 5 are also extremely resistant to degradation by human serum nucleases, Oligonucleotides with 2'-Deoxy-2'-Alkylnucleotide This invention uses 2'-deoxy-2'-alkylnucleotides in oligonucleotides, which are particularly useful for enzymatic cleavage of RNA or singlestranded DNA, and also as antisense oligonucleotides, As the term is used in this application, 2'-deoxy-2'-alkylnucleotide-containing enzymatic nucleic acids are catalytic nucleic molecules that contain 2'-deoxy-2'alkylnucleotide components replacing, but not limited to, double stranded stems, single stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner, Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA transcript.

Also within the invention are 2'-deoxy-2'-alkylnucleotides which may be present in enzymatic nucleic acid or even in antisense oligonucleotides, Contrary to the findings of De Mesmaeker et al. applicant has found that such nucleotides are useful since they enhance the stability of the antisense or enzymatic molecule, and can be used in locations which do not affect the desired activity of the molecule. That is, while the presence of the 2'-alkyl group may reduce binding affinity of the oligonucleotide containing this modification, if that moiety is not in an essential base pair 1 I \vO 95123225 P T("7i B)510i1o5( 123 forming region then the enhanced stability that it provides to the molecule is advantageous. In addition, while the reduced binding may reduce enzymatic activity, the enhanced stability may make the loss of activity of less consequence. Thus, for example, if a 2'-deoxy-2'-alkyl-containing molecule has 10% the activity of the unmodified molecule, but has higher stability in vivo then it has utility in the present invention. The same analysis is true for antisense oligonucleotides containing such modifications, The invention also relates to novel intermediates useful in the synthesis of such nucleotides and oligonucleotides (examples of which are shown in the Figures), and to methods for their synthesis, Thus, in one aspect, the invention features 2'-deoxy-2'alkylnucleotides, that is a nucleotide base having at the 2'-position on the sugar molecule an alkyl moiety and in preferred embodiments features those where the nucleotide is not uridine or thymidine. That is, the invention preferably includes all those nucleotides useful for making enzymatic nucleic acids or antisense molecules that are not described by the art discussed above, Examples of various alkyl groups useful in this invention are shown in Fliure 81, where each R group is any alkyl. The term "alkyl" does not include alkoxy groups which have an "-O-alkyl" group, where "alkyl" is defined as described above, where the 0 is adjacent the 2'-position of the sugar molecule, In other aspects, also related to those discussed above, the invention features oligonucleotides having one or more 2'-deoxy-2'-alkylnucleotides (preferably not a 2'-alkyl- uridine or thymidine); e.g. enzymatic nucleic acids having a 2'-deoxy-2'-alkylnucleotide; and a method for producing an enzymatic nucleic acid molecule having enhanced activity to cleave an RNA or single-stranded DNA molecule, by forming the enzymatic molecule with at least one nucleotide having at its 2'-position an alkyl group. In other related aspects, the invention features 2'-deoxy-2'-alkylnucleotide triphosphates. These triphosphates can be used in standard protocols to form useful oligonucleotides of this invention.

The 2'-alkyl derivatives of this invention provide enhanced stability to the oligonulceotides containing them, While they may also reduce absolute activity in an in vitro assay they will provide enhanced overall 1Y WVO 95,23225 PC'T/I' B5/I0015 124 activity in vivo. Below are provided assays to determine which such molecules are useful. Those in the art will recognize that equivalent assays can be readily devised, In another aspect, the invention features hammerhead motifs having enzymatic activity having ribonucleotides at locations shown in Figure 80 at 6, 8, 12, and 15.1, and having substituted ribonucleotides at other positions in the core and in the substrate binding arms if desired, (The term "core" refers to positions between bases 3 and 14 in Figure 80, and the binding arms correspond to the bases from the 3'-end to base 15.1, and from the 5'-end to base Applicant has found that use of ribonucleotides at these five locations in the core provide a molecule having sufficient enzymatic activity even when modified nucleotides are present at other sites in the motif. Other such combinations of useful ribonucleotides can be determined as described by Usman et al. supra, Figure 80 shows base numbering of a hammerhead motif in which the numbering of various nucleotides in a hammerhead ribozyme is provided, This is not to be taken as an indication that the Figure is prior art to the pending claims, or that the art discussed is prior art to those claims.

Referring to Figure 80 the preferred sequence of a hammerhead ribozyme in a to 3'-direction of the catalytic core is CUGANGAG[base paired with]CGAAA. In this invention, the use of 2'-C-alkyl substituted nucleotides that maintain or enhance the catalytic activity and or nuclease resistance of the hammerhead ribozyme is described. Although substitutions of any nucleotide with any of the modified nucleotides shown in Figure 81 are possible, and were indeed synthesized, the basic structure composed of promarily 2'-O-Me nucleotides weth selected substitutions was chosen to maintain maximal catalytic activity (Yang et al. Biochemistry 1992, 31, 5005-5009 and Paolella et al. EMBO J. 1992, 11, 1913-1919) and ease of synthesis, but is not limiting to this invention.

Ribozymes from Figure 80 and Table 45 were synthesized and assayed for catalytic activity and nuclease resistance. With the exception of entries 8 and 17, all of the modified ribozymes retained at lease 1/10 of the wild-type catalytic activity. From Table 45, all 2'-modified ribozymes showed very large and significant increases in stability in human serum (shown) and in the other fluids described below (Example 55, data not shown), The order of most agressive nuclease activity was fetal bovine

I

WO 95/23225 P1'T/I 95/00()156 125 serum, human serum >human plasma human synovial fluid. As an overall measure of the effect of these 2'-substitutions on stability and activity, a ratio B was calculated (Table 45). This B value indicated that all modified ribozymes tested had significant, >100 >1700 fold, increases in overall stability and activity. These increases in B indicate that the lifetime of these modified ribozymes in vivo are significantly increased which should lead to a more pronounced biological effect.

More general substitutions of the 2'-modified nucleotides from Figure 81 also increased the t1/2 of the resulting modified ribozymes.

However the catalytic activity of these ribozymes was decreased In Figure 86 compound 37 may be used as a general intermediate to prepare derivatized 2'C-alkyl phosphoramidites, where X is CH3, or an alkyl, or other group described above.

The following are non-limiting examples showing the synthesis of nucleic acids using 2'-C-alkyl substituted phosphoramidites, the syntheses of the amidites, their testing for enzymatic activity and nuclease resistance.

Example 53: Synthesis of Hammerhead Ribozymes Containing 2'-Deoxy- 2'-Alkvlnucleotides Other 2'-Modified Nucleotides The method of synthesis used generally follows the procedure for normal RNA synthesis as described in Usman,N.; Ogilvie,K.K.; Jiang,M,-Y.; Cedergren,R.J. J. Am. Chem. Soc. 1987, 109, 7845-7854 and in Scaringe,S.A.; Franklyn,C.; Usman,N. Nucleic Acids Res. 1990, 18, 5433- 5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (compounds 10, 12, 17, 22, 31, 18, 26, 32, 36 and 38), Other 2'-modified phosphoramidites were prepared according to: 3 4, Eckstein et al. International Publication No. WO 92/07065; and 5 Kois et al.

Nucleosides Nucleotides 1993, 12, 1093-1109. The average stepwise coupling yields were The 2'-substituted phosphoramidites were incorporated into hammerhead ribozymes as shown in Figure However, these 2'-alkyl substituted phosphoramidites may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group I or Group II intron catalytic nucleic acids, or into antisense

~_I

WO 95/23225 PCT/B1195/()0015( 126 oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 54: Ribozyme Activity Assay Purified 5'-end labeled RNA substrates (15-25-mers) and purified end labeled ribozymes (-36-mers) were both heated to 95 quenched on ice and equilibrated at 37 separately. Ribozyme stock solutions were 1 mM, 200 nM, 40 nM or 8 nM and the final substrate RNA concentrations were 1 nM. Total reaction volumes were 50 mL. The assay buffer was 50 mM Tris-CI, pH 7.5 and 10 mM MgCI 2 Reactions were initiated by mixing substrate and ribozyme solutions at t 0. Aliquots of mL were removed at time points of 1, 5, 15, 30, 60 and 120 m. Each time point was quenched in formamide loading buffer and loaded onto a denaturing polyacrylamide gel for analysis. Quantitative analyses were performed using a phosphorimager (Molecular Dynamics).

Example 55: Stability Assay 500 pmol of gel-purified 5'-end-labeled ribozymes were precipitated in ethanol and pelleted by centrifugation. Each pellet was resuspended in mL of appropriate fluid (human serum, human plasma, human synovial fluid or fetal bovine serum) by vortexing for 20 s at room temperature. The samples were placed into a 37 °C incubator and 2 mL aliquots were withdrawn after incubation for 0, 15, 30, 45, 60, 120, 240 and 480 m.

Aliquots were added to 20 mL of a solution containing 95% formamide and TBE (50 mM Tris, 50 mM borate, 1 mM EDTA) to quench further nuclease activity and the samples were frozen until loading onto gels, Ribozymes were size-fractionated by electrophoresis in acrylamide/8M urea gels. The amount of intact ribozyme at each time point was quantified by scanning the bands with a phosphorimager (Molecular Dynamics) and the half-life of each ribozyme in the fluids was determined by plotting the percent intact ribozyme vs the time of incubation and extrapolation from the graph.

Example 56: 3',5'-O-(Tetraisopropyl-disiloxane-1,3-diyl)-2'-O-Phenoxythiocarbonyl-Uridine (7) To a stirred solution of 3',5'-O-(tetraisopropyl-disiloxane-1,3-diyl)uridine, 6, (15.1 g, 31 mmol, synthesized according to Nucleic Acid

I

WO 95/23225 P(T/1B95/00 127 Chemistry, ed. Leroy Townsend, 1986 pp. 229-231) and dimethylaminopyridine (7.57 g, 62 mmol) a solution of phenylchlorothionoformate (5,15 mL, 37.2 mmol) in 50 mL of acetonitrile was added dropwise and the reaction stirred for 8 h. TLC (EtOAc:hexanes 1:1) showed disappearance of the starting material. The reaction mixture was evaporated, the residue dissolved in chloroform, washed with water and brine, the organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel with EtOAc:hexanes 2:1 as eluent to give 16.44 g of 7.

Example 57: 3',5'-O-(Tetraisopropyl-disiloxane-1,3-diyl)-2'-C-Allyl -Uridine To a refluxing, under argon, solution of disiloxane-1,3-diyl)-2'-O-phenoxythiocarbonyl-uridine, 7, (5 g, 8.03 mmol) and allyltributyltin (12.3 mL, 40.15 mmol) in dry toluene, benzoyl peroxide (0.5 g) was added portionwise during 1 h. The resulting mixture was allowed to reflux under argon for an additional 7-8 h. The reaction was then evaporated and the product 8 purified by flash chromatography on silica gel with EtOAc:hexanes 1:3 as eluent. Yield 2.82 g Example 58: 5'-O-Dimethoxytrityl-2'-C-Allyl-Uridine (9) A solution of 8 (1.25 g, 2.45 mmol) in 10 mL of dry tetrahydrofuran (THF) was treated with a 1 M solution of tetrabutylammoniumfluoride in THF (3.7 mL) for 10 m at room temperature. The resulting mixture was evaporated, the residue was loaded onto a silica gel column, washed with 1 L of chloroform, and the desired deprotected compound was eluted with chloroform:methanol 9:1. Appropriate fractions were combined, solvents removed by evaporation, and the residue was dried by coevaporation with dry pyridine. The oily residue was redissolved in dry pyridine, dimethoxytritylchloride (1.2 eq) was added and the reaction mixture was left under anhydrous conditions overnight. The reaction was quenched with methanol (20 mL), evaporated, dissolved in chloroform, washed with aq. sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and evaporated. The residue was purified by flash chromatography on silica gel, EtOAc:hexanes 1:1 as eluent, to give 0.85 g of 9 as a white foam.

_1_1 WO 95/23225 I("T111')510015(0 128 Example 59: 5'-O-Dimethoxvtritvl-2'-C-Allyl-Uridine 3'-(2-Cyanoethyl N,Ndiisopropylphosphoramidite) 5'-O-Dimethoxytrityl-2'-C-allyl-uridine (0.64 g, 1.12 mmol) was dissolved in dry dichloromethane under dry argon, N,N-Diisopropylethylamine (0.39 mL, 2.24 mmol) was added and the solution was ice-cooled.

2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.35 mL, 1.57 mmol) was added dropwise to the stirred reaction solution and stirring was continued for 2 h at RT, The reaction mixture was then ice-cooled and quenched with 12 mL of dry methanol, After stirring for 5 m, the mixture was concentrated in vacuo (40 and purified by flash chromatography on silica gel using a gradient of 10-60% EtOAc in hexanes containing 1% triethylamine mixture as eluent. Yield: 0.78 g white foam, Example 60: 3',5'-O-(Tetraisopropyl-disiloxane-1,3-divl)-2'-C-Allyl-N4- Acetyl-Cytidine (11) Triethylamine (6.35 mL, 45.55 mmol) was added dropwise to a stirred ice-cooled mixture of 1,2,4-triazole (5.66 g, 81.99 mmol) and phosphorous oxychloride (0.86 mL, 9.11 mmol) in 50 mL of anhydrous acetonitrile. To the resulting suspension a solution of 1,3-diyl)-2'-C-allyl uridine (2.32 g, 4.55 mmol) in 30 mL of acetonitrile was added dropwise and the reaction mixture was stirred for 4 h at room temperature. The reaction was concentrated in vacuo to a minimal volume (not to dryness). The residue was dissolved in chloroform and washed with water, saturated aq, sodium bicarbonate and brine, The organic layer was dried over sodium sulfate and the solvent was removed in vacuo. The resulting foam was dissolved in 50 mL of 1,4-dioxane and treated with 29% aq. NH 4 0H overnight at room temperature. TLC (chloroform:methanol 9:1) showed complete conversion of the starting material. The solution was evaporated, dried by coevaporation with anhydrous pyridine and acetylated with acetic anhydride (0.52 mL, 5.46 mmol) in pyridine overnight. The reaction mixture was quenched with methanol, evaporated, the residue was dissolved in chloroform, washed with sodium bicarbonate and brine. The organic layer was dried over sodium sulfate, evaporated to dryness and purified by flash chromatography on silica gel MeOH in chloroform). Yield 2,3 g as a white foam.

WO 95123225 I'(I/1195)i(oO S 129 Example 61: 5'-O-Dimethoxytrityl-2'-C-Allyvi-N4-AcetvI-Cytidine This compound was obtained analogously to the uridine derivative 9 in 55% yield.

Example 62: 5'-O-Dimethoxytrityl-2'-C-allyl-N-4-Acetyl-Cytidine Cvanoethyl N,N-diisoorovlphosphoramidite) (12) 2'-O-Dimethoxytrityl-2'-C-allyl-N 4 -acetyl cytidine (0.8 g, 1.31 mmol) was dissolved in dry dichloromethane under argon. N,N-Diisopropylethylamine (0.46 mL, 2.62 mmol) was added and the solution was ice-cooled.

2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (0.38 mL, 1.7 mmol) was added dropwise to a stirred reaction solution and stirring was continued for 2 h at room temperature. The reaction mixture was then icecooled and quenched with 12 mL of dry methanol. After stirring for 5 m, the mixture was concentrated in vacuo (40 OC) and purified by flash chromatography on silica gel using chloroform:ethanol 98:2 with 2% triethylamine mixture as eluent, Yield: 0.91 g white foam.

Example 63: 2'-Deoxy-2'-Methylene-Uridine 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine 14 (Hansske,F.; Madej,D.; Robins,M. J. Tetrahedron 1984, 40, 125 and Matsuda,A.; Takenuki,K.; Tanaka,S.; Sasaki,T.; Ueda,T. J. Med. Chem, 1991, 34, 812) (2.2 g, 4.55 mmol dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo, The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-methylene-uridine (1.0 g, 3.3 mmol, 72.5%) was eluted with 20% MeOH in CH 2

CI

2 Example 64: 5'-O-DMT-2'-Deoxy-2'-Methylene-Uridine 2'-Deoxy-2'-methylene-uridine (0.91 g, 3.79 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2 C1 2 (100 mL) and washed with sat, NaHC03, water and brine. The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes as eluant to yield 15 (0.43 g, 0.79 mmol, 22%).

I

WVO 95123225 C/IB/(O IT1,11105100156 130 Example 65: D MT-2'-Deoxy-2'- Methylene-UQriding 3'-(2-Cya noethyl N,N-diisopropylphosphoramidite) (17) 1 -(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-3-D-ribofu-ranosyl)uracil (0.43 g, 0.8 mmol) dissolved in dry 0H2Cl 2 (15 mL) was placed in a round-bottom flask under Ar. Dlisopropylethylamine (0.28 mL, 1 .6 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.25 mL, 1,12 mmol), The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 The product (0.3 g, 0.4 1 0 mmol, 50%) was purified by flash column chromatography over silica gel using a 25-70% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.42 (0H 2 C1 2 MeOH 15:1) Example 66: 2'-Deoxy-2'-Difluoromethvlene-3',5'-O-(Tetraisop-ropvldisiloxane-1,3-divl)-Uridine 1 5 2'-Keto-3',5'- O-(tetraisopropyldisiloxane-1 ,3-diyl)uridine 14 (1.92 g, 12.6 mmol) and triphenylphosphine (2.5 g, 9.25 mmol) were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 'C0 A warm 00) solution of sodium chlorodifluoroacetate in diglyme (50 mL) was added (dropwise from an equilibrating dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and concentrated in vacuo. The residue was dissolved in CH 2 01 2 and chromatographed over silica gel. 2'- Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)uridine (3.1 g, 5.9 mmol, 70%) eluted with 25% hexanes in EtOAc.

Example 67: 2'-Deoxy-2'-Difluoromethylene-Uridine 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)uridine (3.1 g, 5.9 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (10 mL) for 20 m and concentrated in vacuo, The residue was triturated with petroleum ether and chromatographed on silica gel column.

2'-Deoxy-2'-difluoromethylene-uridine (1.1 g, 4.0 mmol, 68%) was eluted with 20% MeOH in 0H 2 C1 2 Example 68: 5'-O-DMT-2'-Deoxy-2'-Difluoromethylene-Uridine (16' Deoxy-2'-diflIuoromethylene- urid ine (1.1 g, 4.0 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (1.42 g, 4.18 mmol) in pyridine (10 mL) was added dropwise over 15 m, The resulting mixture -YIII I NVO 95/23225 /I'(I,111395!AM15 131 was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction, The mixture was concentrated in vacuo and the residue taken up in CH 2 C1 2 (100 mL) and washed with sat. NaHC03, water and brine. The organic extracts were dried over MgSO4, concentrated in vacuo and purified over a silica gel column using 40% EtOAc:hexanes as eluant to yield 5'--DMT-2'-deoxy-2'-difluoromethylene-uridine 16 (1.05 g, 1.8 mmol, Example 69: 5'-O-DMT-2'-Deoxy-2-Difluoromethylene-Uridine Cyanoethyl N.N-diisopropylohosphoramidite) (18) 1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-3-D-ribofuranosyl)-uracil (0,577 g, 1 mmol) dissolved in dry CH 2 C1 2 (15 mL) was placed in a round-bottom flask under Ar, Diisopropylethylamine (0.36 mL, 2 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.44 mL, 1.4 mmol). The reaction mixture was stirred for 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 oC). The product (0.404 g, 0.52 mmol, 52%) was purified by flash chromatography over silica gel using EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.48 (CH 2 01 2 MeOH 15:1).

Example 70: 2'-Deoxy-2'-Methvlene-3',5'-O-(TetraisoDropvldisiloxane-1,3diyl)-4-N-Acetyl-Cytidine Triethylamine (4.8 mL, 34 mmol) was added to a solution of POC13 (0.65 mL, 6.8 mmol) and 1,2,4-triazole (2.1 g, 30.6 mmol) in acetonitrile mL) at 0 oC. A solution of 2'-deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl) uridine 19 (1.65 g, 3.4 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at room temperature for 4 h. The mixture was concentrated in vacuo, dissolved in

CH

2 C1 2 (2 x 100 mL) and washed with 5% NaHCO 3 (1 x 100 mL). The organic extracts were dried over Na 2 SO4 concentrated in vacuo, dissolved in dioxane (10 mL) and aq. ammonia (20 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (3 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.

NaHCO 3 (5 mL). The mixture was concentrated in vacuo, dissolved in

CH

2 01 2 (2 x 100 mL) and washed with 5% NaHC03 (1 x 100 mL). The WO 95/23225 WO 95/3225 (1/111395/00 156 132 organic extracts were dried over Na 2

SO

4 concentrated in vacuo and the residue chromatographed over silica gel, 2'-Deoxy-2'-methylene-3',5'-O- (tetraisopropyldisiloxane-1 ,3-diyl)-4-N-acetyl-cytidine 20 (1.3 g, 2.5 mmol, 73%) was eluted with 20% EtOAc in hexanes, Example 71: 1 -(2'-Deoxy-2'-Methylene-5'-O-Dimethoxvtrityl-3-D-ribofuranosvl)-4-N-Acetyl-Cvtosine 21 2'-Deoxy-2'-methylene-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-4-Nacetyl-cytidine 20 (1.3 g, 2.5 mmol) dissolved in THF (20 mL) was treated with 1 M TEAF in THF (3 mL) for 20 m and concentrated in vacuo. The 1 0 residue was triturated with petroleum ether and chromatographed on silica gel column, 2'-Deoxy-2'-methylene-4-N-acetyl-cytidine (0.56 g, 1.99 mmol, was eluted with 10% MeCH in CH 2 01 2 2'-Deoxy-2'-methylene-4-Nacetyl-cytidine (0.56 g, 1.99 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI (0.81 g, 2.4 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in 0H 2 01 2 (100 mL) and washed with sat. NaHCO 3 (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes 60:40 as eluant to yield 21 (0.88 g, 1.5 mmol, Example 72: 1 -(2'-Deoxy-2'-Methylene-5'-O-Dimethoxytrityl-3-D-ribofuranosyl'i-4-N-Acetyl-Cytosine 3'-(2-Oyanoethyl-NN-diisopropylnhosphoramidite) (22) 1 -(2'-Deoxy-2'-methylene-5'-C-dimethoxytrityl-p3-D-ribofuranosyl)-4-Nacetyl-cytosine 21 (0.88 g, 1.5 mmol) dissolved in dry 0H 2 C1 2 (10 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine (0.8 mL, mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.4 mL, 1.8 mmol), The reaction mixture was stirred 2 h at room temperature and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 The product 22 (C,82 g, 1 .04 mmol, 69%) was purified by flash chromatography over silica gel using 50-70% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.36 (CH 2 01 2 :MeOH 20:1).

I

Illll~aa~0l3 I WO 95/23225 2P(IC11395/00156 133 Examole 73: 2'-Deoxv-2'-Difluoromethylene-3',5'-O-(Tetraisopropyi disiloxane-1,3-diyl)-4-N-Acetvl-Cytidine (24' Et 3 N (6.9 mL, 50 mmol) was added to a solution of POC13 (0.94 mL, mmol) and 1,2,4-triazole (3.1 g, 45 mmol) in acetonitrile (20 mL) at 0 C.

A solution of 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine 23 ([described in example 14] 2.6 g, 5 mmol) in acetonitrile (20 mL) was added dropwise to the above reaction mixture and left to stir at RT for 4 h. The mixture was concentrated in vacuo, dissolved in CH2C1 2 (2 x 100 mL) and washed with 5% NaHCO 3 (1 x 100 mL). The organic extracts were dried over Na 2

SO

4 concentrated in vacuo, dissolved in dioxane (20 mL) and aq. ammonia (30 mL). The mixture was stirred for 12 h and concentrated in vacuo. The residue was azeotroped with anhydrous pyridine (2 x 20 mL). Acetic anhydride (5 mL) was added to the residue dissolved in pyridine, stirred at RT for 4 h and quenched with sat.

NaHC03 (5mL). The mixture was concentrated in vacuo, dissolved in

CH

2 C1 2 (2 x 100 mL) and washed with 5% NaHC03 (1 x 100 mL). The organic extracts were dried over Na 2

SO

4 concentrated in vacuo and the residue chromatographed over silica gel. 2'-Deoxy-2'-difluoromethylene- 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3,9 mmol, 78%) was eluted with 20% EtOAc in hexanes.

Examole 74: 1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dimethoxytritl--Dribofuranosyl)-4-N-Acetyl-Cytosine 2'-Deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3diyl)-4-N-acetyl-cytidine 24 (2.2 g, 3.9 mmol) dissolved in THF (20 mL) was treated with 1 M TBAF in THF (3 mL) for 20 m and concentrated in vacuo.

The residue was triturated with petroleum ether and chromatographed on a silica gel column. 2'-Deoxy-2'-difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8 mmol, 72%) was eluted with 10% MeOH in CH 2 CI0 2 2'-Deoxy-2'difluoromethylene-4-N-acetyl-cytidine (0.89 g, 2.8 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-Cl (1.03 g, 3.1 mmol) in pyridine mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2 01 2 (100 mL) and washed with sat. NaHCO 3 (50 mL), water (50 mL) and brine (50 mL). The organic extracts were dried over MgSO 4 concentrated in NNIO 95,123225 51J22 t. ll Qk" i 1 111 510 134 vacuo and purified over a silica gel column using EtOAc:hexanes 60:40 as eluant to yield 25 (1 .2 g, 1 .9 mmol, 68%).

Example 75: 1-(2'-Deoxy-2'-Difluoromethylene-5'-O-Dmethoxytrityl-3-Dribofuranosyl)-4-N-Acetylcytosine 3'-(2-cyanoethyl-NN-diisopropylphosphoramiditeL) (26) 1 -(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl-3-D-ribofuranosyl)-4-N-acetylcytosine 25 (0.6 g, 0.97 mmol) dissolved in dry 0H 2 01 2 mL) was placed in a round-bottom flask under Ar. Diisopropylethylamine mL, 2,9 mmol) was added, followed by the dropwise addition of 2cyanoethyl N,N-diisopropylchlorophosphoramidite (0.4 m-L, 1.8 mmol), The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL), After 10 m the mixture was evaporated to a syrup in vacuo (40 00), TheP product 26, a white foam (0.52 g, 0.63 mmol, 65%) was purified by flash chromatography over silica gel using 30-70% EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.48 (CH 2 01 2 :MeOH 20: 1).

Example 76: 2'-Keto-3',5'-O-(Tetraisopropyldisiloxane-1 .3-diyl)-6-N-(4-t- Butylbenzoyfl-Adenosine (28) Acetic ai hydride (4.6 mL) was adde i to a solution of propyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine (Brown,J.; Christodolou, JonesS.; Modak,A.; Reese,0.; Sibanda,S,; Ubasawa A.

J. Chein Soo, Perkin Trans, 1989, 1735) (6,2 g, 9.2 mmol) in DMSO (37 mL) and the resulting mixture was stirred at room temperature for 24 h. The mixture was concentrated in vacuo. The residue was taken up in EtOAc and washed with water. The organic layer was dried over MgSO 4 and concentrated in vacuo. The residue was purified on a silica gel column to yield 2'-keto-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 28 (4.8 g, 7.2 mmol, 78%).

Example 77: 2'-Deoxv-2'-methylene-3'.5'-O-(Tetraisopropldisiloxane-1 .3diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine (29) Under a pressure of argon, sec-butyllithium in hexanes (11.2 mL, 14.6 mmol) was added to a suspension of triphenylmethylphosphonium iodide (7.07 g,17.5 mmol) in THF (25 mL) cooled at -78 00, The homogeneous orange solution was allowed to warm to -30 00 and a .8olution of 2'-keto- 3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine

I

95'232Z5 953225P( I I 1195900 56 135 28 (4.87 g, 7.3 mmol) in THE (25 ml-) was transferred to this mixture under argon pressure. After warming to RT, stirring was continued for 24 h. THE was evaporated and replaced by CH 2 01 2 (250 mL), water was added mL), and the solution was neutralized with a cooled solution of 2%e' HOI.

The organic layer was washed with H 2 0 (20 mL), 5% aqueous NaHCO 3 mL), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

4 the solvent was evaporated in vacuo to give the crude compound, which was chromatographed on a silica gel column. Elution with light petroleum ether:EtOAc 7:3 afforded pure 2'-deoxy..2'-methylene-3',5'-O-(tetraiso- 1 0 propyldisiloxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 29 (3.86 g, 5.8 mmol, 79%).

Example 78: 2'-Deoxy-2'-Methylene-6-N-(4-t-Butylbenzoyl)-Adenosine 2'-Deoxy-2'-methylene-3',5'-O-(tetrasopropydisiloxane-1 1 3-diyl)-6-N- (4-t-butylbenzoyl)-adenosine (3.86 g, 5.8 mmol) dissolved in THF (30 ml-) was treated with 1 M TBAF in THE (15 ml-) for 20 m and concentrated in vacuo, The residue was triturated with petroleum ether and chromatographed on a silica gel column, 2'-Deoxy-2'-methylene-6-N-(4-t-.

butylbenzoyl)-adenosine (1.8 g, 4.3 mmol, 74%) was eluted with MeOH in 0H 2 1 2

I.

Example 79: 5'-Q-DMT-2'-Deoxy-2'-Me t ne-6-N-(4-t-Butylbenzoyl)- Adenosine (29) 2'-Deoxy-2'-methylene-6-N-(4-t-butylbenzoyl)-adenosine (0.75 g, 1.77 mmol) was dissolved in pyridine (10 ml-) and a solution of DMT-Cl (0.66 g, 1.98 mmol) in pyridine (10 ml-) was added dropwise over 15 m.

The resulting mixture was stirred at RT for 12 h and MeoH- (2 mL.) was added to quench the reaction, The mixture was concentrated in vacuo and the residue taken up in 0H- 2 01 2 (100 ml-) and washed with sat. NaHCO 3 water and brine. The organic extracts were dried ovei MgSO 4 concentrated in vacuo and purified over a silica gel column using EtOAc:hexanes as an eluant to yield 29 (0.81 g, 1.1 mmol, 62%).

Example 80: 5'-O-DMT-2'-Deoxy-2'-Methylene-6-N-(4--Butvlbenzoyl).

Adenosine '2-Cyanoethyl N.N-diisopropylphosphoramidite) (31) 1 -(2'-Deoxy-2'-methylene-5'-O-dimethoxytrityl-3-o-ribofu,-anosyl)-6-N.

(4-t-butylbenzoyl)-adenine 29 dissolved in dry CH- 2 01 2 (15 ml-) was placed

I

NVO 95i'23225 XVO 95:23225PCT1!I 1095U() 156C 136 in a round bottom flask under Ar, Dilsopropylethylammne was added, followed by the dropwise addition of 2-cyanoethyl N, Ndilsopropyichiorophosphoramidite, The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 mn the mixture was evaporated to a syrup in vacuc (40 00). The product was purified by flash chromatography over silica gel using 30-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eiuant (0.7 g, 0.76 mmol, 689;). Rf 0.45

(CH

2 01 2 MeOH 20:1) Example 81: Deoxy-2'-Dif luoromethylene-3', (Tetra iso ropyld isil ox- 1 0 ane-1 .3-diyl)-6-N-(4-t-Butylbenzoyl)-Adenosine 2'-Keto-3',5'-O-(tetraisopropyldisioxane-1 ,3-diyl)-6-N-(4-t-butylbenzoyl)-adenosine 28 (6.7 g, 10 mmol) and triphenylphosphine (2.9 g, 11 mmol were dissolved in diglyme (20 mL), and heated to a bath temperature of 160 00, A warm (60 IC) solution of sodium chlorodifluoroacetate (2.3 g, 15 mmol) in diglyme (50 ml-) was added (dropwise from an equilibrating dropping funnel) over a period of -1 h. The resulting mixture was further stirred for 2 h and concentrated In vacuo. The residue was dissolved in CH- 2

CI

2 and chromatographed over silica gel. 2'- Deoxy-2'-difluoromethylene-3',5'-O-(tetrasopropyldsloxane.1 13-diyl)-6-N- (4-t-butylbenzoyl)-adenosine (4.1g, 6.4 mmol, 64%) eluted with hexanes in EtOAc, Example 82: 2'Doy2-ilooehlne6N(--Lt~ezy) Adenosine 2'-Deoxy-2'-difluoromethylerne-3',5'-O.(tetraisopropyldisiloxane-1,3dlyl)-6-N-(4-t-butylbenzoyl)-adenosine (4.1 g, 6,4 mmol) dissolved in THF mL) was treated with 1 M TBAF in THF (10 mL) for 20 mn and concentrated in vacuo. The residue was triturated with petroleum ether and chromatographed on a silica gel column, 2'-Deoxy-2'-difluoromethylene-6-N-(4-t-butylbenzoyl)-adenosine (2.3 g, 4.9 mmol, 770/%) was eluted with 20% MeOH in 01-12012, Example 83: 5'-O-DMT-2'-Deoxy-2'-Difluoromethyene6N(4tButyl.

benzoyl)-Adenosine 2'-Deoxy-2'-difluoromethylene6-N-(4-t-butylbenzoyl)..adenosine (2.3 g, 4.9 mmol) was dissolved in pyridine (10 mL) and a solution of DMT-CI in NVO 95/23225 11( I I 39 510 (15 137 pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction. The mixture was concentrated in vacuo and the residue taken up in CH 2 Cl 2 (100 mL) and washed with sat, NaHC03, water and brine. The organic extracts were dried over MgSO4, concentrated in vacuo and purified over a silica gel column using 50% EtOAc:hexanes as eluant to yield 30 (2.6 g, 3.41 mmol, 69%).

Example 84: 5'-O-DMT-2'-Deoxy-2'-Difluoromethvlene-6-N-(4-t-Butylbenzoyl)-Adenosine 3'-(2-Cyanoethyl N, N-diisopropyvlphosphoramidite) (32l 1-(2'-Deoxy-2'-difluoromethylene-5'-O-dimethoxytrityl- -D-ribofuranosyl)-6-N-(4-t-butylbenzoyl)-adenine 30 (2.6 g, 3.4 mmol) dissolved in dry CH2C1 2 (25 mL) was placed in a round bottom flask under Ar.

Diisopropylethylamine (1,2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.06 mL, 4.76 mmol). The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL). After 10 m the mixture evaporated to a syrup in vacuo (40 oC). 32 (2.3 g, 2.4 mmol, 70%) was purified by flash column chromatography over silica gel using 20-50% EtOAc gradient in hexanes, containing 1% triethylamine, as eluant. Rf 0.52 (CH2C1 2 MeOH 15:1).

Example 85: 2'-Deoxy-2'-Methoxycarbonylmethylidine-3',5'-O-(Tetraiso- Dropyldisiloxane-1,3-divyl)-Uridine (33) Methyl(triphenylphosphoranylidine)acetate (5.4 g, 16 mmol) was added to a solution of 2'-keto-3',5 -0-(tetraisopropyl disiloxane-1,3-diyl)uridine 14 in CH2C1 2 under argon. The mixture was left to stir at RT for h. CH 2

CI

2 (100 mL) and water were added (20 mL), and the solution was neutralized with a cooled solution of 2% HCI. The organic layer was washed with H 2 0 (20 mL), 5% aq. NaHCOs (20 mL), H 2 0 to neutrality, and brine (10 mL). After drying (Na 2

SO

4 the solvent was evaporated in vacuo to give crude product, that was chromatographed on a silica gel column, Elution with light petroleum ether:EtOAc 7:3 afforded pure 2'-deoxy-2'methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine 33 (5.8 g, 10.8 mmol, 67.5%).

NVO 95123225 ')5/23225 I1C'IAB95/O() 138 Example 86: 2'-Deoxy-2'-Methoxycarbonylmethylidine-U riding (34) Et 3 N*3 HF (3 mL) was added to a solution of 2'-deoxy-2'-methoxycarboxylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1 ,3-diyl)-uridine 33 g, 9.3 mmol) dissolved in 0H 2 01 2 (20 mL) and Et 3 N (15 mL). The resulting mixture was evaporated in vacuo after 1 h and chromatographed on a silica gel column eluting 2'-deoxy-2'-methoxycarbonylmethylidineuridine 34 (2.4 g, 8 mmol, 86%) with THF:0H 2 01 2 4: 1.

Example 87: 5'-O-DMT-2'-Deoxy-2'-Methoxycarbonylmethylidine-Uridine 2'-Deoxy-2'-methoxycarbonyl methyl idilne-u ridi ne 34 (1.2 g, 4.02 mmol) was dissolved in pyridine (20 mL). A solution of IDMVT-Cl (1.5 g, 4.42 mmol) in pyridine (10 mL) was added dropwise over 15 m. The resulting mixture was stirred at RT for 12 h and MeOH (2 mL) was added to quench the reaction, The mixture was concentrated in vacuc and the residue taken up In 0H 2

CI

2 (100 mL) and washed with sat, NaHCO 3 water and brine, The organic extracts were dried over MgSO 4 concentrated in vacuo and purified over a silica gel column using 2-5% MeOH in 0H 2 01 2 as an eluant to yield 5'-O-DMT-2'-deoxy-2'-methoxycarbonylmethylidine-uridine (2.03 g, 3.46 mmol, 86%).

Example 88: D MT-2'- Deoxy-2'-Methoxyca rbonylmethylidi-ne-U rid ine 3'-(2-cvanoethyl-N.N-diisopropylphosphoramidite) (36) 1 -(2'-Deoxy-2'-2'-methoxycarbonylmethylidine-'-O-dimethoxytrityl-p- D-ribofuranosyl)-uridine 35 (2.0 g, 3.4 mmol) dissolved in dry CH 2 01 2 mL) was placed in a round-bottom flask under Ar. Dilsopropylethylamine (1.2 mL, 6.8 mmol) was added, followed by the dropwise addition of 2cyanoethyl NN-disopropylchlorophosphoramidite (0.91 mL, 4.08 mmol).

The reaction mixture was stirred 2 h at RT and quenched with ethanol (1 mL), After 10 m the mixture was evaporated to a syrup in vacuo (40 00C), D MT-2'-deoxy-2'-m eth oxycarbonyl methyl idin e- urid in e cyanoethyl-N,N-diisopropylphosphoramidite) 36 (1,8 g, 2.3 mmol, 67%) was purified by flash column chromatography over silica gel using a EtOAc gradient in hexanes, containing 1 triethylamine, as eluant. Rf 0.44 (CH 2

CI

2 :MeOH 9.5:0.5).

I~C~BA PCTI/I B951(l()15( \VO 95123225 Example 89: 2'-Deoxy-2'-Carboxvmethylidine-3',5'-O-(Tetraisopropyldisiloxane-1,3-diyl)-Uridine 37 2'-Deoxy-2'-methoxycarbonylmethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine 33 (5.0 g, 10.8 mmol) was dissolved in MeOH (50 mL) and 1 N NaOH solution (50 mL) was added to the stirred solution at RT. The mixture was stirred for 2 h and MeOH removed in vacuo. The pH of the aqueous layer was adjusted to 4.5 with 1N HCI solution, extracted with EtOAc (2 x 100 mL), washed with brine, dried over MgSO4 and concentrated in vacuo to yield the crude acid. 2'-Deoxy-2'carboxymethylidine-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine 37 (4.2 g, 7.8 mmol, 73%) was purified on a silica gel column using a gradient of 10-15% MeOH in CH 2 C1 2 The alkyl substituted nucleotides of this invention can be used to form stable oligonucleotides as discussed above for use in enzymatic cleavage or antisense situations. Such oligonucleotides can be formed enzymatically using triphosphate forms by standard procedure.

Administration of such oligonucleotides is by standard procedure. See Sullivan et al. PCT WO 94/02595.

Oligonucleotides with 3' and/or 5' Dihalophosphonate This invention synthesis and uses 3' and/or 5' dihalophosphonate-, 3' or 5'-CF 2 -phosphonate-, substituted nucleotides that maintain or enhance the catalytic activity and/or nuclease resistance of an enzymatic or antisense molecule.

As the term is used in this application, and/or 3'dihalophosphonate nucleotide containing ribozymes, deoxyribozymes (see Usman et al., PCT/US94/11649, incorporated by reference herein), and chimeras of nucleotides, are catalytic nucleic molecules that contain and/or 3'-dihalophosphonate nucleotide components replacing, but not limited to, double-stranded stems, single-stranded "catalytic core" sequences, single-stranded loops or single-stranded recognition sequences. These molecules are able to cleave (preferably, repeatedly cleave) separate RNA or DNA molecules in a nucleotide base sequence specific manner. Such catalytic nucleic acids can also act to cleave intramolecularly if that is desired. Such enzymatic molecules can be targeted to virtually any RNA or DNA transcript. This invention concerns 95/23225 PCT/1139510015( 140 nucleic acids formed of standard nucleotides or modified nucleotides, which also contain at least one 5'-dihalophosphonate and/or one 3'dihalophosphonate group.

The synthesis of 1-O-Ac-2,3-di-O-Bz-D-ribofuranose 5+dihalomethylphosphonate in three steps from 1-O-methyl-2,3-Oisopropylidene-B-D-ribofuranose 5-deoxy-5-dihalomethylphosphonate is described for the difluoro, in Figure 87). Condensation of this suitably derivatized sugar with silylated pyrimidines and purines affords novel nucleoside 5'-deoxy-5'-dihalomethylphosphonates. These intermediates may be incorporated into catalytic or antisense nucleic acids by either chemical (conversion of the nucleoside dihalomethylphosphonates into suitably protected phosphoramidites 12a or solid supports 12b, Figure 88) or enzymatic means (conversion of the nucleoside 5'-deoxy-5'-dihalomethylphosphonates into their triphosphates, 14 Figure 89, for T7 transcription).

Thus, in one aspect the invention features 5' and/or 3'dihalonucleotides and nucleic acids containing such 5' and/or 3'dihalonucleotides. The general structure of such molecules is shown below.

o o II II

(R

3 0) 2

PCX

2 R (R 3 0) 2

PCX

2

B

R

2 R1 CX 2

R

1

CX

2 Ri

(R

3 0) 2 P (R 3 0) 2 P O where R 1 is H, OH, or R, where R is a hydroxyl protecting group, e.g., acyl, alkysilyl, or carbonate; each R 2 is separately H, OH, or R; each R 3 is separately a phosphate protecting group, methyl, ethyl, cyanoethyl, pnitrophenyl, or chlorophenyl; each X is separately any halogen; and each B is any nucleotide base.

The invention in particular features nucleic acid molecules having such modified nucleotides and enzymatic activity. In a related aspect the invention features a method for synthesis of such nucleoside dihalo and/or 3'-deoxy-3'-dihalophosphonates by condensing a \WO 95/23225 "'PCT/B195/0015O( 141 dihalophosphonate-containing sugar with a pyrimidine or a purine under conditions suitable to form a nucleoside and/or a 3'-deoxy-3'-dihalophosphonate, Phosphonic acids may exhibit important biological properties because of their similarity to phosphates (Engel, Chem. Rev. 1977, 77, 349-367). Blackburn and Kent Chem. Soc., Perkin Trans. 1986, 913- 917) indicate that based on electronic and steric considerations _-fluoro and _,_-difluoromethylphosphonates might mimic phosphate esters better than the corresponding phosphonates. Analogues of pyro- and triphosphates 1, where the bridging oxygen atoms are replaced by a difluoromethylene group, have been employed as substrates in enzymatic processes (Blackburn et al,, Nucleosides Nucleotides 1985, 4, 165-167; Blackburn et al., Chem. Scr. 1986, 26, 21-24). 9-(5,5-Difluoro-5phosphonopentyl)guanine has been utilized as a multisubstrate analogue inhibitor of purine nucleoside phosphorylase (Halazy et al., J.

Am, Chem. Soc. 1991, 113, 315-317). Oligonucleotides containing methylene groups in place of phosphodiester 5'-oxygens are resistant toward nucleases that cleave phosphodiester linkages between phosphorus and the 5'-oxygen (Breaker et al., Biochemistry 1993, 32, 9125-9128), but can still form stable complexes with complementary sequences, Heinemann et al, (Nucleic Acids Res, 1991, 19, 427-433) found that a single 3'-methylenephosphonate linkage had a minor influence on the conformation of a DNA octamer double helix.

I

i IIIIPI~ I WO 95/23225 P (',TlB 95/(11 142

NH.

O O I OI NN

'O-P-X-P-O-P-

N

N

0' 0' 0' 0 OH OH 1 0 N

H

ND

(H0) 2 0PCF2 NH 2

(ETO)

2

POCF

2 Li 3 One common synthetic approach to a,a-difluoro-alkylphosphonates features the displacement of a leaving group from a suitable reactive substrate by diethyl (lithiodifluoromethyl)phosphonate (Obayashi et al., Tetrahedron Lett, 1982, 23, 2323-2326). However, our attempts to synthesize nucleoside 5'-deoxy-5'-difluoro-methylphosphonates from using 3 were unsuccessful, i.e. starting compounds were quantitatively recovered, The reaction of nucleoside aldehydes with 3, according to the procedure of Martin et al. (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842), led to a complex mixture of products. Recently, the synthesis of sugar a,a-difluoroalkylphosphonates from primary sugar triflates using 3 was described (Berkowitz et al., J. Org.

Chem. 1993, 58, 6174-6176). Unfortunately, our experience is that nucleoside 5 -triflates are too unstable to be used in these syntheses.

The following are non-limiting examples showing the synthesis of nucleoside 5'-deoxy-5'-difluoromethyl-phosphonates. Those in the art will recognize that equivalent methods can be readily devised based upon ~CII~IQ~C I WO 95/23225 P B5/0(015( 143 these examples. These examples demonstrate that it is possible to achieve synthesis of 5'-deoxy-5'-difluoro derivatives in good yield and thus guide those in the art to such equivalent methods. The examples also indicate utility of such synthesis to provide useful oligonucleotides as described above.

Those in the art will recognize that useful modified enzymatic nucleic acids can now be designed, much as described by Draper et al,, PCT/US94/13129 hereby incorporated by reference herein (including drawings).

Example 90: Synthesis of Nucleoside difluoromethylphosphonates Referring to Fig. 87, we synthesized a suitable glycosylating agent from the known D-ribose a,a-difluoromethylphosphonate (Martin et al., Tetrahedron Lett. 1992, 33, 1839-1842) which served as a key intermediate for the synthesis of nucleoside difluoromethylphosphonates.

Methyl 2,3-O-isopropylidene-p-D-ribofuranose a,adifluoromethylphosphonate was synthesized from the according to the procedure of Martin et al. (Tetrahedron Lett. 1992, 33, 1839-1842) (Figure 87). Removal of the isopropylidene group was accomplished under mild conditions (1 2 -MeOH, reflux, 18 h (Szarek et al., Tetrahedron Lett. 1986, 27, 3827) or Dowex 50 WX8 MeOH, RT (about 20-25°C), 3 days) in 72% yield. The anomeric mixture thus obtained was benzoylated with benzoyl chloride/pyridine to afford the 2,3di-O-benzoyl derivative, which was subjected to mild acetolysis conditions (Walczak et al., Synthesis, 1993, 790-792) (Ac20, AcOH, H 2

SO

4 EtOAc, 0°C, The desired 1-O-acetyl-2,3-di-O-benzoyl-D-ribofuranose difluoromethylphosphonate was obtained in quantitative yield as an anomeric mixture. These derivatives were used for selective glycosylation of silylated uracil and N 4 -acetylcytosine under Vorbriggen conditions (Vorbriggen, Nucleoside Analogs. Chemistry, Biology and Medical Applications, NATO ASI Series A, 26, Plenum Press, New York, London, 1980; pp. 35-69. The use of F 3 CS02OSi(CH 3 3 as a glycosylation catalyst is precluded because it is expected to lead to the undesired 1ethyluracil or 9-ethyladenine byproducts: Podyukova, et al., Tetrahedron ~II~Y~illl~- I \VO 95,123225 PCT/I 195/00 144 Lett, 1987, 28, 3623-3626 and references cited therein) (SnCl 4 as a catalyst, boiling acetonitrile) to yield P-nucleosides (62% 6a, 75% 6b).

Glycosylation of silylated N 6 -benzoyladenine under the same conditions yielded a mixture of N-9 isomer 6c and N-7 isomer 7 in 34% and yield, respectively. The above nucleotides were successfully deprotected using trimethylsilylbromide for the cleavage of the ethyl groups, followed by treatment with ammonia-methanol to remove the acyl protecting groups.

Nucleoside 5'-deoxy-5'-difluoromethylphosphonates 8 were finally purified on a DEAE Sephadex A-25 (HC03) column using a 0.01-0.25 M TEAB gradient for elution and obtained as their sodium salts (82% 8a; 87% 8b; 82% 8c).

Selected analytical data: 31P-NMR (31P) and 1 H-NMR (1H) were recorded on a Varian Gemini 400. Chemical shifts in ppm refer to H 3 P0 4 and TMS, respectively. Solvent was CDCI 3 unless otherwise noted. 5: 1 H 8 8.07-7.28 Bz), 6.66 J 1 ,2 4.5, acH1), 6.42 pH1), 5.74 J 2 ,3 4.9, PH2), 5.67 (dd, J 3 2 4.9, J 3 4 6.6, PH3), 5.63 (dd, J 3 2 6.7, J 3 4 3.6, a H 3), 5.57 (dd, J 2 1 4.5, J 2 3 6.7, aH2), 4.91 H4), 4.30 CH 2

CH

3 2.64 (m,

CH

2

CF

2 2.18 PAc), 2.12 aAc), 1.39 CH 2

CH

3 3 1 P 6 7.82 (t, JP,F 105.2), 7.67 JP,F 106.5). 6a: 1 H 8 9.11 1H, NH), 8.01 11H, Bz, H6), 5.94 J 1 4.1, 1H, H 1 5.83 (dd, J5, 6 8.1, 1H, H5), 5.79 (dd, J2',1' 4.1, J2', 3 6.5, 1H, 5.71 (dd, J 3 2 6.5, J 3 4 6.4, 1H, 4.79 (dd, J 4 3 6.4, J4',F 11.6, 1H, 4.31 4H, CH 2

CH

3 2.75 (tq, JH,F 19.6, 2H, CH 2

CF

2 1.40 6H, CH 2

CH

3 3 1 8 7.77 JP,F 104.0). 8c: 31 P (vs DSS) (D20) 5 5.71 JP,F 87.9).

Compound 7 was deacylated with methanolic ammonia yielding the product that showed Xmax (H 2 0) 271 nm and ;min 233 nm, confirming that the site of glycosylation was N-7.

Example 91:Synthesis of Nucleic Acids Containing Modified Nucleotide Containing Cores The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al., J. Am, Chem. Soc. 1987, 109, 7845-7854 and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end (Figure 88 and Janda et al., Science 1989, 244:437-440.). These WO 95/23225 'P'T/Il9510(0156 145 nucleoside 5'-deoxy-5'-difluoromethylphosphonates may be incorporated not only into hammerhead ribozymes, but also into hairpin, hepatitis delta virus, Group 1 or Group 2 introns, or into antisense oligonucleotides. They are, therefore, of general use in any nucleic acid structure.

Example 92: Synthesis of Modified Triphosphate The triphosphate de,ivatives of the above nucleotides can be formed as shown in Fig. 89, according to known procedures. Nucleic Acid Chem,, Leroy B. Townsend, John Wiley Sons, New York 1991, pp. 337-340; Nucleotide Analogs, Karl Heinz Scheit; John Wiley Sons New York 1980, pp. 2 1 1 2 18 Equivalent synthetic schemes for 3' dihalophosphonates are shown in Figures 90 and 91 using art recognized nomenclature. The conditions can be optimized by standard procedures.

The nucleoside dihalophosphonates described herein are advantageous as modified nucleotides in any nucleic acid structure, e.g., catalytic or antisense, since they are resistant to exo- and endonucleases that normally degrade unmodified nucleic acids in vivo. They also do not perturb the normal structure of the nucleic acid in which they are incorporated thereby maintaining any activity associated with that structure.

These compounds may also be of use as monomers as antiviral and/or antitumor drugs.

Oligonucleotides with Amido or Peptido Modification This invention replaces 2'-hydroxyl group of a ribonucleotide moiety with a 2'-amido or 2'-peptido moiety. In other embodiments, the 3' and portions of the sugar of a nucleotide may be substituted, or the phosphate group may be substituted with amido or peptido moieties, Generally, such a nucleotide has the general structure shown in Formula I below: 111 ~UYB~I~II~Clr I WO 95/23225 PC"11I B95/1(0I15(S 146 0

B

0 0 O N N H- RI R 3 0O I.P-0 FORMULA I The base is any one of the standard bases or is a modified nucleotide base known to those in the art, or can be a hydrogen group, In addition, either R 1 or R 2 is H or an alkyl, alkene or alkyne group containing between 2 and 10 carbon atoms, or hydrogen, an amine (primary, secondary or tertiary, R 3

NR

4 where each R3 and R 4 independently is hydrogen or an alkyl, alkene or alkyne having between 2 and 10 carbon atoms, or is a residue of an amino acid, an amide), an alkyl group, or an amino acid (D or L forms) or peptide containing between 2 and 5 amino acids, The zigzag lines represent hydrogen, or a bond to another base or other chemical moiety known in the art. Preferably, one of R 1 R2 and R 3 is an H, and the other is an amino acid or peptide.

Applicant has recognized that RNA can assume a much more complex structural form than DNA because of the presence of the 2'hydroxyl group in RNA. This group is able to provide additional hydrogen bonding with other hydrogen donors, acceptors and metal ions within the RNA molecule. Applicant now provides molecules which have a modified amine group at the 2' position, such that significantly more complex structures can be formed by the modified oligonucleotide, Such modification with a 2'-amido or peptido group leads to expansion and enrichment of the side-chain hydrogen bonding network. The amide and peptide moieties are responsible for complex structural formation of the oligonucleotide and can form strong complexes with other bases, and interfere with standard base pairing interactions. Such interference will allow the formation of a complex nucleic acid and protein conglomerate.

-I

DI \VO 95/23225 PC T/I 95/()I156 147 Oligonucleotides of this invention are significantly more stable than existing oligonucleotides and can potentially form biologically active bioconjugates not previously possible for oligonucleotides. They may also be used for in vitro selection of unique aptamers, that is, randomly generated oligonucleotides which can be folded into an effective ligand for a target protein, nucleic acid or polysaccharide.

Thus, in one aspect, the invention features an oligonucleotide containing the modified base shown in Formula I, above.

In other aspects, the oligonucleotide may include a 3' or 5' nucleotide having a 3' or 5' located amino acid or aminoacyl group. In all these aspects, as well as the 2'-modified nucleotide, it will be evident that various standard modifications can be made. For example, an may be replaced with an S, the sugar may lack a base abasic) and the phosphate moiety may be modified to include other substitutions (see Sproat, supra).

Example 93: General procedure for the preparation of 2'-aminoacyl-2'deoxy-2'-aminonucleoside coniugates.

Referring to Fig. 92, to the solution of 2'-deoxy-2'-amino nucleoside (1 mmol) and N-Fmoc L- (or amino acid (1 mmol) in methanol [dimethylformamide (DMF) and tetrahydrofuran (THF) can also be used], 1ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) [or 1isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ)] (2 mmol) is added and the reaction mixture is stirred at room temperature or up to °C from 3-48 hours. Solvents are removed under reduced pressure and the residual syrup is chromatographed on the column of silica-gel using 1methanol in dichloromethane. Fractions containing the product are concentrated yielding a white foam with yields ranging from 85 to 95 Structures are confirmed by 1 H NMR spectra of conjugates which show correct chemical shifts for nucleoside and aminoacyl part of the molecule.

Further proofs of the structures are obtained by cleaving the aminoacyl protecting groups under appropriate conditions and assigning 1 H NMR resonances for the fully deprotected conjugate.

Partially protected conjugates described above are converted into their 5'-O-dimethoxytrityl derivatives and into 3'-phosphoramidites using standard procedures (Oligonucleotide Synthesis: A Practical Approach, I WO 95/23225 PCTI B 95/00(15(i 148 M.J, Gait ed,; IRL Press, Oxford, 1984). Incorporation of these phosphoramidites into RNA was performed using standard protocols (Usman etal,, 1987 supra).

A general deprotection protocol for oligonucleotides of the present invention is described in Fig. 93.

The scheme shows synthesis of conjugate of 2'-d-2'-aminouridine.

This is meant to be a non-limiting example, and those skilled in the art will recognize that, variations to the synthesis protocol can be readily generated to synthesize other nucelotides adenosine, cytidine, guanosine) and/or abasic moieties.

Exampie 94: RNA cleavage by hammerhead ribozymes containing 2'- 3minoacyl modifications.

Hammerhead ribozymes targeted to site N (see Fig. 94) are synthesized using solid-phase synthesis, as described above. U4 and U7 positions are modified, individually or in combination, with either 2'-NHalanine or 2'-NH-lysine, RNA cleavage assay in vitro: Substrate RNA is 5' end-labeled using [y.

3 2 p] ATP and T4 polynucleotide kinase (US Biochemicals). Cleavage reactions were carried out under ribozyme "excess" conditions. Trace amount 1 nM) of 5' end-labeled substrate and 40 nM unlabeled ribozyme are denatured and renatured separately by heating to 90°C for 2 min and snap-cooling on ice for 10 -15 min. The ribozyme and substrate are incubated, separately, at 37°C for 10 min i a buffer containing 50 mM Tris-HCI and 10 mM MgCl2. The reaction is initiated by mixing the ribozyme and substrate solutions and incubating at 37°C. Aliquots of 5 pl are taken at regular intervals of time and the reaction is quenched by mixing with equal volume of 2X formamide stop mix, The samples are resolved on 20 denaturing polyacrylamid, gels, The results are quantified and percentage of target RNA cleaved is plotted as a function of time.

Referring to Fig. 95, hammerhead ribozymes containing 2'-NHalanine or 2'-NH-lysine modifications at U4 and U7 positions cleave the target RNA efficiently.

I

'95 23225 PC TI1 .5 11 1 149 Sequences listed in Figure 94 and the modifications described in Figure 95 are meant to be non-limiting examples. Those skilled in the art will recognize that variants (base-substitutions, deletions, insertions, mutations, chemical modifications) of the ribozyme and RNA containing other 2'-hydroxyl group modifications, including but not limited to amino acids, peptides and cholesterol, can be readily generated using techniques known in the art, and are within the scope of the present invention, Example 95: Aminoacylation of 3'-ends of RNA I. Referring to Fig. 96. 3'-OH group of the nucleotide is converted to succinate as described by Gait, supra. This can be linked with amino-alkyl solid support (for example: CpG). Zig-zag line indicates linkage of 3'OH group with the solid support.

II. Preparation of aminoacyl-derivatized solid support A) Synthesis of O-Dimethoxytrityl (O-DMT) amino acids Referring to Fig. 97, to a solution of L- (or serine, tyrosine or threonine (2 mmol) in dry pyridine (15 ml) 4,4'-dimethoxytrityl chloride (3 mmol) is added and the reaction mixture is stirred at RT (about 20-25oC) for 16 h. Methanol (10 ml) is then added and the solution evaporated under reduced pressure. The residual syrup was partitioned between 5% aq.

NaHCO 3 and dichloromethane, organic layer was washed with brine, dried (Na 2

SO

4 and concentrated in vacuo. The residue is purified by flash silicagel column chromatography using 2-10% methanol in dichloromethane (containing 0.5 pyridine). Fractions containing product are combined and concentrated in vacuo to yielu white foam (75-85 yield).

B) Preparation of the solid support and its derivatization with amino acids Referring to Fig. 97, the modified solid support (has an OH group instead of the standard NH 2 end group) was prepared according to Haralambidis et al., Tetrahedron Lett. 1987, 28, 5199, (P denotes aminopropyl CPG or polystyrene type support), O-DMT or NHmonomethoxytrityl (NH-MMT amino acid was attached to the above solid support using standard procedures for derivatization of the solid support (Gait, 1984, supra) creating a base-labile ester bond between amino acids ~IIIIIl1RII W\O 95/23225 IPTc1I 950015o 150 and the support, This support is suitable for the construction of RNA/DNA chain using suitably protected nucleoside phosphoramidites.

Example 96: Aminoacylation of 5'-ends of RNA I. Referring to Fig. 98, 5'-amino-containing sugar moiety was synthesized as described (Mag and Engels, 1989 Nucleic Acids Res, 17, 5973). Aminoacylation of the 5'-end of the rnonomer was achieved as described above and RNA phosphoramidite of the monomer was prepared as described by Usman et al,, 1987 supra. The phosphoramidite was then incorporated at the 5'-end of the oligonucleotide using standard solid-phase synthesis protocols described above.

II. Referring to Fig. 99, aminoacyl group(s) is attached to the phosphate group at the 5'-end of the RNA using standard procedures described above, VII. Reversing Genetic Mutations Modification of existing nucleic acid sequences can be achieved by homologous recombination, In this process a transfected sequence recombines with homologous chromosomal sequences and can replace the endogenous cellular sequence. Boggs, 8 International J. Cell Cloning 1990, describes targeted gene modification. It reviews the use of homologous DNA recombination to correct genetic defects. Banga and Boyd, 89 Proc. Natl, Acad. Sci. U.S.A. 1735, 1992, describe a specific example of in vivo site-directed mutagenesis using a 50 base oligonucleotide. In this methodology a gene or gene segment is essentially replaced by the oligonucleotide used.

This invention uses a complementary oligonucleotide to position a nucleotide base changing activity at a particular site on a gene (RNA or genomic DNA), such that the nucleotide modifying activity will change (or revert) a mutation to wild-type, or its equivalent. By reversion or change of a mutation, we refer to reversion in a broad sense, such as when a mutation at a second site which leads to functional reversion to a wild type phenotype. Also, due to the degeneracy of the genetic code, a revertant may be achieved by changing any one of the three codon positions.

Additionally, creation of a stop codon in a deleterious gene (or transcript) is defined here as reverting a mutant phenotype to wild-type. An example of 11111~1---1 \VO 95,23225 P("T/IB 1 151 this type of reversion is creating a stop codon in a critical HIV proviral gene in a human.

Referring to Figures 100 and 101, broadly there are two approaches to causing a site directed change in order to revert a mutation to wild-type.

In one (Fig. 100) the oligonucleotide is used to target RNA specifically.

RNA is provided with a complementary (Watson-crick) oligonucleotide sequence to that in the target molecule. In this case the sequence modifying oligonucleotide would (analogously to an antisense oligonucleotide or ribozyme) have to be continuously present to revert the RNA as it is made by the cell. Such a reversion would be transient and would potentially require continuous addition of more sequence modifying oligonucleotide. The transient nature of this approach is an advantage, in that treatment could be stopped by simply removing the sequence modifying oligonucleotide (as with a traditional drug).

A second approach targets DNA (Fi.g 101) and has the advantage that changes may be permanently encoded in the target cell's genetic code. Thus, a single course (or several courses) of treatment may lead to permanent reversion of the genetic disease, If inadvertent chromosomal mutations are introduced this may cause cancer, mutate other genes, or cause genetic changes in the germ-line (in patients of reproductive age).

However, if the base changing activity is a specific methylation that may modulate gene expression it would not necessarily lead to germ-line transmission, See Lewin, Genes,1983 John Wilely Sons, Inc. NY pp 493-496.

Complementary base pairing to single-stranded DNA or RNA is one method of directing an oligonucleotide to a particular site of DNA. This could occur by a strand displacement mechanism or by targeting DNA when it is single-stranded (such as during replication, or transcription).

Another method is using triple-strand binding (triplex formation) to doublestranded DNA, which is an established technique for binding polypyrimidine tracts, and can be extended to recognize all 4 nucleotides. See Povsic, Strobel, Dervan, P. (1992). Sequence-specific doublestrand alkylation and cleavage of DNA mediated by triple-helix formation.

J. Am. Chem. Soc. 114, 5934-5944 (1992). Knorre, Valentin, V.V., Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk,

I

~l~aYRI~R WO 95/23225 PCT/i 1395100 152 1993) describe conjugation of reactive groups or enzyme to oligonucleotides and can be used in the methods described herein.

Recently, antisense oligonucleotides have been used to redirect an incorrect splice into order to obtain correct splicing of a splice mutant globin gene in vitro, Dominski Z; Kole R (1993) Restoration of correct splicing in thalassemia pre-mRNA by antisense oligonucleotides. Proc Natl Acad Sci U S A 90:8673-7, Analogously, in one preferred embodiment of this invention a complementary oligomer is used to correct an existiing mutant RNA, instead of the traditional approach of inhibiting that RNA by antisense, In either the RNA or DNA mode, after binding to a particular site on the RNA or DNA the oligonucleotide will modify the nucleic acid sequence, This can be accomplished by activating an endogenous enzyme (see Figure 102), by appropriate positioning of an enzyme (or ribozyme) conjugated (or activated by the duplex) to the oligonucleotide, or by appropriate positioning of a chemical mutagen, Specific mutagens, such as nitrous acid which deaminates C to U, are most useful, but others can also be used if inactivation of a harmful RNA is desired, RNA editing is an naturally occurring event in mammalian cells in which a sequence modifying activity edits a RNA to its proper sequence post-transcriptionally, Higuchi, Single, Kohler, M, Sommer, and Seeburg, P. (1993) RNA Editing of AMPA Receptor Subunit GluR-B: A base-paired intron-exon structure determines position and efficiency Cell 75:1361-1370. The machinery involved in RNA editing can be co-opted by a suitable oligonucleotide in order to promote chemical modification.

The changes in the base created by the methods of this invention cause a change in the nucleotide sequence, either directly, or after DNA repair by normal cellular mechanisms. These changes functionally correct a genetic defect or introduce a stop codon, Thus, the invention is distinct from techniques in which an active chemical group an alkylator) is attached to an antisense or triple strand oligonucleotide in order to chemically inactivate the target RNA or DNA.

Thus, this invention creates an alteration to an existing base in a nucleic acid molecule so that the base is read in vivo as a different base.

1181~8~81111 \VO 95/23225 I 1"T111! 195!00 150, 153 This includes correcting a sequence instead of inactivating a gene but can also include inactivating a deleterious gene, Thus, in one aspect, the invention features a method for altering in vivo the nucleotide base sequence of a naturally occurring mutant nucleic acid molecule, The method includes contacting the nucleic acid molecule in vivo with an oligonucleotide or peptide nucleic acid or other sequence specific binding molecules able to form a duplex or triplex molecule with the nucleic acid molecule. After formation of the duplex or triplex molecule a base modifying activity chemically or enzymatically alters the targeted base directly, or after nucleic acid repair in vivo, This results in the functional alteration of the nucleic acid sequence.

By "alter", as it is used in this context, is meant that one or more chemical moieties in a targeted base, or bases, is altered so that the mutant nucleic acid will be functionally different. Thus, this is distinct from prior methods of correcting defects in DNA, such as homologous recombination, in which an entire segment of the targeted sequence is replaced with a segment of DNA from the transfected nucleic acid, This is also distinct from other methods that use reactive groups to inactivate a RNA or DNA target, in that this method functionally corrects the sequence of the target, instead of merely damaging it, by causing it to be read by a polymerase as a different base from the original base. As noted above, the naturally occurring enzymes in a cell can be utilized to cause the chemical alteration, examples of which are provided below.

By "functionally alter" is meant that the ability of the target nucleic acid to perform its normal function transcription or translation control) is changed. For example, an RNA molecule may be altered so that it can cause production of a desired protein, or a DNA molecule can be altered so that upon DNA repair, the DNA sequence is changed.

By "mutant" it is meant a nucleic acid molecule which is altered in some way compared to equivalent molecules present in a normal individual, Such mutants may be well known in the art, and include, molecules present in individuals with known genetic deficiencies, such as muscular dystrophy, or diabetes and the like. It also includes individuals with diseases or conditions characterized by abnormal expression of a gene, such as cancer, thalassemia's and sickle cell anemia, and cystic

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\VO 95/23225 PCI/I1I95/00150 154 fibrosis. It allows modulation of lipid metabolism to reduce artery disease, treatment of integrated AIDS genomes, and AIDs RNA, and Alzeimer's disease. Thus, this invention concerns alteration of a base in a mutant to provide a "wild type" phenotype and/or genotype. For deleterious conditions this involves altering a base to allow expression or prevent expression as is necassary. When treating an infection, such as HIV, it concerns inactivation of a gene in the HIV RNA by mutation of the mutant non-human gene) to a wild type no production of a non-human protein). Such modification is performed in trans rather than in cis as in prior methods.

In preferred embodiments, the oligonucleotide is of a length (at least 12 bases, preferably 17 22) sufficient to activate dsRNA deaminase in vivo to cause conversion of an adenine base to inosine; the oligonucleotide is an enzymatic nucleic acid molecule that is active to chemically modify a base (see below); the nucleic acid molecule is DNA or RNA; the oligonucleotide includes a chemical mutagen, the mutagen is nitrous acid; and the oligonucleotide causes deamination of methylcytosine to thymidine, cytosine to uracil, or adenine to inosine, or methtylation of cytosine to In a most preferred embodiment, the invention features correction of a mutation, rather than inactivation of a target by causing a mutation.

Using in vitro directed evolution, it is possible to screen for ribozymes with catalytic activities different than RNA cleavage. Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences. Science 261:1411-1418. Using these methods of in vitro directed evolution, an enzymatic nucleic acid molecule, or ribozyme that mutates bases, instead of cleaving the phosphodiester backbone can be selected. This is a convenient method of obtaining an enzyme with the appropriate base sequence modifying activities for use in the present invention.

Sequence modifying activities can change one nucleotide to another (or modify a nucleotide so that it will be repaired by the cellular machinery to another nucleotide). Sequence modifying activities could also delete or add one or more nucleotides to a sequence. A specific embodiment of adding sequences is described by Sullenger and Cech, PCT/US94/12976

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WO 95/23225 ic"'/II 915/00(15( 155 hereby incorporated by reference herein), in which entire exons with wildtype sequence are spliced into a mutant transcript. The present invention features only the addition of a few bases (1 3).

Thus, in another aspect, the invention features ribozymeb or enzymatic nucleic acid molecules active to change the chemical structure of an existing base in a separate nucleic acid molecule, Applicant is the first to determine that such molecules would be useful, and to provide a description of how such molecules might be isolated.

Molecules used to achieve in situ reversion can be delivered using the existing means employed for delivering antisense molecules and ribozymes, including liposomes and cationic lipid complexes. If the in situ reverting molecule is composed only of RNA, then expression vectors can be used in a gene therapy protocol to produce the reverting molecules endogenously, analogously to antisense or ribozymes expression vectors.

There are several advantages of using such an expression vector, rather than simply replacing the gene through standard gene therapy. Firstly, this approach would limit the production of the corrected gene to cells that already express that gene, Furthermore, the corrected gene would be properly regulated by its natural transcriptional promoter. Lastly, reversion can be used when the mutant RNA creates a dominant gain of function protein in sickle cell anemia), where correction of the mutant RNA is necessary to stop the production of the deleterious mutant protein, and allow production of the corrected protein.

Endogenous Mammalian RNA Editing System It was observed in the mid-1980s that the sequence of certain cellular RNAs were different from the DNA sequence that encodes them. By a process called RNA editing, cellular RNA are post-transcriptionally modified to a) create a translation initiation and termination codons, b) enable tRNA and rRNA to fold into a functional conformation (for a review see Bass, B. L. (1993) In The RNA World, R. Gesteland, R, and Atkins, J.

eds. (Cold Spring Harbor, New York; CSH Lab. Press) pp. 383-418), The process of RNA editing includes base modification, deletion and insertion of nucleotides.

Although, the RNA editing process is widespread among lower eukaryotes, very few RNAs (four) have been reported to undergo editing in

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pllllar~ WO 95/23225 PC(I 1 95I 1111156 156 mammals (Bass, supra). The predominant mode of RNA editing in mammalian system is base modification (C U and A The mechanism of RNA editing in the mammalian system is postulated to be that C--eU conversion is catalyzed by cytidine deaminase. The mechanism of conversion of A--G has recently been reported for glutamate receptor B subunit (gluR-B) in rat PC12 cells (Higuchi, M. et al, (1993) Cell 75, 1361- 1370), According to Higuchi gluR-B mRNA precursor attains a structure such that intron 11 and exon 11 can form a stable stem-loop structure. This stem-loop structure is a substrate for a nuclear double strand-specific adenosine deaminase enzyme. The deamination will result in the conversion of Reverse transcription followed by double strand synthesis will result in the incorporation of G in place of A.

In the present invention, the endogenous deaminase activity or other such activities can be utilized to achieve targeted base modification.

The following are examples of the invention to illustrate different methods by which in vivo conversion of a base can be achieved. These are provided only to clarify specific embodiments of the invention and are not limiting to the invention. Those in the art will recognize that equivalent methods can be readily devised within the scope of the claims.

Example 97: Exploiting cellular dsRNA dependent Adenine to Inosine converter: An endogenous activity in most mammalian cells and Xenopus oocytes converts about 50% of adenines to inosines in double stranded RNA. (Bass, B. Weintraub, H. (1988). An unwinding activity that covalently modifies it double-stranded RNA substrate. Cell, 55, 1089- 1098.), This activity can be used to cause an in situ reversion of a mutation at the RNA level. Referring to Figures 102 and 104, for demonstration purposes a stop codon is incorporated into the coding region of dystrophin, which is fused to the reporter gene luciferase. This stop codon can be reverted by targeting an antisense RNA which is long enough to activate the dsRNA deaminase, which converts Adenines to Inosines. The A to I transition will be read by the ribosome as an A to G transition in some cases and will thereby functionally revert the stop codon.

While other A's in this region may be converted to I's and read as G, converting an A to I cannot create a stop codon. The A to I transitions ~D II~LII I- \VO 95/23225 P("7/IB' 5/O00156 157 in the region surrounding the target mutation will create some point mutations, however, the function of the dystrophin protein is rarely inactivated by point mutations.

The reverted mRNA was then translated in a cell lysate and assayed for luciferase activity. As evidenced by the dramatic increase in luciferase counts in the graph in figure 103, the A to I transition was read by the ribosome as an A to G transition and the stop codon has successfully been reverted with the lysate treated complex. As a control, an irrelevant noncomplementary RNA oligonucleotide was added to the dystrophin/luciferase mRNA. As expected, in this case no translation (luciferase activity) is observed because of the stop codon. As an additional control, the hybrid was not treated with extract, and again no translation (luciferase activity) is observed (Figure 103).

While other A's in the targeted region may have been converted to I's and read as G, converting an A to I cannot create a stop codon, so the ribosome will still read through the region. Dystrophin is not generally sensitive to point mutations if the open reading frame is maintained, so a dystrophin protein made from an mRNA reverted by this method should retain full activity.

The following detail specifics of the methodology: RNA oligonucleotides were synthesized on a 394 (ABI) synthesizer using phosphoramidite chemistry. The sequence of the synthetic complementary RNA that binds to the mutant dystrophin sequence is as follows to

CCCGCGGTAGATCTTTCTGGAGGCTTACAGTTTTCTACAAACCTCC

CTTCAAA (Seq. ID No. 1) Referring to Figure 104, fifty-nine base pairs of a human dystrophin mutant sequence containing a stop codon was fused in frame to the luciferase coding region using standard cloning technology, into the Hind III and Not I sites of pRC-CMV (Invitrogen, San Diego, CA). The AUG of luciferase was deleted. The sequences of the insert from the Hind III site to the start of the luciferase coding region is to

GCCCCTGAGGAGCGATGGAGGCCTTGAAGGGAGGTTTGTGGAAAA

CTGTAAGCCTCCAGAAAGATCTACCGCGG (Seq ID No. 2) ISI~ DI14~SIII WO 95/23225 PCT/Ii95/(0015(0 158 This corresponds to base pairs 3649-3708 of normal dystrophin (Entrez ID 311627) with a Sac II site at the 3' end. This plasmid was used as a template for in vitro transcription of mRNA using T7 polymerase with the manufacturers protocol (Promega, Madison, WI).

Xenopus nuclear extracts were prepared in 0.5X TGKED buffer Tris (pH 12.5% glycerol, 25 mM KCI, 0.25mM DTT and 0.05mM EDTA), by vortexing nuclei and resuspended in a volume of 0.5X TGKED equal to total cytoplasm volume of the oocytes. Bass, B.L. Weintraub, H.

Cell 55, 1089-1098 (1988).

The target mRNA at 500ng/ul was pre-annealed to 1 micromolar complementary or irrelevant RNA oligonucleotide by heating to 70°C, and allowing it to slowly cool to 370C over 30 minutes. Fifty nanograms of mRNA pre-annealed to the RNA oligonucleotides was added to 7ul of nuclear extracts containing 1mM ATP, 15mM EDTA, 1600un/ml RNasin and 12.5mM Tris pH 8 to a total volume of 12ul. Bass, B.L. Weintraub, H.

supra. This mixture, which contains the dsRNA deaminase activity, was incubated for 30 minutes at 25°C. Next, 1.5ul of this mixture was added to a rabbit reticulocyte lysate in vitro translation mixture and translated for two hours according to the manufacturers protocol (Life Technologies, Gaithersberg, MD), except that an additional 1.3 mM magnesium acetate was added to compensate for the EDTA carried through from the nuclear extract mixture. Luciferase assays were performed on 15ul of extract with the Promega luciferase assay system (Promega, Madison, WI), and luminescence was detected with a 96 we!, .uminometer, and the results are displayed in the graph in figure 102.

Example 98: Base changina activities The chemical synthesis of antisense and triple-strand forming oligomers conjugated to reactive groups is well studied and characterized (Knorre, Valentin, Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993) and Povsic, Strobel, S. Dervan, P.

Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation J. Am. Chem. Soc. 114, 5934-5944 (1992). Reactive groups such as alkylators that can modify nucleotide bases in targeted RNA or DNA have been conjugated to oligonucleotides.

I gI yp~aa~a~-~ra WO 95/23225 PC'T/I/1195/0015 159 Additionally enzymes that modify nucleic acids have been conjugated to oligonucleotides. (Knorre, Valentin, Valentina, Lebedev, A.V. Federova, O.S. Design and targeted reactions of oligonucleotide derivatives 1-366 (CRC Press, Novosibirsk, 1993). In the past these conjugated chemical groups or enzymes have been used to inactivate DNA or RNA that is specifically targeted by antisense or triple-strand interactions. Below is a list of useful base changing activities that could be used to change the sequence of DNA or RNA targeted by antisense or triple strand interactions, in order to achieve in situ reversion of mutations, as described herein (see figure 100-104).

1. Deamination of 5-methylcytosine to create thymidine (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

Also, nitrous acid or related compounds promote oxidative deamination of C to be read at T(Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.). Additionally hydroxylamine or related compounds can transform C to be read at T (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226- 230.) 2. Deamination of cytosine to create uracil (performed by the enzyme cytidine deaminase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993) or by chemical groups similar to nitrous acid that promote oxidative deamination (Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.) 3. Deamination of Adenine to be read like G (Inosine) (as done by the adenosine deaminase, AMP deaminase or the dsRNA deaminating activity Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

4. Methylation of cytosine to Transforming thymidine (or uracil) to 0 2 -methyl thymidine (or 0 2 -methyl uracil), to be read as cytosine by alkynitrosoureas (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).

s s- I II~L111 WO 95/23225 PC-T/1195/0015(0 160 6. Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521:770-778 (1978) which can be done with the mutagen ethyl methane sulfonate (EMS) Microbial Genetics, David Freifelder, Jones and Bartlett Publishers, Inc., Boston,1987, PP.226-230.

7. Amination of uracil to cytosine (as performed by the cellular enzyme CTP synthetase (Bass, B.L. in The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1993).

The following are examples of useful chemical modifications that can be utilized in the present invention. There are a few preferred straightforward chemical modifications that can change one base to another base. Appropriate mutagenic chemicals are placed on the targetting oligonucleotide, nitrous acid, or a suitable protein with such activity. Such chemicals and proteins can be attatched by standard procedures. These include molecules which introduce fundamental chemical changes, that would be useful independent of the particular technical approach. See Lewin, Genes,1983 John Wilely Sons, Inc. NY pp 42-48.

The following matrix shows that the chemical modifications noted can cause transversion reversions (pyrimidine to pyrimidine, or purine to purine) in RNA or DNA. The transversions (pyrimidine to purine, or purine to pyrimidine) are not preferred because these are more difficult chemical transformations. The footnotes refer to the specific desired chemical transformations. The bold footnotes refer to the reaction on the opposite DNA strand. For example, if one desires to change an A to a G, this can be accomplished at the DNA level by using reaction #5 to change a T to a C in the opposing strand. In this example an A/T base pair goes to A/C, then when the DNA is replicated, or mismatch repair occurs this can become G/C, thus the original A has been converted to a G.

ISR matrix Reverted Base Mutant base A T(U) C G ICsPU I8 \VO 9523225 PT'I B95L00 150 161 A j Transversion ITransversion D NA,3'/RNA 3 T(U) Trasversion n DNA5/RNA 7 Transversion C Tsro R DN Llversion GC Tran s version nss svTransversion 1 Deamination of 5-methylcytosine to create thymidine.

2 Deamination of cytosine to create uracil.

3 Deamination of Adenine to be read like G (Inosine).

4 Methylation of cytosine to Transforming thymidine (or uracil) to 0 2 -methyl thymidine (or 0 2 -methyl uracil), to be read as cytosine (Xu, and Swann, Tetrahedron Letters 35:303-306 (1994)).

6 Transforming guanine to 6-O-methyl (or other alkyls) to be read as adenine (Mehta and Ludlum, Biochimica et Biophysica Acta, 521:770-778 (1978)).

7. Amination of uracil to cytosine. Bass supra. fig. 6c.

In Vitro Selection Strategy Referring to Figure 105, there is provided a schematic describing an approach to selecting for a ribozyme with such base changing activity. An RNA is designed that folds back on itself (this is similar to approaches already used to select for RNA ligases, Bartel, D. and Szostak, J. (1993) Isolation of new ribozymes from a large pool of random sequences.

Science 261:1411-1418). A degenerate loop opposing the base to be modified provides for diversity. After incubating this library of molecules in a buffer, the RNA is reverse transcribed into DNA (that is, using standard in vitro evolution protocol. Tuerk and Gold, 249 Science 505, 1990) and then the DNA is selected for having a base change. A restriction enzyme cleavage and size selection or its equivalent is used to isolate the fraction of DNAs with the appropriate base change. The cycle could then be repeated many times.

)5 2322 P' I *in'SJ n 1 162 The in vitro selection (evolution) strategy is similar to approaches develcped by Joyce (Beaudry, A. A. and Joyce, G.F, (1992) Science 257, 635-641; Joyce, G. F. (1992) Scientific American 267, 90-97) and Szostak (Bartel, D. and Szostak, J. (1993) Science 261:1411-1418; Szostak, J. W.

(1993) TIBS 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein, each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence.

The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (the region flanking the mutant nucleotide), 2) complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their base modifying activity, 3) introduction of restriction endonuclease site for the purpose of cloning. The degenerate domain can t~e created to be completely random (each of the four nucleotides represented at every position within the random region) or the degeneracy can be partial (Beaudry, A. A. and Joyce, G.F, (1992) Science 257, 635- 641). In this invention, the degenerate domain is flanked by regions containing known sequences (see Figure 105), such that the degenerate domain is placed across from the mutant base (the base that is targeted for modification). This random library of nucleic acids is incubated under conditions that ensure folding of the nucleic acids into conformations that facilitate the catalysis of base modification (the reaction protocol may also include certain cofactors like ATP or GTP or an S-adenosyl-methionine (if methylation is desired) In order to make the selection more stringent).

Following incubation, nucleic acids are converted into complimentary DNA (if the starting pool of nucleic acids is RNA). Nucleic acids with base modification (at the mutant base position) can be separated from rest of the population of nucleic acids by using a variety of methods, For example, a restriction endonuclease cleavage site can either be created or abolished as a result of base modification. If a restriction endonuclease site is created as a result of base modification, then the library can be digested with the restriction endonuclease The fraction of the population that is cleaved by the RE is the population that has been able to catalyze the base modification reaction (active pool), A new piece of DNA (containing oligonucleotide primer binding sites for PCR and RE sites for cloning) is ligated to the termini of the active pool to facilitate PCR amplification and subsequent cycles (if necessary) of selection. The final pool of nucleic acids with the best base modifying activity is cloned in to a plasmid vector WO 23225 icr'ni,* 163 and transformed into bacterial hosts. Recombinant plasmids can then be isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques.

Base modifying enzymatic nucleic acius (identified via in vitro selection) can be used to cause the chemical modification in vivo.

In addition, the ribozyme could be evolved to specifically bind a protein having an enzymatic base changing acitivity, Such ribozymes can be used to cause the above chemical modifications in vivo. The ribozymes or above noted antisense-type molecules can be administered by methods discussed in the above referenced art.

VIII. Administration of Nucleic Acids Applicant has determined that double-stranded nucleic acid lacking a transcription termination signal can be used for continuous expression of the encoded RNA. This is achieved by use of an R-loop, an RNA molecule non-covalently associated with the double-stranded nucleic acid and which causes localized denaturation ("bubble" formation) within the double stranded nucleic acid (Thomas et al., 1976 Proc. Natl. Acad, Sci, USA 73, 2294), In addition, applicant has determined that that the RNA portion of the R-loop can be used to target the whole R-loop complex to a desirable intracellular or cellular site, and aid in cellular uptake of the complex, Further, applicant indicates that expression of enzymatically active RNA or ribozymes can be significantly enhanced by use of such Rloop complexes.

Thus, in one aspect, the invention features a method for introduction of enzymatic nucleic acid into a cell or tissue. A complex of a first nucleic acid encoding the enzymatic nucleic acid and a second nucleic acid molecule is provided, The second nucleic acid molecule has sufficient complementarity with the first nucleic acid to be able to form an R-loop base pair structure under physiological conditions, The R-loop is formed in a region of the first nucleic acid molecule which promotes expression of RNA from the first nucleic acid under physiological conditions. The method further includes contacting the complex with a cell or tissue under

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W\O 23225 K' 1 l'5,101156 164 conditions in which the enzymatic nucleic acid is produced within the coil or tissue, By "complex" is simply meant that the two nucleic acid molecules interact by intermolecular bond formation (such as by hydrogen bonding) between two complementary base-paired sequences. The complex will generally be stable under physiological condition such that it is able to cause initiation of transcription from the first nucleic acid molecule.

The first and second nucleic acid molecules may be formed from any desired nucleotide bases, either those naturally occurring (such as adenine, guanine, thymine and cytosine), or other bases well known in the art, or may have modifications at the sugar or phosphate moieties to allow greater stability or greater complex formation to be achieved. In addition, such molecules may contain non-nucleotides in place of nucleotides.

Such modifications are well known in the art, see Eckstein et al,, International Publication No, WO 92/07065; Perrault et al,, 1990 Nature 344, 565; Pieken et al., 1991 Sciece 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci, 17, 334; Usman et International Publication No, WO 93/15187; and Rossi et al,, International Publication No. WO 91/03162, as well as Sproat,B, European Patent Application 92110298,4 which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules, All these publications are hereby incorporated by reference herein, By "sufficient complementarity" is meant that sufficient base pairing occurs so that the R-loop base pair structure can be formed under the appropriate conditions to cause transcription of the enzymatic nucleic acid, Those in the art will recognize routine tests by which such sufficient base pairs can be determined. In general, between about 15 80 bases is sufficient in this invention, By "physiological condition" is meant the condition in the cell or tissue to be targeted by the first nucleic acid molecule, although the R-loop complex may be formed under many other conditions, One example is use of a standard physiological saline at 370C, but it is simply desirable in this invention that the R-loop structure exists to some extent at the site of action so that the expression of the desired nucleic acid will be achieved at that site of action. While it is preferred that the R-loop structure be stable under e I- WVO 95123225 P(I I iWS IlilllI 165 those conditions, even a minimal amount of formation of the R-loop structure to cause expression will be sufficient, Those in the art will recognize that measurement of such expression is readily achieved.

especially in the absence of any promoter or leader sequence on the first nucleic acid molecule (Daube and von Hippel, 1992 Sencee. 258, 1320).

Such expression can thus only be achieved if an R-loop structure is truly formed with the second nucleic acid. If a promoter of leader sequence is provided, then it is preferred that the R-loop be formed at a site distant from those regions so that transcription is enhanced, In a related aspect, the invention features a method for introduction of ribonucleic acid within a cell or tissue by forming an R-loop base-paired structure (as described above) with the first nucleic acid molecule lacking any promoter region or transcription termination signal such that once expression is initiated it will continue until the first nucleic acid is degraded.

In another related aspect, the invention features a method in which the second nucleic acid is provided with a localization factor, such as a protein, an antibody, transferin, a nuclear localization peptide, or folate, or other such compounds well known in the art, which will aid in targeting the R-loop complex to a desired cell or tissue, In preferred embodiments, the first nucleic acid is a plasmid, e.g., one without a promoter or a transcription termination signal the second nucleic acid is of length between about 40-200 bases and is formed of ribonucleotides at a majority of positions; and the second nucleic is covalently bonded with a ligand such as a nucleic acid, protein, peptide, lipid, carbohydrate, cellular receptor, nuclear localization factor, or is attached to malelmide or a thiol group: the first nucleic acid is an expression plasmid lacking a promoter able to express a desired gene, it is a double-stranded molecule formed with a majority of deoxyribonucleic acids; the R-loop complex is a RNA/DNA heteroduplex; no promoter or leader region is provided in the first nucleic acid; and the Rloop is adapted to prevent nucleosome assembly and is designed to aid recruitment of cellular transcription machinery, In other preferred embodiments, the first nucleic acid encodes one or more enzymatic nucleic acids, it is formed with a plurality of \\WO 95123225 K 1 1111)"11114(' 166 intramolecular and intermolecular cleaving enzymatic nucleic acids to allow release of therapeutic enzymatic nucleic acid in vivo.

In a further related aspect, the invention features a complex of the above first nucleic acid molecules and second nucleic acid molecules, R-loop complex An R-loop complex is designed to provide a non-integrating plasmid so that, when an RNA polymerase binds to the plasmid, transcription is continuous until the plasmid is degraded, This is achieved by hybridizing an RNA molecule, 40 to 200 nucleotides in length, to a DNA expression plasmid resulting in an R-loop structure (se fiure 106). This RNA, when conjugated with a ligand that binds to a cell surface receptor, triggers internalization of the plasmid/RNA-ligand complex. Formation of R-loops in general is described by DeWet, 1987 Methods jinEnzymol. 145, 235; Neuwald et al,, 1977 J. Virol. 21,1019; and Meyer et al., 1986 JJ, JIt. Mol.

Str, Res. 96, 187, Thus, those in the art can readily design complexes of this invention following the teachings of the art.

Promoters placed in retroviral genomes have not always behaved as planned in that the additional promoter will serve as a stop signal or reverses the direction of the polymerase. Applicant was told that creation of an R-loop between the promoter and the reporter gene increased the transfection efficiency. Incubation of an RNA molecule with a doublestranded DNA molecule, containing a region of complementarity with the RNA will result in the formation of a stable RNA-DNA hetroduplex and the DNA strand that has a sequence identical to the RNA will be displaced into a loop-like structure called the R-loop. This displacement of DNA strand occurs because an RNA-DNA duplex is more stable compared to a DNA- DNA duplex. Applicant was also told that an 80 nt long RNA was used to generate a R-loop structure in a plasmid encoding the B-galactosidase gene, The R-loop was initiated either in the promoter region or in the leader sequence. Plasmids containing an R-loop structure were microinjected into the cytoplasm of COS cells and the gene expression was assayed. R-loop formation in the promoter region of the plasmid inhibited expression of the gene, RNA that hybridized to the leader sequence between the promoter and the gene, or directly to the first nucleotides of the mRNA increased the expression levels 8-10 fold, The WO Q)5S23225 PICTIJ("i1i)5, (I .b 167 proposed mechanism is that R-loop formation prevents nucleosomo assembly, thus making the DNA more accessible for transcription.

Alternatively, the R-loop may resemble a RNA primer promoting either DNA replication or transcription (Daube and von Hippel, 1992, as ra}.

One of the salient features of this invention is to generate R-loops in expression vectors of choice and introduce them into cells to achieve enhanced expression from the expression vector. The presence of an Rloop may aid in the recruitment of cellular transcription machinery. Once an RNA polymerase binds to the plasmid and initiates transcription, the process will continue until a termination signal is reached, or the plasmid is degraded, This invention will increase the expression of ribozymes inside a cell, The idea is to construct a plasmid with no transcription termination signal, such that a transcript-containing multiple ribozyme units can be generated, In order to liberate unit length ribozymes, self-processing ribozymes can be cloned downstream of each therapeutic ribozyme (see figure 107) as described by Draper supra.

Ligand Targeting Another salient feature of this invention is that the RNA used to generate R-loop structures can be covalently linked to a ligand (nucleic acid, proteins, peptides, lipids, carbohydrates, etc.). Specific ligands can be chosen such that the ligand can bind selectively to a desired cell surface receptor. This ligand-receptor interaction will help internalize a plasmid containing an R-loop. Thus, RNA is used to attach the ligand to the DNA such that localization of the gene to certain regions of the cell is achieved, One of several methods can be used to attach a ligand to RNA.

This includes the incorporation of deoxythymidine containing a 6 carbon spacer having a terminal primary amine into the RNA (see figure 108). This amino group can be directly derivatized with the ligand, such as folate (Lee and Low, 1994 J, Biol. Chem, 269, 3198-3204), The RNA containing a 6 carbon spacer with a terminal amine group is mixed with folate and the mixture is reacted with activators like 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC). This reaction should be carried out in the presence of 1-Hydroxybenzotriazole hydrate (HOBT) to prevent any undesirable side reactions.

I I WVO 95.'23225 'PCT I 1 'r Il1 15c1 168 The RNA can also be derivatized with a heterobifuctional crosslinking agent (or linker) like succinimidyl 4-(pmaleimidophenyl)butyrate (SMPB). The SMPB introduces a maleimide into the RNA. This maleimide can then react with a thiol moiety either in a peptide or in a protein. Thiols can also be introduced into proteins or peptides that lack naturally occurring thiols using succinylacetylthioacetate.

The amino linker can be attached at the 5' end or 3' end of the RNA. The RNA can also contain a series of nucleotides that do not hybridize to the DNA and extend the linker away from the RNA/DNA complex, thus increasing the accessibility of the ligand for its receptor and not interfering with the hybridization, These techniques can be used to link peptides such as nuclear localization signal (NLS) peptides (Lanford et al,, 1984 Cell 37, 801-813; Kalderon et al., 1984 Cell 39, 499-509; Goldfarb et al,, 1986 Nature 322, 641-644)and/or proteins like the transferrin (Curiel et al., 1991 Proc, Natl. Acad, Sci, USA 88, 8850-8854; Wagner et al., 1992 roc. Natl.

Acad, Sci. USA 89, 6099-6103; Giulio et al., 1994 Cell. Signal, 6, 83-90) to the ends of R-loop forming RNA in order to facilitate the uptake and localization of the R-loop-DNA complex, To link a protein to the ends of Rloop forming RNA, an intrinsic thiol can be used to react with the maleimide or the thiols can be introduced into the protein itself using either iminothiolate or succinimidyl acetyl thioacetate (SATA; Duncan et al., 1983 Anal, Biochem 132, 68), The SATA requires an additional deprotection step using 0.5 M hydroxylamine.

In addition liposomes can be used to cause an R-loop complex to be delivered to an appropriate intracellular cite by techniques well known in the art, For example, pH-sensitive liposomes (Connor and Huang, 1986 Cancer Res. 46, 3431-3435) can be used to facilitate DNA transfection.

Calcium phosphate mediated or electroporation-mediated delivery of the R-loop complex in to desired cells can also be readily acomplished, In vitro Selection In vitro selection strategies can be used to select nucleic acids that a) can form stable R-loops b) selectively bind to specific cell surface receptors. These nucleic acids can then be covalently linked to each other, This will help internalize the R-loop-containing plasmid efficiently using receptor-mediated endocytosis. The in vitro selection (evolution) strategy is

I

L -~ILIII I 5')'23225 I 1l95 0 15)(1 169 similar to approaches developed by Joyce (Beaudry and Joyce, 1992 Science 257, 635-641; Joyce, 1992 Scientific American 267, 90-97) and Szostak (Bartel and Szostak, 1993 Science 261:1411-1418; Szostak, 1993 TLBa. 17, 89-93). Briefly, a random pool of nucleic acids is synthesized wherein each member contains two domains: a) one domain consists of a region with defined (known) nucleotide sequence; b) the second domain consists of a region with degenerate (random) sequence.

The known nucleotide sequence domain enables: 1) the nucleic acid to bind to its target (a specific region of the double strand DNA), 2) complimentary DNA (cDNA) synthesis and PCR amplification of molecules selected for their affinity to form R-loop and/or their ability to bind to a specific receptor, 3) introduction of a restriction endonuclease site for the purpose of cloning. The degenerate domain can be created to be completely random (each of the four nucleotides represented at every position within the random region) or the degeneracy can be partial (Beaudry and Joyce, 1992 Science 257, 635-641), In this invention, the degenerate domain is flanked by regions containing known sequences.

This random library of nucleic acids is incubated under conditions that ensure equilibrium binding to either double-stranded DNA or cell surface receptor. Following incubation, nucleic acids are converted into complementary DNA (if the starting pool of nucleic acids is RNA), Nucleic acids with desired characteristics can be separated from the rest of the population of nucleic acids by using a variety of methods (Joyce, 1992 supra). The desired pool of nucleic acids can then be carried through subsequent rounds of selection to enrich the population with the most desired traits. These molecules are then cloned in to appropriate vectors.

Recombinant plasmids can then be Isolated from transformed bacteria and the identity of clones can be determined using DNA sequencing techniques.

Other embodiments are within the following claims.

II ~I I I 9J5/23225 I 111' mi 151.

170 Characteristics f Pibcrzvmeo Group I Introns Size: -200 to >1000 nucleotides.

Requires a U in the target sequence immediately 5' of the cleavage site.

Binds 4-6 nucleotides at 5' side of cleavage site.

Over 75 known members of this class, Fcuna in Tetrahymena thermcphila rRNA, fungal mitcchondria, cniorcplasts, -nage T4, oluegreen algae, and others.

RNAseP RNA (M1 RNA) Size: -290 to 400 nucleotides.

RNA portion of a ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature tRNA.

Roughly 10 known members of this group all are bacterial in origin.

Hammerhead Ribozyme Size: -13 to 40 nucleotides, Requires the target sequence UH immediately 5' of the cleavage site.

Binds a variable number nucleotides on both sides cf the cleavage site, 14 known members of this class, Found in a number of plant pathogens (virusoids) that use RNA as the infectious agent (Figures 1 and 2) Hairpin Ribozyme Size: -50 nucleotides.

Requires the target sequence GUC immediately 3' of the cleavage site, Binds 4-6 nucleotides at 5' side of the cleavage site and a variable number to the 3' side of the cleavage site, Only 3 known member of this class. Found in three plant pathogen (satellite RNAs of the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle virus) which uses RNA as the infectious agent (Figure 3).

Hepatitis Delta Virus (HDV) Ribozyme Size: 50 60 nucleotides (at present).

Cleavage of target RNAs recently demonstrated.

Sequence requirements not fully determined.

Binding sites and structural requirements not fully determined, although no sequences 5' of cleavage site are required.

Only 1 known member of this class. Found in human HDV (Figure 4).

Neurospora VS RNA Ribozyme Size: -144 nucleotides (at present) SUBSTITUTE SHEET (RULE 26)

M

NN 95132211; 171 Cleavage of target RNAs recently 'Xi-mcrotrated, Seouence requirements not fully otermneu.~ 6-incincg sites and structural requirements nct fully aeter-ninerj. Orniv known member f this class. Founa in Nourc2scra VS PRNA Figquro SUBSTITUTE SHEET IRULE 26)

I

N N 0 1) 5 .13 225 N 1 11195 172 'a Ie 2 -,,rnan lCAIM .HHTarget sequence 2.Position Target Sequences rit. Position Target Sequences 2. CCA C =G C ;C U G 3 8 9C S C A4G A ;6 GC-=C C t:GCUACU 42 cXcz0 C 2. CCUGU ACUCAGAC, 425 34 UGCJAUCJ AGAGUCC 427-^ ~lG UCAGAGU U Gr AC 4 50 lA?.CC AC kC CC 48 GCAACCU C AGCCOXG 452 AAZU CUC 54 UrCAGCC t j C GCtLGG 456 LLC C-='GCC 58 CCUCGCJ A UGGCUCC 495 OcAACCU C ACCG,'UGG 64 UAUGCG 1 J C' CAC G 510 UG=GCU' C C G G 96 CCGCACJ C GG -TC C 5-6 4 ZGc CA 102 UGGU C- CUGCUCG- 592 GCA 108 'UCCUCU C GGc-GCrUC 607 AGCCA 12.5 CGG=rj' C UTCUCCC 608 GCCAAUU ZUC'GC i2.9 GCUCUwGU U CCCAGGA 609 CCAALtU C UC Z m G C 1270 CUCUGLU C CCAGGAC 'il: A:~tc 146 CAGACAU C UGU='C 65;6 GAGCUGU UGGAAC 152 UCLGUGU C CCCct'CA 657, AGCUjGZj U AGAAC-A 158 UCCCCCU C Alx;AGUC 668 ?~cCUC GCCCCC- 165 CAAAAGU C AUCCUGC 677 CCCC A CCAGIC1C 18AAGUCAU C CUGCCCC- 684 ACCAGCU C ACU GGAGGCJ C CGUGCUG 692 CA G ACC UGUCCJG 209 AGCACCU C CUGUGAC 693 AG ACCU= U G C =G C 227 CCCAAGU U GUGGGO 696 CtG r-C CAG 230 AAGUGU U GGGCAUA 709 AG.CCCACU C C- -C 237 UGG-GCAU A GAGACCC 720 CACAACU U Tt;CAGCC- 248 ACCCCGU U GCCtJAAA 723 AACUUGU C A-C-CCC 253 GUL'GCCTU A AAAAGGA 735 0 CU;AGAGC- 263 AAGGAGU U GCUCCUG 738 GGGCV&a A GAGGUGG 26"1 AGUTJGCU C C UG C CUG 765 CCUC GUCCC- 293 AAGGUGU A VGAACUG 769 GCUtUOCGA 319 AGAAGAU A GCCAACC 770 GUCUTUU C CCUGGAC 335 AUGUGCU A UUCAAAC 785 C-GGC'UGU U CCCAGUC 3 37 GTJCTAU U CAAACUG 786 GGCUGUU C CCAGUCU 338 UGCUAUU C AACUGC 792 UCCCAGU C =C-GAG 359 GGGCAGU C AACACU 794 C CA G IC U z GG AGG C 367 AACAGC A AAACCUUJ 80,7 CCCAGGUQ =1 2CG G 37 4 AAAC=U U CCU'CACC 833 AGAGCU U GAACCCC AAACCU C CliCACC 816 CcG CUU 378 CCUUCCTU C ACCGUGU 852. TC~cz A U-CAA SUBSTITUTE SHEET (RUL.E 26) 95123225 (I13)uIS 173 863- AACMU c =1tU=3 C4~ s 86 5 ACC=C sr cc- q. 4 A 885 C=CAGUT, C A~TGUGA 1'444 3A~~A cU AAGWGA 93 3 G' GCAGJ A AUJACUGG -'455 3GG;c zGG.

936 CkTJAAU A CUGGGGA i482 VZ:GlGC'- C U'C Cc 978 UGACCAU UACAGCJ 1484 Gucc 980 ACC.Auc'J A cAcc'juu 1'493 CC CCA MAGAUU 985 UACAGCWJ ';UCGGCG -1500 AL'CAGALU G UCAUCA 9871 A~C-CJU u CC-GGCGC 1503 AC-Z'~jCACUCA 988 CAGCU-UU c CGGCGCC 1505 =G:A AU'CACU:G 1005 ACMUGAU U CUG-ACGA i509 U AUCAJ3 C ACUGUGG 1005 CGUGAU3 C UGACGAA is5:8 =GGL A 103CAGAGGU C UCAAG 1530 CCGCAGU AUAG 1025 GAGGU~i C AG?.AGGG 1533 CAGUCAU A AUGGG-'A 1056 CCACCCU A GAGC~kA 1551i CAGGCCUJ C ACCA=7%U 1092 AtUGG~ U CCAGCCC 1 55 9 AGCAC-2. A CJcTcAU 1093 UGGG7T3 C CAGCCCA i563 CLC-'- AAC 1125~f CCAC UGUA16JACCC A UAACC-GC i163 CGCACU U Ct7CCTJGC 1-567 CCUCLUAU A AC=GCA 1164 CGCAGI-JU C UCCUGICU 1584 GGAAGAL' C AAGAvAAU 1165 GCUUCU C CUGCUCU i592 AAGAAAU A CAGACJA 1172 UCCUC-CU C UGCAACC 1599 ACAGACU A CAACAGG 1200 C-AGCUJ U AUACAC-k 1651 ACGCC-U CCUGAC 1201 CGAGCOU A UACACA 1561 UGAACCU A UCCCGG 1203 ACUUAU A CACAAGA 1563 AAC=)AU C CC-GGGAC 1.227 GGGAGCU U CGUGTJCC 1678 AGGGCCUJ C UUCG 1228 GCGACUU C GUGUCCI 1580 GC=J~ U C~CLGC 1233 UUCGUGU C CUGUtAG 1581 G COC=U~ C CLCGGCC 123 8 G U C CJGU A UGGCCCC 1684 UUC GCC 1264 GAc-GGAU U GUCCGGG 1690 'jGCr UC"CcAUX.

1257 GG-AUUGU C CGGGAAA 1591 CGGCCUU C CCAUAUU 1294 AG~AUT U CCCAGCA 1695 UUCCCAU A MZ'GUGG 1295 G-AAAAUU C CCAGCAG 1598 ICCAMYU U GGUGGCA 1305 GCAGACU C CAAUGUG 1737 GCAAUGAC 1321 CCAGGCrJ U GGGGGAA 1750 UGCCU A CA CCUMAC 1334 LkCC V kU U GCCCGAG 1756 UACAC=J ACGCC 1344 CCGAGCU C AAGUGUC 1787 AGGGCAU U GUCCUcAk 1351 CA-AGUGTJ C UAAAGGA 1790 GCAtUUGU C CUCAGJC 1353 AGUGUCU A AAGGAUG 1793 UUGUCCU C AGUCAGA 1356 UGGCACUJ U UCCCACU 1797 CCUCAGU C AGALTAM.

1367 GGCACULU U CCCACUG 1802 GUCAGAU A CAACAGC 1368 GCAL7.'U C CCACUGC 18 *12- ACAGOC! t.G;GCC '380 UGC-CA: C CGGC-G~t 1813 CU UG3CA :388 G-GGAAU C AGUGACU 1825 oCAuGGU A OCUcC '398 UG-ACUGU C ACUCGAG 1837 CAC.ACGU- A AAAc-cC 1402 UGI.;CU C GAGAUCU 1845 1- 'AGGCC SUBSTITUTE SHEET tRULE 26)

I

p( '11 I A NN 0 9)5/23225 174 2.902.

i9=8 i 93 0 1996 2005 202.3 20i.5 2020 2039 2040 2057 2062.

2071 2076 20971 2098 2130 2145 2152 215 2160 2162 22.63 2166 2167 22.7 2171 21.73 22.74 2175 22.76 28.3 c~c~cA0 =7AC C AGt:C 3A7U=1QMU A GICACAU CZUG1U G"C C ACAL'GAC CAUGACU A AGCCAAG CAGACU C AA-AC kcAkuGAU G-AuGAu UGGAUGU U AAAGUCUJ ~GAUGUUA AUC _jG"Z: C U GCCUG ?LAAGUCU-% A CZU'2AU GAGACAU A GVCZ-CCAC AC-GACAU A% CAACt,'GG C;GAAA A CUGAAAC UGCAAACJ U GCtUGCCtJ G G=GCCUJ A U'CCGGUA U:GCCtJ7U U GGGtAUG AUUGGTU A UGCUGAG ACAGACJ U ACAGAAG CAGACUU A CAkGAAG-A -UGC-CCU C C7AAGAC CCAU A GACAUGU CAUGUGU A GCUCAA GLAGCAU C AAAACAC C C. %CAC U riuCrUGACG CACAC'U C C*UGACGG GCCAGCUJ U GGGCACU C'UGC'tT C UACUGAC 22C5 220 22-24_ 2248 2254 2259 2260 22764 227 9 2282 2288 2291 2321 2338 2339 2342.

2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2402.

2412.

242.7 2418 2425 2426 2433 21434 2448 2449 UA'xU AG UGU Cl CGAGU:GiU1_

UUAGU

GUC=X'U A UG"Wt GC" GUAWGCU A C-=UGAAA "GAACAU A GGL==U CAUAcU UAGGUCU C 'UGCCXU G C= C AC=-.AGC C-GGAGCZJ C C ^AkGXC UCCAGU c CAUGUCCA UCCLAUGU, C kAL'2JA

GCUG'CU

CAACCICU

UG-AUGAU

GAUAUGU

UAL:'UAU

A7CTUAUU

UGUAUUU

UAUUUAU

ALTUCAUU

WAU'TJGUU

UjUGUUAU UGumJuu

GUUAUU

1t 5 'AU~tX A CCA GC U CAC2.AU

AGCUA~TJ

GCJALLU

A ClUG.ACCCe U GAUGAUA A UGUAUUU A Ut~tj'AUUC u UAL'tTCAU U AUVCAUU A UUCAUUU U CAUUtJGU C AUUUGUU U UGUEJAtU U GUM=UU U AUUUUAC A UUUUACC U UUACCAG U UACCAGC U ACCAGCU A CCAGCUA A =UAUCG U UrAUtGAG U AUUGAGU A UUGAGtUG GUCACAku U UCACAUU c ,UCAAGGUJ C ACCAGG-j A GUACAG~j U CAGtUUJGU A LUACAGGU U AGGLjUGU A AAAC2AUC Z2GGC-ACU U GACUUCU C LCUCAU U CCUGCCUj U CUGCCUjU U Uc*C=tU C GAGUG(AUj rU AGUjGAUU U UCGAUUU u GAUUUU C UMtM'UCU A UMUCAU C AAGCACU A GCACUAU A GA CUGGU A UAAUJGGU U AAUGUTU c CACAGGU U ACAGMTU C CAGAGAUJ U AGAGAUUT A GAGGCCU U AGGC=tT A

C.%AGGUC

AACGuOak

ACCAGGU

C-AGUUMU

3TJACACG

CACGCY

AAAtZGGG

CUCAUUG

tUCAtXCG AUjUGGCC

GGCCAAC

UCCCCAG

CCCAGA

UUCUAUC

UCLAUCG

tIAUCC-GC

UCGGCAC

GGCACA

UAUGGAC

U:C-GACtUG

AL'GGUU

CACAGU

ACAGGUU

CAGAGAU

AGAGAUU

OCCAGUG

UUCUCC

SUBSTITUTE SHEET (RULE 26) VLfl 4)rll 1,)15 Il' I II'5 I Z,,kvZ:- 2 7,6 U C c U 24'0CC= CCCCCCAA 27 69 A2A :4-9 GAA~U Gw1.AGC AU~ GUcAA M "U22 2420 ACACZUU U TUUAGCC- 28C3 UAX~U V '433: 2CUUUrGt. AGCCAC2 2804- GCV .,484 rCUUUGL7 A GCZkCC'J 2813 2UGGACCU 2504 CCCACAU A CAlucJ 2182: tXGACU 2kUAOC.V U U:CUGCZA 28 22 U:GAC2UL7U U.G=GUC 2509AUACAU U CUG22AG 2823 GAC~'tU~-Cc 2510 UACAUUU C UGCAtGUO 1282119 UUGGG7CU AA~GUGAU 252 0 CCAGUGU U CACAAUG 282- AACUGA--U 2CUC CA C CAGUGUU C ACAAUGA 2840 ZUGAtUCZU- cC CAICU C 2 53--3 UGAcCUC C AGCCG,-UC 2847 22ACU AGCCC 21540 C A GCGG U C AUGUCUG 2853 !CaGCU 2CUGAGUA 2545 GUCAUGTJ C UJGGACAU 2860 C-1UlGAGU GZ GG 2568 AGGGAU A UGCCCAA 2872 GGACCAU A GGC U=-C 2579 C2C.;.GCU A UGCC UUG 2877 AUA=GC ACAAC 2585 '.AUGCCU U GUCC=c 2899 GG T UAU' 2588 GCCUUGU C CUCUUGU 2900 GCAA r: GALM= 2591 UTJGUCCU C UUCGUCC 1 J 2904 AUUUGAU UUML'UU 2593 GCCUCU U GUCCUGU 2905 UUUGACU U U'MUU 2596 C'UCUUG;U C CUGUUUG 2906 iuUGAtl= uUL'U=t 2 601 GUCCUGU U UGCAUUU 2907 UGALMTJUu U UUMTJt' 2602" 'UCCUGUU U GCAUUUC 2908 GALMuU't Ui UUUU 2607 UUCAU U UCACUGG 2909 AUUUUU u UUM.tiU 2608 UCG2AUU U "'ACUGGG 2910 ULM'UUU U =UUUU 2609 UGCAUUU C ACUGGGA 2911 UUUUT u UtjtJ'E7 2620 GGGAGCU U Gr-ACUAU 2 91.2 UUUmmut U UUUUUL'C 2626 UUGCACU A UUGCAGC 2913 i~mmmU U UX~iurUC 2628 GCACUAU U GCAGCUC 29:4 LUUU U UUUUCAG 2635 UGCAGCU C CAGUUUC 2915 t JM7UU7t U UUUCAC-A 2640 CUCCAGU U UCCUGGA 2916 T UUUU U UUCAGAG 2 64: UwCAGUU U CCUGCAG 2917 UUUUUU U UCAGAGA 2642 C2AGUUU C CUGCAGU 2918 UUUUTJ U CAAA 2653 CAkGUGAU C AGGGUCC 2919 UUUUUU' C AGAGACG 2659 UCAGGGU C CUGCAAG 2931 ACGGGGU C tUCGCAAC 2689 CCAAGGU A UUJGGAGG 2933 CGGGUCU C G2AACAU 269: AAGGUAU U GGAGGAC 2941 G-CAACkTJ U GC2OAGA 2700 GAGGACU C CCUCCCA 2951 CCAGACU U C UUGli 2704 ACUCCCU C CCAGCUU 2952 CAGACMC C C'UMCGUG 272 2CAGCU U UGGA)AGG 2955 ACIWUCCU- 1 u =UGGCA 172 CCAGC--U U GGAAGGG 2956 Cl-ucCU U GUGUUjAG 272: GAAGG C AUCCGCG- 2961 UUUGGU ATU-AXL 27241 GJAU C CGCGUGU 2962 uuGuGuu A GUUAAU'A 27441 UGUGUGU A UGUGUAG 2965 CGUMAGU U AAULAAAG SUBSTITUTE SHEET (RULE 26) \k 095 23225 0 23225PCI/I 1195/001 56 176 ,XAGLUU A AL-AAAGC G=.UtAU A AAGCrwUU AAAGCU U UCUCAAC

AAAGC

T

'U CCA

T

AACtjt'U C UcAACUG SUBSTITUTE SHEET (RULE 26) 'lmtlik 1'11" 0 095/23225 N( 1 1119; (1156 177 Table 3 Mouse 10AM HH Target Sequence nPosition Target Sequence Pt ositior Targe Squnc r-"UgG C acCGuUG 36 0'r' uCA 23 CaGuC,,U u CUCJC' 374;A1C' U CZgCCC 26UGgJCUj C UGC'jCCU 375 AAgC=ZX C: CgcC-cz 3-1 "jtGt c CUCcaca 378 acCaU C ACW1UGUv 34 Uu~tcaU a A-GUcG 3186 AC:-TGt A u~cGuuLV gCAcAct7 U GILAgC'J 394 C:r GAC u 'UcGAuCu.

48 aggACCU C AGCgG 4'l2^0 aaCz ccccccg 54 UggGCCtJ C GucjAUGG 425 a^ c-caGC.

58 \Cat~gc,:,7 u UaGCUCC 4127 0gUUc GA 54 cAcccCUJ C CCAGCAG 450 AGgACCUj c ACC-'tgC OucuigC3 C CULGGCCC 452. GAAaCz-U u ucCljuuG 102 UgCcaGtJ a CUGCt~gG 45 6 UUACCCU c aGCcCm 108 cCLC'jw C cuGGCCC 495 0,!Ac,-aU C ACCSCu 11.5 tUGGUUCt3 C TJGctCCU 32.10 tc GG 19 C-gaaU=t c aCCAGGA 5641 -t GU a iCcAuC c 120 CUJCUGcU C CugGccC 592 C-APaAGAU C ACaugOG 146 CAGUCgu C c'7JUCC 607 AGCCAAU U U CUC a CG 152 UCt'UGU C agcCaCu 608 3C A AU U =CcaUGC 158 UCICuguU u AAAAacC 609 :CCvAzUU UCat;GCC 155 CAgAAGU U gtUUtJGC AUUUCJ aLUGCCGC 158 AAGcCUtJ C CUGCCCC 556 ,-GCUTJ U t:CAGcug 185 GGUGGgU C CGUGCaG 557 AGCUGUU U GAGcugA 209 gcCACUU C CTJGgC 568 cgagCCU a GGC'CaCC 227 CagAAGU U GtJUuuGC 677 GaCCuCtU A CCAGCCu 230 AAGtj'GU U UUGCucc 684 UCAGCU C CgGuCCU 237 IJGuGCuU u GAGAaCu 592 CgGACU-U U cGauCUu 248 AaCCCaU c uCCtJAAA 693 AGgaCcU c acCCUGC- 253 ccUJGCCtJ A AggAaGA 595 CCTgUuU C CUGCCuic 263 AgGGuutJ c uCUaCUG 709 gGCG~vCt C CaCCuCA 257 AGCgGCU C OUGCCUa 720 U =ACJ U uUCAG~u 293 A.AGcUGU u UJGAgCUJG 723 C~uU C AGcuccg 319 AGgAGAU A cugAgCC 735 aCCaGaG C C-j9GAGa 335 cUJGUGCtJ U TgagAAC 73 8 uGC-gCC'J c GuGaL'GG 337 Gt~cCaAU U CAcACUG 765 CaGUcGU C cGcUuCC 338 aGCUgUtJ u gAgCUGa 759 GGcCUGTJ U uCCtJGcc 359 GuGCAGU C guCcGCtJ 770 UULGcU C CCUGGAa 785 CG~cCUGtI U UCCUGCC 1353 AG'UGggU c gAaGgL;G 786 GC CLUUt U CCUGCCU 1366 UaaCAgU c CaCaACU 792 UggagGU C UCGG-AG 1367 aG~CACU C C-CACCU 794 CugGgCU L, GGAGa~u 1356a GUACUgU a CCACUcu 807 CUCgGaU a uACCUGG 1380 I I -CA" C GGG.,ugg, 833 CAaAGcU c GAcaCCC 2.388 G Ga GA cU C- AGVC,,CU 846 *:ccugGU C ACCg-uUG 1398 UGSCUGU; C A~g..

851 G a gACC U c UacC-kgC 14c02 U c U A-a SUBSTITUTE SHEET (RULE 26) 1.78 863 AgCcACIT u. CcUCU-IG 408AA Z GaX 866 GgCUUCuC 141 GAGst' c~ ZqaaGqr 867 AUE'CgUTJ u cClGagA 4 2 c cCAC =1 A C'uUUMGU 869 ,CuUcCrJ C augCAAG 1.42S aC'JgCCV u4 gGaGaG s8 AuGC-CuU Aac,-cGI 1429 Cu, ,3 CU 885 CM'ugGJ a gagGUGA 1444 aAagg=t C Agj'-aGC-A 933 ct~auAat3 c AL'uCUGG 1455r GGaAuTU C AC-aGga 936 uAaUt7ctJU CtGGuGc 1.482 AgutGuu u U g ca C 9718 UaACagU C LUArAaCU 1484 clUGuUCU u C^-CauG 980 ACagUCJ A CAaCUCU 1493 zuguGcUu L GAGAac 986 UACAaCU U LUu.CaGCU 1500 AUGA-:U c allggCC- 987 ACAaCTX U uCaG~uC i503 gGAcUaU a Al,,CAt'uc 988 c:aC7t5U u CaGCuCC i5306 tXatgtiU u AL'aACcG.

1005 ACcaGAU c CUGgaGA 1509 cucCAU C ACceGu 1006 uGaGAgU C UGggGAA 152.8 ucaU~GGU c CAGgCG- 1.023 ugG?.GGU C LUCgG.AAG 1530 Cuau~aU C AUucUGG 1025 GAGGUL'f C gGAAGGG i533 ugGUCAU u gUGGGCc- 1066 CCAC-CU c aAaauAA 1551 C A uGCCU u AGCAgcU 1092 AcuGGatJ c uCAGgC'. 1559 AGCACcU c C-IcaccU 1093 UGGacc. u CAGCCaA 1563 CUtJAugU u LrUAACC- 1125 C.CkaCUJ C uUcu(GA 1565 UAugUuU A UAPACCGC 11 63 CGaAGCU U CUUUUGC 1567 UgUuUAU A ACCGCCA 1164 GaAGCU C UuuUGCU 1584 GaAAGA3 C AgGAuAU 1166 AG=tCU u uUGcrj'CLT 1592 AgGAuAU A C-baguUA 1172 tUCCtJGuU u aaaAACC'- 1599 ACAaguU A CAgaAGG 1200 cuCuGCtJ c cUcCACA 1651 CcCaCCU C Czt'GAgC 1201 g~uGC-UU u~ UgaACAg 1661 gaAACCU u L'CCuuuG 120 AcuUMU u CACcAGu 1663 cutcCuGa 1227 GGuAcaU a CGL'GtgC 1678 AGGaCCU C agCCUgG 1228 GaAGCt)U C uUut~gCU 1680 aGCCaCU U CCLCuGg 1233 LT7CGt~uU C CgGagaG 1681 GCCaCUtJ C CtJCuGgC 1238 GUgCUGU A UGGUCCu 1684 aCUTJCCU C uGgCUgu 1264 GAaGGgU c GUgCaaG 1690 cCGGaCtJ U uCgAUcU 1267 uGAgaGU C UGC-GgAA 1691 CGGa=t u CgAUcUU 1294 AGgAgAU a CugAGCc 1696 UgCCCAU c ggGG--UGG 1295 GAggggU C UCAGCAG 1698 CggAUAU a cctUGGag 1306 GCAGACU C ugAaaUG 1737 gAGACcJ c UaCCAgc 1321 gaAGGCtJ c aGGaC-gA 1750 gGCgGCUJ c CACCUca 1334 AACCCAU c UCCuaAa 1756 gAagCCU u C^cuGCCC 1344 auCGAGCU C gAGaGt~g 1787 gaGaCAU U GUCCcCA 1351 ugAaUGU a UAAguuA 1790 GC.AUUGU u CUCuaau 1793 UgGUCCU C gGcugGA 2173 UUagagU U UUACCAG 1797 CacCAGt) C AcAUAaA 2174 Uagaguu U TUACr-AGc 1802 acCAGAU cC uggAGa 2175 agag'TCU U ACCAGCU 2.22A~~A UaGC2176 gagUUUU A CCAGCUA 8K3 CAGCAtJU U acccucAk 2183 ACCAGC' A ULTUALUG 1825 -CC-cGcU A CC-UcugC 2185 CAGCtJAU U UALUGAG 1837 CAugCCU u uAgCuCc 2186 AGCtA;t U AUUGAGU 1845 cgAgcCU A GGCCACc 2127 GC[JAUtJU A UUGAGt~a SUBSTITUTE SHEET (RULE 26) %NO 95,23225 J) 11If9.;M)O 1 179 1256 Cgt cGC=Ju 2189 UAUtAZU AGL~acC: :81AcatGAU a TWCcANT.,a Z~ c aAc u cltjgAt'G '863= cAcuUGtI A Gcc-,-CAg -1198 gcaGcCU 4 L-tJALGZXu i868 CacAGkU C AC.%UaAa 2199 GcC=C a LUgUuUru i8877 CATJGcCU u AGCagca 2200 Ucl-uccz~ CJCcAaG -iuAACJ C AAGggAc 2201 aagULtU A UGUcGGC 1912 At'AUagU a GAUcagUJ1 20 5 UGA'UTj c GGCaugA UGaAL'GU a uAACtUua 220GgAGaCr- a AgUGgcu 12 3 uGAtGcU c AgGTJc 2220 culggCAU u GuCCrUV:J i9248 tUAgAGtJ u UuaCCaG 2224 Cr7U a UCcauCC UUU u aCCaGcU 2226 UgGaUC'-7 C aC-GC-CC 1964 Ua CAU u GuCC"--za 2233 C.;GaC=J C c'GGAGg .1383 AGGAUAJ A CAAgUua 2242" I-GGCAGCU- a gCgaCC- 1996 aGCGAgAU A CUCAgcC 2248 UauCcaU C C-AUczCk 2005 t'G-gAgC'J a GCgGaCa 2254 U'CCAauU C- ACA c U A 20:3 GCtiauuU A UUGaGUA 2259 alUCACAU U CAcG-GUg 2015 UGCCcAU c GGGgugG 2260 UCkCAkU C AcGGUgc 2020 ggUGGuU c UuCtJGAG 2266 ggAAuGU C ACCAGGa 2039 g-CuGgCU a gCAG; gG 2274 ACCAGaU c CuCgaGa 2040 CuGACcJ c CuGgA-g 2279 Ga-kggGU c CGUgCAaG 2057 UGcuCCJ C CAcAUCC 2282 aAGcUGUO u ugaGcUG 206: CuaCCAU c acCgUGU 2288 UAUAaGU U allggcCU 207:' CAcuUGU A GCcUCAg 2291 caGUgGU u CuCtJGCu 2076 GtJA.GCcU C AgAlua 2321 gAAGAU C- AcAUGGG 2097 CaA~uCU U CuUGAuG 2338 UGaGACTJ c CtgccL'G 2098 CACA-'CUU C CcccCc-G 2339 GaaACcU u UCcIUUUG 21 1 5 GCCAGCtJc C GGaggaU 2341 GACcL'CU a ccaGcCu 2128 CaGC'JaU u UAuUGAg 2344 tJUucgAuJ c uuCC.AgC 2130 ccUuuu c CUGCQ-iC 2358 CCcagCUJ c UCagCAG 2145 CAQCU U cuUGAUg 2359 CUGCUUU U gaaCAGA 2152 UauUaAU u UagAgUU 2360 aaCC'U C CiuuGAA 2156 uugAUGtJ A UtXItUUa 2376 agGUGgU U clUjUCUga 2158 gALUGUATJ U UAtXJa.U 2377 gGUGgUUJ c UUCL'gag 2159 AUGTJAUU U AUUaAUjU 2378 agGgULTU c CUtAcuG 2160 UGUAUUU A UUaAUUUT 2379 UGctUUU c ucAUaaG 2162 tIAtUtJAU U aAUUUag 2380 aAgUUU a UgUCGGC 2 16 3 AUgUAUjU u AUUaatJU 2382 aUlUcUCU A UuGcCcc 2166 acUUCAU U cucUAUU 2384 allcCagU a GaCAC;A 2167 AUguAUU U alUAaU 2399 AAaCACU A UgU'GGAC 2170 uAUUUaUJ U AaUUUAg 2401 aagCUgUu UGagCUG 2171 AgUUGUU u UgcUcCC 2411 UACUGGU c AgGaUgC 2417 gAAUGGU a CkuAcGU 2691 AAuGUcU c cG-AGGcC 2418 ActJGGatT C UCAGGcc 2700 GAaGcCU u CCUgCCc 2425 cAbugGGU c gAGgGuU 2704 gacl-uCU a CCAGCcU 2426 AuuaaUU u AGAGUrtJ 271. CCC-AGCU c UcagcaG 2433 uA2GAGuU U uaCCAGc 2712 gagGucUc GGAAGGG 2434 AGAGuLU u aCCAGcu 2721 GA.GGU C gL~gCaaG 24418 G-AaGCCtJ U ccUgCcC 2724 GGuaCAU a CGuGUGc 2449 AaC-CCUU c cUgCcCC 2744 gGUGgGU c cGCGcAG SUBST[TUTE SHEET (RULE 26) %k 0 95,213225 11 1,2111195,100156 180 2 45:Z -C=gTiUC U gCU 275Z g g a=' 2455 gAagCMCU 11 CCUgC-CC 27 aga C c 1UG c 2459 C-CaCaCUJ U CCcCCCCc 2 2460 CaCaCU C C -C CCc g 27169 a2 CUGU aaA 2479 GAgACCU c UaccAGC 127-9 7 atiGAU C AUGGLICC 2480 uC-ACCgU U GUgAuCC 2803 UCAUGGU:: :zagGCg 2483 CCaaUGU c AGCcCC 26804 gguGGCU Zc u; ,G 2484 =JUUuUU c aCCAgtic 281-3 CUCC c CGC-- -c 2492 agCACCIJ C CCcCC-u 2 82.'5 aOAG'' a cAacU= 2504 CC-CACCzu A CUUtgu 2 82 c~c.z UGz=7cUGCGagg 2508 uAUcC-AU c caUcCCA 2822 9GAgccU c GGacl-u 2509 uUAgAgU U ut~aCCAG 2823 ugCcUUU a GcuCcCa 2510 tJAgAgUU u UaCCAGc 2829 cLUGGaCUi a u*aUcAt: 2520 CuuUGU U CcCAALIG 2837 kgGuGgUu .1Cuc.ga 2521. CG c aU Mu ACcctr-A 2840 UGAgaCUi C,,gCC--g 2533 UG-AugCtJ C AGguaUC 2847 CCaAugtJ AGC--aCC 2540 CAGCaGU C cgcUgUG 2853 gCAGCCU C uUauGtu 2545 GUgcUGU a UGGuCcU 2860 gCcaAGU A aCtUGuGA 2568 gtxGaAgU c UGuCaAA 2872 GCGAC'-uU a aGCcaAg 2579 auAAGuU A UGg~cU'G 2877 uuccZu a CcAuCAC 2585 cugGCaU U GUuCUCU 2899 cGgAcUX U cG-AUcUU 2588 GCaUtJGt u CUCt~aaU 2900 uuA3~ut~a a G.g=~Z 2591 UgGUuCU C UgcUCCtJ 2904 ACEU~CAU U cucEuauu 2593 cUuCt~uU U GcuCUJGc 2905 cUUCAkU c UIc~au-ug 2596 CUuUUGU u CccaaUG 2906 UjUGAUg U a UU-Xa .U a 2601 acCgUGU a UuCgUtJU 2907 UGuatU'tJ a at U U 2602 UCCaGcU a cCAUccC 2908 GAagclUU c UZUUUgcU 2607 cUcGgAtJ a UacCUGG 2909 AgcUUcu U uugcucrJ 2608 caGCAgtJ c CgCUGuG 2910 UgUaUUU a TjTaaUTj 2609 gGaAUgU C ACcaGGA 2912. UgUaUUU a UjUaaEJu-u 2620 aGG-AcCtJ c aCcCt~gc 292.2 UUgUUcU c UaaUgUC 2626 UT~uCgaU c tJUcCAGC 292.3 UUUcucU a cuggUcA; 2628 CACacU U Gu.AGCcu 2914 UgcbM"u c Uca~aAkG 2635 UUCAGCU C CgGUccu 2915 aUU~aUjU a alUfuAGA 2640 ggCCuGU U UCCUGCc 2916 UaUUcgU UccgGAG 2641 cCr-AGcU c uCaGCAG 2917 alUucgU-U u cCgGAGA: 2642 CCuGUUU C CUGCcuc 2918 UWcg=U c OgGAGAg 2653 uAcUGgU C AGGaUgC 2919 UUcUcaU a ;AC-gGucG- 2659 gaAGGG,-U CgUGCAAG 2932. ugGaC-GU C =-GgAAg 2689 CuAAuGU c UccGAGG 2933 C-aGGUCU c C-cAAggg 2941 GagACAU U GuC~cc-A 2951 CCAcgCU a CCUcUGc 2952 CAGcagU C CgcUGUG 2955 AgUgaCU c UGUGUcA 2956 UUUICCUU U GaaUcAa 2962. UcUGUGU c AGccAcU 2962 allGUaUjU u aUUAAUu 2965 UuUgAaU c AAUAAAG SUBSTITUTE SHEET (RULE 26) WO 95/23225 181 296i GcUgGc,. A gcAgAGg 1;9 6 AaUCAAU A Ai.~Xh~tj 29-15 tAgAGUU t7 'JacCkgC 2976 gAgGgLU(J U CUOUACJ 2977 kAGC'JgU u UgAgCUG 2979 uCaUUCO' C uAuUGCC- IC I( I 1).r Ifl SUBSTITUTE SHEET (RULE 26)

I

XN 0 95 232125 PC 1111139SAM) 156 182 Taole 4 Human lOAM HH Ribozyme Sequences nt. Position Ribozyme Sequence ii CAGCGTJC Ct7GCAL'GAGGCCGAAAGGCCG-AA ACUGGGG- 23 AGCAGAG CG.AUGAGGCC--AAA; C-GCCGA-k AG0tJCAG 26 AGtAGCA Ct;GAUGAC-GrCC-A.AAGC-CCGA-A AGGAGZCJ 31 GG CGATJGAC-GCCGAAAC-CCCAA AGCAGAG 34 CAACJCU Ct7GAUGAGGCC-GA AAGGCCGAA AGUAGCA AC-TCUCC A L'GAG GC SAAGC -G CAA kCU~jC.-: 48 CGAGGCJ C'jG A UG A =G AA A GG--CG AA AC-GU'UC-C 54 -CAUAC-C C CUG A G G CCGA AA GG C C %A AGG-CUGA 5Z8 GGGA CtAGGCG G-CG?.A AGCGAGG 64 Crt;GCJGG CUGAUGAGGCCGAAAGGCCGAA AGCC-kTj-A 96 GGACCAG CZMAUGAGGCcGAAAGGCGAA AGUGCGAG 102 CG-AGCAG CUGAUGAC-GCCG.A-AGGCCCG?. ACCAGGA 108 G-AGCCCC CtGAUGAGGCCGXAAGGCCGAA AGCAGCA 11.5 GGGPAACA C CAtJGAGGC'CGAAAGGCCG.AA AGCCCCG 119 UCCUGGG =UG AUG AG G C C -A AAG G C CGAA ACAGAGC 120 GUCCUGG C1UGAUGAGGCCG-AAGC'AA AACAGAG 146 G-ACACAk C G AUG AG G C -GkAG GC C G AA AUGTCTUG 152 UGAGGGG CUG AUG AG GC CGA AAG GC CG AA AC-7ACA 158 GACUUUU C UG AUG AGG CC GA.A A GG CC G AA AGGGGGA 165 GCAGGAU CUjGAUGAGGCCCGAGGCCAA ACUMtJUG 168 GGOCAG CUGAUGAGGCCGA.AAGGCC-GAA AUGACUU 185 CA=CCG CUGAUGAGGCCGAAAGGCCGAA AGCCUCC 209 GUCACAG CUGAUGAGGCCCGAAAGGCCGAA kGGUGCU 227 GCCCAAC CUJG AU GAG G CC-,AAC-GC C G AA ACUtJGGr, 230 UAUGCCC CUGAUGAGCCCAAAGGCCG-AA ACAAC~tJ- 237 C-GG-,UCUC CUG-AUGAGGCCCGAAAGC-CCGAA AU'GCCCA 248 UUUAGGC CUG-AUGAGGCCGAAAGGCCCGAA ACGGGGtJ 253 UL'-tU= CUGAUGAGGCCGAAAGGCCCAA AGGCAAC 263 CAkGGAGC CUGAUGAGGCCGAAAGGCCGAA ACL'CCttj 267 CAGGCAG CUiGALGAGGCCGAAAGGCCGAA AGCAACU 293 CAGUUCA CrJGAUGAGGCCGAAAc-GCCGAA AC-ACCUU 319 GGUUGGC CUGAUGAGGCCG--AAGGCCGAA AUCUUTCU 335 GUUUGAA CUGGAUGAGGCCG-AAAGGCCGAA AGCACAU 337 CAGUUUG CCG A UG AG GC C-AAGG C CAA AUAGCAC 338 C-CAGUt7U CUGAUGAGGCCGAAAGGCCGAA AAUAGCA 359 AGC-UGUU CUG-ALGAGGCCGuAAGGCCGAA ACUGCCC 367 :,AC-GUUrJ CUGAUG-AGGCCGAAAGGCCGAA AGCUGUU 374 GGUGAGG CUGAUGAGGCCG-AAAGGCCGAA AG,-,U 375 CCGGUGAG CUGAUGAGGCCCGAAAC-GCCCG.3k AAGGUUUT 378 ACACC-GU CUCGAUGAGGCCG e. GGCCCGAA AGGAAC-C.

386 AGUCCAG C UG AUGA G GCCG A AA C-ZCCGA.A. ACrA CGC.TU 394 CGUUCUG C U GA UG AG GC C AA G G C G AGUCCAG 420 AAGAGGG C UG A UGACGG CC GA.; AG GC CGAA AGGGGC.

425 CT-GCCAA CUG-AUG-AGGCC -AC-GCCGAA ACGGGA-G SUBSTITUTE SHEET (RULE 26) NNO 95,23225 183 4 27 G ~cu G CUZ.GAUG-AGGCC-GA-AAGGCC-GA AGAGG 3G 450 -,MAGGG L C UG A UG AG GC CG AG C C GAA AGGtCUC'J 451 CM.AGGG Ct'GAUGAGGCCAAGGCC-GA AAGG-UUC 456 GCAGCG CU'GAUGAGGCCGAAAGGCC-GAA AGGGtAA 495 CCACGGU CUJGAUGAGGCCGAAAGGCC-GAA AGGtJUGG CCCCACG CUGAUGAGGCCGAAAGGCCGAA AGCAGCA 564 UGGjC-,U CUGALGAGGCCGAAAGGCCGAA A C C CA G 921 cAt;GGU CUGAt7GAGGCCGAAAGGCCGAA AUCUCtIC 607 CAkCGAG-A CtUGAUGAGGCCGAAAGGCCGAA AU;G-U 608 GCACGAG CUGAUGAGGCCGAAAGCGAA AAUUGGC 609 C-GcA!CG-A C,:GAtIGAGGCCG-AAGGC CGAA AAA!UUGG 6112 C-C-,GcA"C CUGALGAGGCCGAAGGCCG-AA AGAAAUU 656 GUUr-k CtIGAL'GAGGCCGAAAGGCCGAA ACAZCU 657 UGU!CUC CU.GAtJGAGGCCGAAAGGCCGAA ?AAGCU 668 GGGGGCC CUGAUGAGGCCGAAAGGCCG-AA AGGUGUU 677 GACUGG CUGAUGAGGCCGAAGGCCGA. AGGGGGC 684 AGGUCUJG CUGAUGAGGCCGAAAGGCCCGAA AGCUGGU 692 CAGGACA CUGAUGAGGCCGAAAGGCCGAA AGGUCtIG 693 GCAGGAC CUG-AUGAGGCCGAAAGGCCG-AA AAGGUJCU 696 CGGGCAG CUGAUG-AGGCCGAAAGGCCGAA ACAAAGG 709 L'GUGGc-G CUCAUGAGGCCGAA.GGCCGAA AGUCGC-U 720 GGC-UGAC CUJGAUGAGGCCGAAAGGC:CGAA AGUUItJG 723 GGGGGCtJ CUGAUjGAGGCCGAAAGGCCGAA ACAAGU 735 C,-CrUAG CUGAUGAGGCCGAAAGGCCGAA ACCCGGG 738 CCACCUC CUG=UGAGGCCGAAAGGCCGAA AGGACCC 765 GGGAACA CUG-AUGAGGCCGAAGGCCGA ACCACGG 769 UCCAGGG CUGAUJGAGGCCGAAAGGCCGAA ACAflACC 770 GUCCAGG CUGATJGAGGCCGAAAGGCCGAA AACAGAC 785 GACUGGG CUGAUGAGGCCGAAAGGCCGAA AC.AGCCC 786 AGACUGG CtTGAUGAGGCCGAAAGGCCG-AA AACAGCC 792 CCUCCG-A CUGAUGAGGCCGAAAGGCCGAA ACtJGGGA 794 GGCCUJCC CUJGAUGAGGCCGAAAGGCCG-AA AGACUGG 807 CCAGG%-UG CUG-AUGAGGCCGAAAGGCCGAA ACCb'GGG 833 GGGGUUC CUG-AUGAGGCCGAAAGGCCGAA ACCtJCUG 846 CAUAGGU CtJGAUGAGGCCGAAAGGCCGAA ACGGUGG 851 GUUGCCA CUGAUGAGGC-CGAAAC-GCCGA AGGUGAC 863 CGAGAAG CUGAUGAGGCCGAAAGGCCGAA AGUCGUU 866 GGCCGAG CUGAUGAGGCCGAAAGGCCGAA AGGAGUC 867 UGGCCG-A CUGAUGAGGCCGAAAGGCCGaA AAC-GAGTJ 869 CUUGGCC CUG-AUGAGGCCGAAAGGCCGAA AGAAGGA 881 ACUGACtJ CUGAUGAGGCCGAAAGGCCGAA AGGCCUU 885 UCACACU CUGAUCGAGGCCGAAAGGCCGAA ACUGAGG 933 CCAGUAtJ CUGAUCGAGGCCGAAAGGCCGAA ACUGCAC 936 tJCCCCAG CUGAUGAGGCCGAAAGGCCGAA AUUACEJG 978 AGCtJGUA CUGAtJGAGGCCGAAAC-GCCGAA AUGGUCA 980 2 AA G C UG CUGAUGAGGCCGAAAGGCCGAAk AGAUGGU 986 CGCCGG-A CUGAUGAC-GCCGAAAGGCCGAA AGCUGUA 987 GCGCCGG CUG-AUGAGGCCGAAAGGCCGAA AAGCUGU 988 G-GCGCC-G CUJGAUGAGGCCGAAAGGCCCGAA: AAAC-CUG N 1195/001 SUBSTITUTE SHEWET (RULE 26) N N 0 9 5, 2 32 25 N 1,1105,001.5b 184 :006 'tCGUCA C ;AtUGAGGC- 'AGGCCGAA AXc.kco 2.02 3 CM= M. LGAULGAGGCCGAAAGGCCGAA ACCL'CUG C.2 CCJ =GAU7GA G GC-1G A AA G G CCG AA AGA C=C' 2.066 LUGGCUC CtJUG'G GCCGAAAGGCCGAA ACCt:GG 1092 GGGC-UGG CUGAUGAGGCCGAAAGGCCGAA ACCCCAU 1093 L:GGGCUG CUGA!GAGGC C CAAAGC-CC GAA AACCCCA UCAGCAG CUGAUGAGGCCGAAAPGGCCG-AA ?.c-CLGG 62 GCGGAG CL7GAUGAGGCCGAAAGGCCGAA AGC-C 2.16 AGCGGA CUGAUGAGGCCGAAAGGCCGAA ;~AGCr:GC .266 AGAGCAG CUGALGAGGCCGAAAGC-CGAA G; ACU 1:72 C-TtUUGCA% CUGAtC-AC-GCCGAAAC-GCClGAA k-ACCA 2.2 00 UGUGUAU CUG-AUGAGGCCGAAAGGCCGAA AC-CUGG!C 1201 UUGUtA CUGAUGAGGCC-GAAAGGCCGAA AAGCL-GG 1203 UCUUGUG CUGAUCAGGCCGAAAG-GCCGAA ALUAAC-CU 2.227 GGACACG CUGAUGAGGCCGAAAGGCCGAA AGCUCCC 1228 AGGACAC CtJGAUGAGGCCGAAAC-GfCCGAA ALG3:'-TJ C C 223 CAUACAG Ct7GAUGAGGCCGAAAGGCCG-AA ACACGA.A 1238 GGGGCCA CTJGAL'GAGGCCG.AAAGGCCGAA ACAGGAC :264 C"CGGAC CUGAUGAGGCCGAAAGGCCGAA AUCCC 1267 UUUCC-CG CUGAUGAGGCCG-AAAGGCCGAA ACAAUCC- 1294 UGC-UGGG CUGAUGAGGCCGAAAGGCC-GAA At7'UUUCU 1295 CUGCUGG CJGAUGAGGCCGAAAC-GC'-CGA AAM'=tC 1206 CACAUUG CUGAUGAGGCCGAAGGCCGAA AGUCUGC 1321 TUUCCC-CC CUGAL'GAGGCCGAAGGCCGA AG-CCUGG 1334 CUC=GGC CUGAUGAGGCCGAAAGGCCG-AA 1-344 GACACtJU CUGAUGAGGC-CGAAAGGCCGA AGCtJCGG 1351 UC=UUU CUGAUGAGGCCGAAGGCCGAA ACA=tiG 1353 CAUCCUU C UG AU'G A GGC CG AA G GC CGAjLA AGACACU 1366 AGUGGGA CUGAUGAGGCCGAAGGCCGAA AGUGCCA 1367 CAGUGGG CUGAUGAGGCCGAAAGGCCGAA AAGUGCC 1368 GCAGUGG CUGAUGAGGCCGAAAGGCCGAA AAAGUC-C 1380 AUUCCCC CUGAUGAGGCCGAAAGGCCGAA AUG-GGCk 1388 AGUCACUJ CUGAUGAGGCCGAAACGCCGAA AuTJCCCC- 1398 CUCGAGU CUGAUJGAGGCCGAAAGGCCGAA ACAGUCA 1402 AGAUCUC CUGAUGAGGCCGAAAGGCCGAA AGUGACAk 1408 CCCUCAA CUGAUGAGGCCGAAAGGCCG-AA AUCUCGA 1410 UGCCCUC CUGAUrGAGGCCGAAAGGCCGAA AG-AtUC 1421 ACAGAGG CUGAUGAGGCCGAAAGGCCG-AA AGC-GUGC 1425 CCCG-ACA CUGAUGAGGCCGAAAGGCCGAA AG,-GUrAGG 1429 CtJGGCCC Ct7GAUGAGGCCGAAAGGCCG;A ACAGAGG 1444 UCCCCtJU CUGAUGAGGCCGAAAGGCCGAA AGUGCUC 1455 CGCGGGU CUGAUGAGGCCGAAAGGCCGAA ACCCCC 1482 CGGGGGA CUGAL'GAGGCCGAAAGGC:CGAA kGAcAU 1484 CCGGGGG CUGAUG-AGGCCGAAAGG-CCAA AGAGGAC 1493 AUCUCk CUGATUGCAGGCCGAGGCCGk A C CGG 1500 UGAUGAC CUGAUGAGGCCGAAAGGCCGA'A AUCUCAU 2.503 UGAUGAU CUGAUGAGGCCGAAAGGCCGAA AC-AAUCU 1506 CAGUGAU CUG-AUGAGGCCGAAAGGCCGAA

AUGACAA

SUBSTITUTE SHEET (RULE 26) WO!( 95/23225 11( 'I1 105)5(115o 185 i5C^9 CACAGI' CUGAt;GAGGCCGAAAC-GCV.GAA AGAG :5:18 CGGCUGC CL~GAUGGC C GAAAGGC CGAA ACCACAG 153 0 CCAUJUAU CUGAUGAGGCCGAAAGGC:CGAA AC"UGC-%r3 .533 L'GCCCAU CUGAUGAGGC-GGWGGCC"GAA AtJGACU G 1551 ACGLGCJ CUGACGAG7GCCGP.AAGG-CC-AA AGGCCLG i3-99 AUAGAGG CCGAUGAGGCCGAAGGCCAA ACGUGZj 1563 G~TJUUA C UG AMJGAGC GA A AG GC C GAA AGGUACG3 1565 GCGGUUA CtJGAUGAGGCCGAAAGC-CCGAA AGAGGUA 15r67 Uc-GCGGU CUG AUG G C C AAAG G CC A AL7'GG 1584 AUTXJCUtJ CUGAUGAGGCCGAG C "GA A Aur'CuLCC 1592 UA~TJG CGALGAGGCCGAAGGCCCGAA AT=UCL .599 C~C 'GTJUG CT.JGAUGAGCCGAAAGGC'CGAA AtUt 16 .GLVCAGG Ct'GAUGtAC-GCCGAAAGCCGAA AGGCG7JG 1661 CCCGGGA CU'7GAUGAGGCCCGAAAGGCCGAA AGGUUCA 1663 GUCCCGG CGAUGAGGCCGAAAGGCCGA6A AUAGGUUJ 1678 CGA7GAA CUG-AUGAGGCCGAAAZGCCGAA AGGCCCU 1680 GCCG-AGG CUGAUGAGGCCGAAGGCC*G?%A AGAGGCC 1681 GGCCGAG CUGAtr-uGGCCGAAAGGCCGAA AAGAGGC 1684 GAAGGCC CUGATJGAGGCCGA.AGGCCG-AA AGGAAG.A 1 690 AUAUGGG CGAUGAGGCCGAAGG'7CGAA A-GCCGA 1691 ;LAAUGG CUGAUGAGGCCGAAGGCCGAA AAGGCCG 1696 CCACCAA CUGAUGAGGCCGAAAGGCCGAA AUGGAA 1698 UJGCCACC CUC-AUGAGGCCGAGGCCGAA AUAUGGG 1737 CAUGGCA CrJGATJGAGGCCGWAAGCC-AA AUGUMU 1750 GUAGGUG CUGALGAGGCCGAAXAGGCCGAA AGCUGCA i756 GGGCCGG CUGAUGAGGCCGAAAGGCCGAA AGGTJGUA 1787 UGAGGAC CUGAUGAGGCCGAAAGGCCGAA AUGCCCU 1790 GACUGAG CUGAT)GAGGCCGAAAGGCCGAA ACAAUGC 1793 UCTJGACU CUGAUGAGGCCGAAAGGCC-GAA AGGACAA 1797 UGLTAUCU CtJGAUGAGGCCGAAAGGCCGAA ACUGAGG 1802 GCUGUUG CUGAUGAGGCCGAAAGGCCGAA AUCtIGAC 1812 GGCCCCA CUGAUGAGGCCGAAAGGCCGAA AL'GCUGU 1813 UJGGCCCC CUGAUGAGGCCGAAAGGCCGAA AAUGCUG 1825 GUGCAGG CUGAUGAGGCCGAAAGGCCGAA ACCAUGG 1837 AG;UGUUU CUGAUGAGGCCGAA.AGGCCGAA AG,,GUGG 1845 CGUGGCC CUGAUGAGGCCGAAAGGCCGAA AGUGUUUt 1856 CAGAUCA CtJGAUGAGGCCGAAAGGCCGAA AUGCGtJG 1861 GACUACA CUGAUGAGGCCGAAAGGCCGAA AUCAGAU 1865 AUGUGAC CUGAUGAGGCCGAAAGGCCGAA, ACAGAUc 1868 GUCAUGTJ CUGAUGAGGCCGAAAGGCCGAA ACUACAG 1877 CUUGGCtJ CUGAUJGAGGCCGAAAGGCCGAA AGUCAUG 1901 AUGUCUU CUGAUGAGGCCGAAAGGCCCAA AGUCtIUG 1912 AUCCAUC CUGAU AGGCCGAAAGGCCGAA AUCAUGU 1922 AGACUUU CUGAUGAGGCCGAAAGGCCGAA ACAUCCA 1923 UAGACUU CUGAUGAGGCCGAAAGGCCGX\ ACALCC 1928 CAGGC-UA CUGAUGAGGCCGAAAGGCCGAA ACUUUAA 1930 AUCAGGC CUGAUGAGGCCGAAAVGGCCG-AA AGACUUU 1964 GUGGGGC CUGAUGAGGCCGAAAG-GCCGAA AUGUCUC 1983 CCAGUG CUJGAUJGAGGCCGAAAGGCCG-AA AuGuccu SUBSTITUTE SHEET (RULE 26)

I

WO 95,23 22 5 ll('l 1111).t% 01mr (I 186 2005 AG~cACLA C :GAGC--GAAAC-=--GAA Atuc 2 013 UACWA ~GLA GAC2CGGC C-AA ACGCAGC 20154 CATJACCC CLUGAUGAGG C C"-AAc-CC CGAA AtAGA .020 CCrk :GUCA-C A G=C-AA A C C CtL t 2039 C~UCUGU C',;GA UG A G CCAAG G CC G AA AGkUCL'G 2040 tUCtCG =CAGCGCAA=-A AATUC7:G 57- TLCJAtLG CMAUGAGGCC ~GAAG AGGCA' 2Q6i ACAUTUC CUCZAGAGG -CAAAGGCC GAA AUGGAG 207. C'ALIGC CUGATJGAGGCCGAA.AGCCCAA PACACAZUG 20716 GTGZ't ttAGC AGC ALUGCJAC- 2097 =wCAGG CGUAGZAAcCGAG,GUc-G 2098 CGCAG CUC-AtGAGGC---AAGGCCG-AA AAGUGUG 2115 i AGIMCCC CG'GGCAAGCA AG.;CGC 2128 TUCAGUA CtGAUGAGGCCGAACCGAA ACAGcA G 21.30 GGGUCAG CUCGAlC'GA-GCCGAAA-GCCCGAA AGACAGC 2145 UAL'CAUC CUGAI AGGCCGAAAGC-CCGAA AGGTG 2 152 AAAOACA Ct7GAUGAG-CCG? AGCCG AL'CAUCA 2156 C-AAUAAA, CUGALGAC-GCC'G c-cCCGA ACAtJAUC 2158 AUGAAUA CUjGAL'G.A~C -CCGAAAGC-CGAA AUACAUA 2159 AAUGA.U CL'GAL'GGGCC~CAA-GCCCAA AtJACAkU 2160 AAAGAA CLGA=C-GGCC-GAA CCAA AAAUACA 2162 ACAAAUG CL'GALG-A~C:-CC-GAAAGCCGA"A AUAA.AL-A 2163 AACAAAU CjAGGC"-ACGCA AAUAU 2166 AAUAACA CL-tGGCC--AGCrk AUGAA'CA 2167 AAAUJAAC CtGCAUC-AC-CCGIAAC-GC-C-AA AAUGA.AU 2170 GUAAAAU CtGACGAC-GCGAA-GCCCGAA AC-kAUG 217 GGUAAA CUGAtGAGGCCCGAAAGC-CGC-A AACAAAU 2173 CUGGUAA CUGAtLC-AGGCCGAAAC-GCCG-AA AU~ACAA 2174 GCUGGMJ CUGAUGAGGCCGAAAGGCCGAA AAtJAACA 2175 AGCtJGGtJ CUGAUGAGGCCGAAAGrCCCGAA AAAUrAAC 2176 ACUGG CtGUAG,;GCCGAAAGGCCGAA AAAAUAA 2183 CAAUAAA CUGAL:GAG-C-CCGAAAC-GCCG-AA ACtJGG' 2185 CUCAAUA CUGAUGGCCCGAAAG-GCCCGAA AUAGCUG 2186 ACUCAAU CUGAUGAG-CCGAAAGGCCGAA AAUAGCO.

2187 CACUCAA Ct3GAUGAGGCCGAAAG=CGAA AAtAC-C 2189 G-ACACtC CtJG-AtGAGC-CCCGAMGC-CGAA AtJAAALTA 2196 CAUAAAA CUGAUGAGGCCGvAAGGCCCAA ACAkCLCA 2198 UACAUAA CEJGAUGAGGCCGAAAGC-CCGA-A AGACACJ 2199 CUACAtJA CrUGAUGAGGC.C-AA=GCC-A-A AAGACAC 2200 CCCU CUGAUGAGCAAAC-GCCGAA AAAGACA 2201 GCCUACA CUGAUGAGGCCZAAAGGCCG-AA AAAAGAc 2205 =UUGC'- CtGAUGAGGCCGAAAGGCCGAA ACAUAAA 2210 TGUAUU CUtGAUGAGC--ccA;z.GcrcG AGcC-UAc 2220 AGAGACC Ct'GAUGA-GGCCGAAGGC'-C-AA AL'GtUCA "124 ~GCCAGA CUGAtGAGCCC-AAAC-C-CGA- ACZ'JAUG '26 GAGGCCA CUGALGAGCCAAAGGCC-GAA AGACCUA 2233 cGCC-=tJ CGAUGAGcCC AGGcCGAA kGGOcAG 2242 GGACUGG CUiGAtLGAC-CCG,-AAAc-GCcGL, AGCUCCG SUBSTITUTE SHEET (RULE 26) 95,23225 K( 11119.;,'5(I.

A87 2248 ;GACAZG -,:-AAG~A 2254 ucmaut C~~UAc-C~2AGC~ ACALGG 2259 GACcr.,,G GLGA 'GCA AUGUGAc 226 UGAC=Ut GX.:tC-GGC GAAAGG C CGAA AAL~U(Y.G*A 2 66 ACCUGGU C-t;G,-s:CGGCCGAAAGGC-C2AA AC7JUGA 2 274 CkACUG CGUGAtJGAGGCCGAAAGGC'GAA ACCUGG: 2279 CC':,,GUAC CuGAuGAGCrCGAAAGGCCGAA ACUGtJAC 22 82 CACCLG GUCGAUGAGGCGC-GCCGAA ACAAC'JG 2288 A~TUGUAC CUGAUGAGGC -CAAAC-GCCGAA A C UG'A 2291 tGCAGtG CUG-AUGAGGCCGAAAGGCCGAA ACAACG-U 23=.%M CCAU UGAL'GAG-GCCGAAAGGC-CAA AUCULU 233 8 CA~.UGAG CUGAUGAGCGCCGAAAGCGAA AGUCC=A 2329 CCAAUGA CUG-AUGAGCCGAGGCCGAA AAGUCCC- 2341. c-GCAAU CUG-AUL'GAGGCC-GAAAGGCCGAA AGAAGUC 2344 Gtm~GcC WUAGGc GAAAGGCCGAA AUGAGAA 2358 CUjGGGGA C'JGAtJGAGGCCGAAAGGCCGAA AGGCAGG 2359 UCtJGGGG CtJGAUGAGGCCGAAAG-GCCGAA AAGGCAG 2360 L'tCUGGG CUGAUGAGGCCGAAAGGCCGAA AAAGGCA 2376 AtJAG7AA CUGAUGAGGCCGAAAGGCCG-AA AUCACUC 2377 GAUAGPA CUG-ATJGAGGCCGAAGGCCGAA AUCACUJ 23718 CGAUAGA CUGAUGAGGC.-GAAAGGCCrGAA AAAUC 2379 CCGAUAG CtJGAUGAGGCCGAAAGGCCGAA AAAAUCA 2380 GCCGAUA CUG-AUGAGGCCGAAAGGCCGAA AAAAAL'C 2382 GUGCCG-A CUGAUGAGGCCGA.AAGGCCCGAA AGAAAAA 2384 UUGUGCC CUG-AUGAGGCCGA.AGGCC-GAA AUAGAA 2399 GVCCAUA CLUGAUGAGGCCGAAAGGCCGAA AGUGCLTJ 2401 CAGUCCA CUGAUGAGGCCGAAAGGCCGAA AUAGtJGC 2411 GAACCAU CUGAUGAGGCCGAAAGGCCGAA ACCAGUC 2417 ACCtJGUG CUGAUGAGGCCGAAAGGCCGAA ACCAUUA 2418 AACCUGU CUGAUGAGGCCGAAAGGCCGAA ?AACCAUU 2425 AUCUCUG CUJGAUGAGGCCGAAAGGCCGAA ACCUGUG 2426 AAUCUCU CUGAUGAGGCCGAAAGGCC'-GAA AACCTJGU 2433 ACUGGGU CUGAUGAGGCCGAAAGGCCGAA AUCUCUG 2434 CACUGGG CUGAUGAGGCCGAAAGGCCGAA AAUCtJCU 2448 GAGGAAU CUGATJGAGGCCGAAAGGCCG-AA AGGCCUC 2449 GGAGGAA CrUGAUGAGGCCGAAAGGCCGAA AAGGCCU 2451 AGGGAGG CUG7AUGCAGGCCGAAAGGCCGAA AUAAGGC 2452 AAGGGAG C!JGAUGAGGCCGAAAGGCCGAA AAtJAAcG 2455 GGGAAGG CUGAUGAGGCCGAAAGGCCGAA AGGAAUA 2459 UGGGGGG CUGAtTGAGGCCGAAAGGCCGAA AGGGAGG 2460 UUGGGGG CTUGAUGAGGCCGAAAGGC:CGAA AAGGGAG 2479 GCMTACA CUGAUGAGGCCGAAAGGCCGAA AGGUGtJC 2480 GGCUAAC CUGAUGAGGCCGAAAGGCCGAA AAGGUJGU 2483 C-GUGGCU CUGAUGAGGCCGAAAGGCCGAA ACAAAGG 2484 AGGUGGC- CUGAUGAGGCCGAAAGfCCGAA AACAAAG 2492 GGGUGGG CUG-AUGAGGCC-GAAAGGCC-GAA AGG?-UGGC 2504 AGAAAU-G CUGAL'G-AGGCCGAAAGGCCGAA AUGUGGG 2508 UGGCAG-A CUGAUG-AGGCCGCAAAGGCCGAA AUGUAUG 2509 CUGGCAG CUG-AUGAGGCCGAAAG-GCCGAA AAUGtJAU SUBSTITUTE SHEET (RULE 26) N 9 5 2 322.0 VC 1 1130 mJ5:) I 188 ACU=%=GAGG CG ACA--C-AA AAAUL-A.

CAUGt :G GAL'GAGGC-CGAAAGGCCGAA AaCA=GG 2533 GACCCU CUGACUGAGCGCCGAAGGCCGAA AGJGUCA% 2540 CAGACU CG A U kIGGG C. c- C r A AC M=G 2545 AUGL'CCA CUGA UGAGGC'GAAGGCCGA ACAUGAC 2568 UGGGCA CL'GAUC3AGGCCGAAAGGC-CAA kCUC---- 2579 CA6AGGCA CUG-ALCGAGGCCGAAAGc-CCGAA AGCMMG 2.585 AGAGGAC CCA~lGAGGCCSG.AAGGCCGAA %C-GCALrA 2588 ACAAGAG CAU G AG G CC G z'WAX-CC G AA AC-AGGC 2591- AGGAC-AA CUG A UG AG C- CG A A A C C G AA AC-ACAA 2593 ACA G GAC C UG A ;G A GGCCCGA AA G-CC GAA AZ-AGGAC 96 CA?..C-AG CtGU.UG AG GC:C G AA AGG -C G AA ACAAGAG 26 60 A.AUG!-- CGAUGAGGCCGAAAGCCGAA ACAGGAC 2602 GAAAUJGC CtJGAUGAGGCCCGAAAG-GCCG-AA AACAGGA 2607 CC.AGUGA CUGAUG-AGGCCGAAACGGCCGAA ALC'AA 2608 CCCAGUG CUGAUJGAGGCCGAAAGC-CC-GAA AAUGC~k 2609 UCCCAGU CU-ALCAGGCCGAAAG-GCCGAA AAAUGCA 2620 AUAGL'GC: CUGAUGAGGCCAA~c-GcCGAA AGCUCCC 2626 GCU~ck. CUGAUGAGGCCGAAAG=CCGAA AGbrrZA.

2628 W.GCUjGC CUG-AU~GAGGCClGA?.C-GC-CGAA AUAGtJGC 2635 GAACUG CUGAL'GAGGCCGXAGCGC CGAA AGCUGCAk 2640 tJGC:AGGA CtJGAUG-AGGC'CGAAAG-GC CGAA. ACEJG 264:. CtJ'GCAGG CUGACAGGCCGAAAGCCCAA AACtJGGA 2642 ACUGCAG CUGAt:GA6GGCCGAAAGGCCGAA AAACUGG 2 65 3 GGACCCU C GAtnAGGCCGAAAGCCGAA AGCACUG 2659 CUUGCAG CUG-ALGAGGCCCGAAAGGCCGAA ACCEJGA 2689 CCUCCAA CUGAUGAGGCCGAAAGGCCGAA ACCUUGG 2691 GUCCtJCC CUGAUCAGGCCGAAAGGCCGAA AUACCUU 2700 UGGGAGG CUGAUGAGGCCGAAAGGCCGAA AGLCCUC 2704 AAGCUGG CUGAtr-AGGCCGAAAG-GCCGAA AGGGAMt 2711 CCUUCCA% CtJGALGAGGCCGA.AAG-GCCG-AA AGC-~ 2712 CC=tJCC CUGALCGAGGCCGAAAC-CCGAA AAGCUGG 2721 CGCGGAU CUGAtrGAGGCGAAAGGCCGAA ACCCUUC 2724 ACACGCG CUGAtnAGGCCGAAAGGCCGAA AUGACCC 2744 CUACACA CUGALIaAGGCCGAAAGGCCGAA AC-ACACA 2750 GCUUGtJC CUGAtLAGGCCG-AAAGGCCGAA ACACAUA 2759 AGAGCGA CUGAUG-AGGCCGAAAGGCCGAA AGCUUtGUd 2761 ACAGAGC CUGAUGCAGGCCGAAAGGCCGA6A AGAGCU 2765 GGUGACA CUGAUGAGGCCGAAAGGcCGAA AGCGAGA 2769 CCUJGGGtJ CUGAUJGAGGCCGAAAGC-CCGAA ACAGAGC 2797 GAACCAU CUGATJGAGGCCGAAAGGC:CGAA AtJUGCAC 2803 UGCAGUG CUJGAUGAGGCCGAAAGC-CCGAA ACCAUGA 2804 CUJGCAGU CUGAUGAGGCCGAAAGGCC-GAA AACCAUG 2813 AGGUCAA CUGAUGAGGCCG-AAAGGCCGAA ACUGCAG 28 I5 AAAGGU-C CUGAUG-AGGCCGAAAGGCCGAA AG-ACUGC 2821 AGCCCAA CUiGAUGAGGCCGAAAGGCCGAA AGG;UCA 2 842 GAGCCCA CUGAUG-AGGCCGAAAGGCCA AAGGUCA 2823 IJGAGCCC CUGAUG-AGGCCGAAAGGCCGAA AAAGGWC SUBSTITUTE SHEET (RULE 26) 0 951,2322.1; N 1 111951f)(1156 189 289AUCACL'U CU-GAUGAG7GCZ-SAAA7'z-G AGcCAAC--CAA 28237 GU.GGGAG CUGAUGAGGCCAA-GC-G;A~ AUC.AcUU 2840 GAGGUGG CUGALGAGGC--AGACGCCG AGGAUCA 2847 GGAGGC CUwGAL'GAGCCGAAAGGCCA AGGUGc-G 2853 UJACTUCAG CUGALGAGGCCGAAcGrCGvAA G- 2860 U CCCA GrC CUGAUGAGGCCGAAAGGCC-GAA ACcr-c-G 2.87112 GU:GAGCC CUGAUGAGGCCGAAGGCC-GAA 2877 GTJGUUGU CUG-AtGAGGCCCGAAAGGCCCGAA AGC=tAU 2899 AAAAUCA Ct' GAUGAGGCCGAAAGGCCGX-% kL=C-C-- 2900 AAAAAL'C C JG AUG AG G CC GA A A= C G AA AtC-C 2904 AAAAAAA CUGAL'GAGGC~AAGGC--CAA AtuCAjA-;t 2905 AAAAAAA CJ GAUG AG G CC GA; C- C C G AA AAUC-AAA 2906 AAAAAAA CUGALGAGGCCGAAAG-GCCGAA AAAtCA 2907 AAAAAAA CUGAUCAGGCCGAAAGGCCG;LA AAAA.

2908 AAAAAAA CUGALGAGGCCGAAAGGCCC-AA AAAALUC 2909 AAAAAAP. CTGAUGAGGCCG-AAAGGCCGAA AAAAAAL7 2910 AAA CUGAUGAGGCCGAAACG-cc AAAA 2911 AAAAAAA C UGCAU GA G GCC CA A GGC CG AA AAAAAAA 2912 GAAAAAA CUGAUGAGGCCGAAAGGCC-GAA AAAAAAA 2912 UGXAAAA C'UGAUGAf-GCCGAAAGGCCGAA AAAAAAA 2914 CUGAAAA CUGAUGAGGCCGAAAGGCCG-AA AAAAAAA 2915 UCUJGAAA CUGAUGAGGCCGAAAGGCC"AA AAAAAAA 2916 CUCUGAA CUGAUGAGGCCGAAGGCCG-AA AAAAAAA 2917 UCUCUGA CtJGATJGAGGCCCGAAAGGCCG;%A AAAAAAA 2918 GUCUCUG CUGAUGAGGCCGAAAGGCCGAAk AAAAA;A 2919 CGUCUCU CUGCAUGAGGCCG-AAAGGCCGAA AAAkAA 2931 GUUGCGA CUGAUGAGGCCGAAAGGCCG-AA ACCCC~TJ 2933 AUGUUGC CUGAUGAGGCCGAAAGGCCGA'%A AGACCCC 2941. UCUGGGC CUGAUGAGGCCGAAAGGCCGAA AUG"U~C-C 2951 ACAAAGG CUGAUGAGGCCGAAAGGCCGAA AGTICUGG 2952 CACAAAG CUGAUGAGGC-CGAAAGGCCGAA AAGUCLG 2955 UAACACA CUGAUGAGGCCGAAAGGCCG-AA AGGAAGU 2956 CUAACAC CEJGAUGAGGCCGAAAGGCCGAA AAGGAAG 2961 AUUAACU CUGAUIGAGGCCGAAAGGCCGAA ACACAAA 62 UAUUAAC CtJGAUGAGGC-CGAAAGGCCGAA AACACAA 4965 CUJEUAUU CUGAUGAGGCCGAAAGGCCGA.x ACtJAACA 2966 GCUUAU CtUGAUGAGGCCGAAGGCCGA AACUAAC 2969 AAAGCUU CUGAUGAGGCCGAAAGGCCG.AA AUUAACU 2975 GUtJGAGA CUGAUGAGGCCGAAAGGCCGAJA ACUUUA 2976 AGUUGAG CUGAUGAGGCCGAAAC-GCCCGAA A-AC-CtJL' 2977 CAGtJUGA CUGAUGAGGCCGAAAGGCCrGAA AAAGCU 2979 GGCAGUU CUGALGAGGCCGAAAGGCCGAA AGAAAGC SUBSTITUTE SHEET (RULE 26) 5 2225PC] I A 156 Table Mouse ICAM HH Ribozyme Sequence tPosition Ribozyme Sequence C- AACCGTU CUCGAUGAGGCCGAAA=GCG?.A ACCAGG 23 AGC)AGAG ',UAUAC:C- AAC C GAA ACAU 6 AGGAGCA CZGALGAGGCC CAAtAGGC C AA AG-AACCA" -AG CtGA0GAGGCGAAAGGCCG-A.A AGCAGAG 34 CGACCCU CCGAUGAGGCCGAA.AGGCCGAA AL'GAGAA AGGCTIAC CUGAUGAGGCCGAAAGGCC-GPAA AGUGL'GC 48 CC-AGGT CUG-AUGAGGCCGAAAGGCCGAA AGGUCCU 54 CCAUCAC CUGAIJGAGGCCGAAAGGCCGA AGGCCCA 5Z8 GGAGCrJA CUCGAUGAGGCCGAAAGGCCGAA AGGCAUG 64 CUGCUGCG CUGAUJGAGGCCGAAAGGCCGAA AGGGGL'G 96 GGGCCAG CUGAUGAGGCCAAAGGCCGAA AGCAGAG 102 CCAGCAG CUGAUGAC-GrCCGAAAGGCCG-AA ACJGGCk :.08 GGGCCAG CUC-AUGAGGCCGAAAGGCCGA-k AGOAGAG "s AGGAGCAk CUCAUGAGGCCGAAAGGCCGAA AGAACCA 'a -19LCCUGGU CUGAUGAGGCCGvAAAGGCCGAA ACAUUCC 2.20 C-GGCCAG CUG-AUGAGGCCGAAAGGCCGIAA AGCAGAG 2.46 GGAAGCG CL'GAUGAGGCCGAAAGGCCGAA ACGACUjG 152 AGUGGCU CUGAUGAGGCCGAAAGGCCG.ZA AOAkCAGA GG'-UUUUU CL'GAUGAGGCCGAAGGCCGIL ACAGGA GCAAAAC CUGAUGAGGCCGAAAGGCCGAA ACtJUCUG 168 GGGGCAG CUG-AUGAGGCCGAAAGGCCGAA AALGGCU 2.85 CUGCACG CUGAUGAGGCCGAAAGzGCCGAA ACCCACC 209 GCCAGAG Ct3GAUGAGGCCGAAAGGCCGAA AAGUGGC 227 GCAAC CUGAUGAGCCGAGGCCGAA ACUUCUG 230 GGAGCA. CUGAUJGAGGCCGAAGGCCGAA ACACEU 237, AGUUCUC CUGAUGAGGC-CGAAAGGCCGAA AAGCACA 248 UUUAGG-A CtJGAUG-AGGCCGAAAGGCCGAA AUGGGUTJ 253 UCUUCCtJ CUGAUGAGGCCGAAMGGCCGAA kGGOAGG 263 CAGUAGA CUGAUGAGGCCGAAAGGCCGAA AAACCCU 267 UAGGCAG CtJGAUGAGGCCGAAAGGCCGA AGCCCCU 293 CAGCUCA CtJGAUGAGGCCGAAAGGCCGAA ACAGCtU 32.9 GG=CAG CUGAUGAGGCCGAAAGGCCG-AA AtJCUCCU 335 GUUCUCA CUGAUGAGGCCGAAAGGCCGAA ACAcAG 337 CAGUGUG CUGAUGAGGCCGAAAGGCCG-AA AUUGGAC 338 UCAGCUJC CLIGAUGAGGCCGAAGGCCGA AACAGCU 359 AGCGGAC GUC-AUW.GGCCG-AAAGGCCGWA AGUYGC-AC CGG(:jtJC- CTJC-AUGAGGCCGAAAGGCCGAA ?.GCCAtU 3 '4C-G-CAGG CUGAUGAGGcCCAAGGCCGAA AGG~CU 3 5GC-GGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 378 ACACGGU CUG;LUGAGGCCGAAAGGCCGAA AUGGUAG 326 AAACGAA CUGAUGAGGCCGAAAGGCCGAA ACACGGU AA CGA GAUGAGC -CCGAAAGGCC GA'A i=GGG 425 CC"C- C-AZUGAGrGCC GA;ACCGAA AGGGGUG SUBSTITUTE SHEET (RULE 26) WVO 95/23225 P( 3 JJ195Ifl0 I S 191 4 5 C c--AGGGU Ct;GAUGAGGCCGAAAGGCCA GCC 451 CAAGGA CtG3AtGAGGCCGAAAG-ZGAA AGCL'ttC 456 AGUGGCUJ =GAt:C- GCC GAAGCCA AGCtjUA 495 ACACG=t C UGAtGAGGCC-"AA A GGC C GAA At;GGAG 32.0 C -CC Crj'G A ;G A G G C CA A G G CCA AkGC:AGCA 5;64 SAUGGA CU~:A-CCAAGCACGA 921 -,kUCt3 CZGAUGAGGCC-GAAAGGC--GAA AZt:C*U 607 CAtUG3GA C'UGAUGAGGCCGAAAGGCCSAA AUCGGC' 608 Grt;GAG jGAUGAGGCCGAAAGGCCGAA AAWC-GC 609 GGCAL'A CUGAt;GAGGCCGAAGGC--AA AAUUGG 611i CGCGGCU Ct'GAUGAC,3CCGAAAC-GCCCGAA AGAAAU 656 CAGCUCA CUGAUGAGGCCGAAAGGCCGAA ACAGC'TJ 657 tCAGCUC CLtGAUG-AGGCCGAAAGGCCGAA AACAGCU 668 G G G CC- CUGAUG-AGGCCGAAAGGCCC-AA A G GCUC G 677 AGCUGG CUAGGCGAAGCA AGAGGTJC 684 AGC-ACCI CUGAUG AM~C CAAAGGC C AA AGCUGA 692 AAG-AUC- Ct7GALGAGGCCGAAGGCCGAA AAGUCCG 693 GCAGGTJ CMGAUGAGGCCGAAAGGCCGAA AGGLCC3 696 GAGGCAG cUGATJGAGGCCGAAAGGCC-GAA AAACACG 709 UtGAGGT.G CtGCAUC-AGGCCGAAAGGCCGAA AGCC-GCC 720 AGCUJGAA C'MAUJGAGGC C AAAGGCC GAA AGUCtGUA 723 C-GAGCU Ct:GAUGAGGCCGAAAGGCCGA AAAGLU 735 UCJCCAG CUGAUGAGGCCGAGGCCGA AUCUJGGU 738 CCAUC-AC CUGAt7GAGGCCGAAAGGCC-GAA AGGCCCA 765 GGAAGCG CLjGAUG-AGGCCGAAAGGCC-GAA ACG-ACUG 769 GGCAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGCC 770 UTJCCAGG CUGALC-AC-C-CCGAAAGC;CCC-.% AGCAAAA 785 C-GCAGGA CUGAUGAGGCCGAAAGGCCGAA ACAGGCC- 786 AGGCAC-G CUGAUGAGGCCGAAAGGCCGAA AACAGGC 792 CUJUCCGA CUGAUGAGGCCGAAAGGCCGAA ACCU~CCA 794 AGUCUCC CUGAUGAGGCCG-AA-AC-GCCGAA AGCCCAkG 807 CAC-GUA CtJGAUGCAGGC'-CAAAGGCCGAA AUCCGAG 833 GGGUGtC Ct7GAUGAG-GCCGAAAGGCCG.ZA AGCt=~tG 846 C.AACCGJ CUGAUG-AGGCCGAAAGG;CCGAA ACCAGGG~ 851 GCL'C-GUA CUJGAt;GAGGCCGAAAGGCCGAA AGGTJCtLC 863 CCAGAGG CUGAUGAGGCCGAAAGGCCGAA AGUGGCU 866 GGGCAGG CrJGAUG-AGGCCGAAAGGCCGAA AGGCUUC 867 UCt7CCC-G CTJGAtJGAGGCCGAAAGGfCCGAA AACGAAU 869 CJUGCAt7 CL~GAUGAGGCCGAAAC7GCCGAA AGGAAGA 881 ACCGt't3 CUGAVGAGCC GAAAGGC C GA AAC-CCAtI 885 tjCACC'c CUGAGAC-CCGAAGGCCGAA ACCAAGG 933 CC.AGAAU Ct;GATLGAGCC-GAAAGGCCGAA AUUAt7AG 936 GCCAG CUtGAUGAGCC"GAAAGGCCGAA AUG-AUUA 978 A~TJU~Tu"A CUGArTC- AC-Gr-CGAAAGGC CGAA ACUGTIUAJP 980 AAAGL'G CjGAUGAGGCCGAAAGGCC-GAA AG-ACL'GU 986 AtG CrUGAUL'GAC-GCCG- SAAAGSC C GAA AGUXUtA SUBSTITUTE SHEET (RULE 26) WO 95123225 P C '1;1119$l)01.1;o 192 105st 'CUC Z 6G C'UGAUGAGGCGAC-GC--

AUCUGGTV

1006 UL'CCCCA CUGAL'GA=GCGAAAGGCCAX

ACUCA

1023 CUTUCCG-A CUGAUGAGGCCGAAAGGCCGAA

ACCUCCA

1025 CCUC~ CtGAL'GAGGCCGAAAGGCCGAA AGAC=c'.

1066 tdUAUUUU CGAt:GAGGCCGAAGGCCGAA AGAGtGG 1092 GGCCaGA CGAUGAGGCCGAAAGGCCGAA AUCCAGI2 1093 IM G! CUG CtJGAL'GAGGCCGAAAGGCCGAA

AGGUCCA

1125 L;CAAGAA CUGAUGAGGCCGAAAGGCCGAA

AGUTJGGG

1163 CGCAAAAG CUGAUGAGGCCGAAAGGC'CGAA AGCUlUC-3 1164 AGC-AAAA CUGAUGAGGCCGAAAGGCCGAA AAGCt3UC 1166 ACAGCAA CtJGAUGAGGCCGAAAGGCCGAA AGAAGCtJ 1172 GG,-UUEJUU CUGAt7GAGGCCGAAAGGCCGAA

AACAGGA

1200 UGUGGAG CTJGAUGAGGCCGAAAGGCC-GAA

AGCAGAG

1201 CLGUUCA CUGAUGAGGCCGAAGGCG

AAGCAGC:

1203 ACUGGUG CIJGAUGAGGCCGAAAGGCC"GAA

AAAAAGU

1227 GCACACG CUGAUGAGGCCGAAAGGCCGA AUGUACO- 1228 AGCAAAA CUGAUGAGGCCGAAAGGCCGAA AAGCUL'C 1233 CUJC

T

CCG CUGAUGAGGCCGAAAGGCCGAA

AAACGAA

1238 AGGACCA CUGAUGAGGCCGAAAGGCCGAA ACAGCAC 1264 CUUGCAC CUGAUGCAGGCCGAAAGGCCGAA

ACCCC

1267 UtJCCCCAk CUGAUGAGGCCGAAAGGCCG-AA ACtJCUCA 1294 GGCUCAG CUGAUGAGGCCGAAAGGCCGAA At7CUCCTJ 1295 CUGCUGA CUGAUGAGGCCGAAAGGCCGAA ACCCCUC 1306 CAMUCA CUGATJGAGGCCGAAAGGCCGAA AGUCUGC 1321 UCCUCCU CUGAUGAGGCCGAAAGGCCGAA AGCCtUc 1334 UtJUAGGA CUJGAUGAGGCCGAAAGGCCGAA AUGGGLU 1344 CANCtCUC CUGAUGAGGCCGAAAGGCCGAA AGCUCAtJ 1351 UAACUUA CLIGAUGAGGCCGAAAGGCCGAA ACAUUCA 1353 CACCUUC CUGAUGAGGCCGAAAGGCCGAA ACCCACU 1366 AGUJUGUA CUGAUGAGGCCGAAAGGCCGAA ACUGUUA 1367 AGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGCU 1368 AGAGUGG CUGALTGAGGCCGAAAGGCCGAA ACAGtJAC 1380 CCACCCC CUGAUGAGGCCGAAAGGCCGAA AUGGGCA 1388 AGCCACtJ CUGAUGAGGCCGAAAGGCCGAA AGUCUCC 1398 GUUCUGU CUGAUGAGGCCGAAAGGCCGAA

ACAGCCA

1402 AGUUCtUC CtJGAUGAGGCCGAAAGGCCGAA AAGCACA 1408 CCUCCCC CUGAUGAG7GCCGAAAGGCCGAA AUCUCGC 1410 CCCUUCC Ct7GAUGAGGCCGAAAGGCCGAA AGACCUC 1421 ACAAAAG CUGAUGAGGCCGAAAGGCCGAA

AGGUGGG

1425 CUCtIACC CUGAUGAGGCCGAAAGc-CCGAA AGGCAGU 1429 CAGGGGC Ct'GAUGAGGCCGAAAGGCCGAA AUAGAGA 1444 UCCLUCCU CUGAUGAGGCCGAAAGGCCGAA AGCC 1455 UCCUGG,-U C(UGAUGAGGCCGAAAGGCCGAA

ACAUJUCC

1482 GGGAGCA GUGAUGAGGCCGAAAGGCCGAA AACAACU 1484 CAUGAGG CUGAUGAGGCCGAAAGGCCGAA AIAACAG 1493 GLLCUJCA CUGAUGAGGCCGAAAGGCCGAA A.GCACAG 1500 GGACCAU CU'GAUGAGGCCGAAAGGCC-GA At')TUCAtU 1503 GAAUCRAU GUGAGA-.GGCCCAAAG;.CGCCGA.. AUAGUCCi506 CGGUUCAU CUGAUGAGGCCGAAAGC-C&3.- AACAUA SUBSTITUTE SHEET (RULE 261, WO 95/23225 I W*!00 150 193 i509 ACkCGGtJ CUGAUGAGGC CG AAAGGCCGA AL'GG 1518 CGCUGG CUC- AUGAGGC CGA.ZGC CGAA ACCAUGA 1530 CCAGAAU CrUGAUGAGGCC-AAC-GCCGVz; AtUtJ2UAG 1533 GGCCCAC CUJGAUGAGGCCGAAAGGCCcGAA AUGACCA 1551 AC-UGCU CUAt'GAGGCCCG AGGCCCAA AGGC-kUG 1.559 AGGL'GGG CGALGAG-GCGAGGCCGA.A ACGUtGCU 1563 GG'-UUAUA C;C-AUGAGGCZGCAAAt~GCCGAA %C-kUAAG 1565 GCGGUUA CUC-AUWZC-CCGAAAC-GCC-GAA AACAUA 1567 LGGC-G' CUG-AUG ZGC-C-AAG- CGCCGAA AUAAACA 1584 AUAUCCU CLUGAUGAGCCGuAAGCCGAA AUC'JULC 1592 UAACUUG CUCAUGAGGC-GAA.GGCCG AUALTCC 1599 CCUUCUG CUjGAUGAGGCCGA2G-WGCCCGA AACUGU 1651 GCUCAGG CUGAUG-AC-GCCGAAAGGC03GAA AGGUGGG 1661 CAAAGGA CtJGAUGAGGCCGAAAC-GCC.A.A AGGUUUC 1663 UCCAAAG CUGAUGAGGCCGAAA.C-GCCGAA AAAGGUU, 1678 CCAGGCtJ CUGALCAGGCCGAAAGGCCGAA AGGUCCU 1680 CCAGAGG CUGAUG-AGGCCGAAAGGCCGAA AGUGGCU 1681 GCCAGAG CUC-AUrGAGGG-CCGAAAGCCGAA AAGUGGC 1684 ACAGCCA CUGAUGAGGCCGAAAGGCCGAA AGGAAGU 1690 AGAUCGA CUGAUGAGGCCCAAAGGCCGAA AGtJCCGG 1691 AAGAUCG CU'GAUGAGGCCGAAAGGCCGAA AAGUCCG 1696 CC-ACCCC CUC-ATUGAGGCCGAAAGGCCC-AA AUGGGCA 1698 CUCCAGG CUGAG GCCGAAAGGCCGAA AUAUCCG 1737 GCUGGUA CLGAUGAGGCCGAAAGG!CCGAA %CGGUCUC 1750 UGAGGUG CUGAUG-AGGCCGAAAG-GCCGAA AGCCGCC 1756 GGGCAGG CUGAUGAGGCCCGAGGCCGA-,A AGGCtJUC 1787 UGGGGAC CUGAUG-AGGCCGAAAG-GCCGAA AUGUCUC 1790 AUUAGAG CUGAUGAGGCCGAAAGGCCGAA AC.AAUGC 1793 UCCAGCC CUGAUG-AGGCCGAAAGGCCGAA AGGACCA 17 97 UUUAUGU CUGAUGAGGCCGAAAGGCCGAA ACtJGGUG 1802 UCUCCAG CUGAUGAGGCCGAAAGGCCGAA AUCtJGGU 1812 GGCCUG-A CUGAUGAGGCCGAAAGc-CCGAA AUCCAGU 1813 UGAGGGU CUGAUGAGGCCGAAAGGCCCGAA AAUGCUG 1825 C-CAGAGG CUGAUGAGGCCGAAAC-GCCG-AA AGCGUGG 1837 GGAGCUA CUGAUGAGGCCGAAAC-GCCGAA AGGCAUG 1845 GGUGGCC CUGAUG-AGGCCGAAAGGCGAA AGGCUCG 1856 AAGAUCG CUGAUJGAGGCCGAAAGGCCGAA AAGUCCG 1861 UACUGGA CUGAUGAGGCCGAAAGGCCGAA AUCAUGIJ 1865 CUGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAAGUG 1868 UtJUAUGU CUGAUCGAGGCCGAAAG-GCCG-AA ACTIGGUG 1877 AGOUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCAUG 1901 GUCC=tJ CUGAUGAG-GCCGA.CGCCGAA AGUUUA 1912 ACUGAUC CUGAUGAC-GCCGAAAGCCCAA kCUAUAU 1922 UAACUUA CUJGAUGAGGCCGAAAGGCCG-AA ACAUUCA 1923 GAUACCtI CUGATUGAC-GCCGAA.GGCCCGA AGCAUCA 1.928 CUGGUAA CUGAUGAGC-CCCGAAACGCCG-A-A ACjUEAA 1930 AGCUGGU CUG-AUC-AGGCCG-AAAC-GC A.P.A.CUCU 1964 UGGGGCuaCUGUACC GCA AU!GUCUCC 2.983 UAACUUG A AU.CC SUBSTITUTE SHEET (RULE 26) WVO 95'23225 11("I'l 150 194 1996 G=CAG CUGAUGAGC AAGCC AA ALIUC =7'Z 2005 G-GUCCGC CU G AU-AGG-C C GA AAGGC C GAA A GC-UC CA 2013 UACUCAA C UGALU'GA G GC GA AA GC GA.CA AAAUAC-C 2015 CCACCCC CUGAUGAGGCC-AAGGCCGAA AUGGc-c 2020 CLCAGAA GrCM AGGCC C.AGGC CCA-A AACCLAC 2039 CCtJCtGC CUjGAUGAGGCZCAAAC-GCCCZAA AkGCCAC 2040 CCUCC-AG C'JAUAGCCAGGC AA:CC AA AGGU-CAG 2057 GGAUGUG CUG-AL'GAGGCC GAA.GC-C G AA AC-GAGCA 2061 AC-XCGGU CUJGAU-AGGC- CAAAGGC CGAA AUGGtJAG 2071 CUGAGGC Ct7GAUGAGG0C C-AAAGGC C AA ACAAGUG 2076 UAGCUCtJ CUGAL'GACGGCCCAAAG-GCC-GAA AGGCUC 2097 CAUCAAG CUGAUGAGGCCGAAAGGCCGAA AGAGZUUG 2098 CGGGGGG CUT i.7GAC-GC CGAAAGC CCGA A AA(YGUTG 2115 AUCCUCC CU M-UGAGGCCGAAAGGCCGAA AGCU[GGC 2128 CUCAAUA CUGAUGAGGCCGAAAGGCCCGAA AUACUG 2130 GAGGCAG CUG-AUGAGGC CAAAtvCl-c-CCGA AA.C-kC-G 2145 CAkUCAAG CUAUGAC-GCCGAAAC-GCCaAA AGAGUUG 2152 AACUCUA CUGAUGAGGCCG-AAAGGCCCGAA AUUAAUJA 2156 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA; ACAUU~k 2153 AUUTAUA CUGAUGAGGCCGA.GGCCGA. AUACAkUC 2159 AAUtJAAU CUGAUGAGGCCCAAAGGCCGCAA AAUACAU 2160 AAAUUAA CUG-AUGAC-GCCG-AAAG-GCCG.A-A AAAUACAk 2162 CUAAAUU CUGAUGAGGCCGAAAGGCCG-AA AUAA 2163 AAUUAAU CUGAUGAGGCCAAGGCCGAA AUACAU 2166 AAUAGAG CUGAUGAGGCCG-AAAGG%-CCAA AUGAAGU 2167 AAUtJAAU CUGAUGAGGCCCAAAGGCCGAA AAUACATJ 2170 CtYAAAUU CUGAUGAGGCCG AAGC-CCGAA AUAAAUA 2171 GGGAGCA CUGATJGAC-GCCAAAC-GCCCGA AACAALtJ 2173 CUGGtJAA CUGAUGAGGCCG-;lAAGC-CCCAA ACUCUAA 2174 GCUGGUA CUGAUGAGGCCGAAAGGCCGAA rUCUCUA 2175 AGCUGGU CUGAUr-AGGCCG-AAAGGCCCGAA AAACUCU 2176 UAGCUGG CUGAUGAGGCCGAAAGGCCGAA AAA.ACUC 2183 CAAUA CUGAUGAGGCCGAGCGCCG-.'. ACUGGtJ 2185 CUCAAUA CUGA UGAGGCCGAAZ-AC-CCCG-Z-: AUAGCLIG 2186 ACUCAAJ CUGAUGAGGCCGAA.AGCCC-AA AAUTAGCU 2187 UACUJCAA CU GAUGAGGCCGAAAGGCCGA.A AAAUAC-C 2189 G.3GJACUC CUG-AUGACGCCG-AAAGGCCGAA AUAAAUA 2196 CAUCAAG CUGAUGAGGCCGAAkAGGCCGA-A AGAGUtJG 2198 AACAUAA CUGAUJGAGGCCG-AAAGG-CCAA AGGCUGC 2199 AUAAAC-A CUGAUG-AGGC,-CAAAGGCCGazA ?.AGGGC 2200 CtJUGCAU CUGAUC-AGGCCCGAAAGGCCGI. AGGAAGA 2201 GCCGACZA CUGAUGAGGCCG-AAAGGCC G AAAACTU 2205 tJCAGGCC CUGAGGCC-A.AAGG-CC-AA ACAUAA.A 2210 AGCCACU CUGAUGAGGCCGAAAGGCCA AGUCUCC 2220 AGAGAAC CUGAUGAG-GCCGAAAGC-U. AUGCCAG 2224 GGAUGGA CUGAUCAC-GCCG;AAGGCCCAA ACCtGAG 2226 GCGGCCU UGCAGc~ AGAUCCA.

2233 CCUCCAG CUAGCCC aGucAs 2242 GTUCCGC CUAGC-CCACCC.:AACUCCA't SUBSTITUTE SHEET (RULE 26) WVO 95/23225 11CIA 11951100150 195 2248 UC-GGACG CtGAL'GGGCCGAAAGC-CCGAA AUGGAUA 2254 UCAkGUGU CCEGAUGAGGCCG-AAAGGCCGAA AAUUGGA 2259 CAkC CGU G C UG A UGA G G C CG AA G G C C GA AUGUGAU 2260 GCACC-GU CUGAUGAGGCCGAAAGGCCuA.A AAUGUGA t2 66 UCCtJC-CGU CUGAUGAGGCCGAAAGGCZ-GAA ACAUUCC 2274 UCUCCAG C UG AL'G AG G CC G AA AGG C CG; AUCUGG-.U 2279 CUUGCAC CUGAUG-AGGCCAAGGCCGAA ACCrCU.UC 2282 C?%GCLCA CiAU G A G G C CG AAG C C G A ACAGCUU 2288 kC-GC~kU CCG A UGA G G CCA A A GGC GA A ACUUAUA 2291 AGC-kGA.G CCUGAUC-AGGCCCGAAAGGCCGAA kCr~kCUG 2321 CCCAUGU CUC-AUC-AGGCCGAAAGGCC GAA AUCUUUC 2338 CAGGCAG CtJGAUGAGGCCGAAAGGCCGAA AGUCUC.A 2339 CAAAGGA CtJGAUG-AGGCCGAAAGC-CCGAA AGGtJUUC 2341 AGC-CUGG CUGAtJGAGGCCGAAAC-GC"CAA AGAGGUC 2344 GCtJGGA A CGUGAC-CCAAAC-GCCG-A-A AUCG-AAA 2358 CUCCGA CUGATJGAGGCCCGAAAGGCCG-AA ACUGGG 2359 UCGU CUAGAGCAAAGGCCGA-k AAAGCAkG 2360 UtJCAAAG CUGAUGAGGCCGAAAGGCCG-AA AAGGUU 2376 UCAGAAG CUG-AUGAGGCC-GAAAGGCCGuAA ACCACCtJ 2377 CUCAGAA CUGAUGAGGCCGAAAGGCCGAA AACCACC 2378 CAGUAGA CUGAUG-AGGCCGAAAGGCCGAA AAACCCU 2379 CUUMAUGA CUGAUGAGGCCGAAAGGCCGAA AAAAGCA 2380 GCCGACA% CUGAUGAGGCCGAAAGGCCGAA AAAACUU 2382 GC-GCAMA CUGAUGAGGCCGAAAGGCCGAA AGAGAAU 2384 UtJGUGUC CUGAUGAGGCCGAAAGGCCGAiA ACL'GGAU 2399 GUCCACA CUGAUGAGGCCGAAAGGCCGAA AGUGUUU 2401 CAGCUCA CUGAtJGAGGCCGAAAGGCCGAA ACAGCUIJ 2411 GCAUCCU CUC-AUGAGGCCCGAAAC-GCCGAA ACCAGUA 2417 ACGUAUG CUGAUGAGGCCGAAAGGCCGAA ACCAUUC 2418 GGCCUGA CUGAtUGAGGCCGAAAGGCCG-AA AUCCAGU 2425 AACCCUC CUGAUGAGGCCGAAAGGCCGAA ACCCAUG 2426 AAACUCU CUGAUG-ACGCCGAAAGGCCGAA AAUUAAU 2433 GCUGGUJA CUGAUGAC-GCCG-AAAGGCCGAA AACUCUA 2434 AGCUGGU CUGAUGAGGCCGAAAGGCCGAA AAACUCU 2448 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGCUUC 2449 GGGGCAG CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 2451 AGGCAGG CUGAUGAGGCCGAAAGGCCGAA AACAC-GC 2452 GAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGG 2455 GGGCAGG CUGAUGAGGCCGAAAGGCCGAA AGGC=UC 2459 GGGCGGG CUGAUGAGGCCGAAAC-GCCGAA AGUGUGG 2460 CGGGGGG CUGAUGAGGCCGAAAGC-CCGAA AAGUGUG 2479 GCUGGtJA CUGAUJGAGGCC-GAAGGCCGAA AGGTJC 2480 GGAUCAC CUGAUGAGC-CCGAAAGGCCGAA ACGGUGA 2483 GGUGGC CtJGAUGAGGCCGPIAAGGCCGAA ACAUUGG 2484 GACUGGU CUGAUGAGGCCG-AAAGGCCzAA AAAAAAG 2492 AGGUGGG CUGAUGAGGCCGAAAGGCCGA.A AGGUGG-U 2504 ACAAAA G CUC-AUGA-GC-CG.LAAGGCCGA; A AGGUGGGC 2508 UGGGAUG CUAGC-C A--CA UCGGAUA 2509 CUGGCUA-A CUGAUGA-C-G--CAA-C-CCGA.A ACUCU A SUBSTITUTE SHEET (RULE 26) m NVO 95/23225 11C1,111B95100150 196 2 5 10 GCUGG7JA CtTGAUGAC-GC C CAAAGGC CGAA AACJCUA 2520 C-tUGGG CUGAUGAGGCCGAA.GGCCGAA ACAAAAG 2521 UGAGGGU G AUG-AGGC CGC-AGGC C-A AtGCt:G 2533 GAUACCU CrJGAUG-AGGC C GAAGGC CGAA AGCAUCA 2540 CACAGC G CtJGAUGAGGC CGA AA GGC C GA ACUjGCUG 2545 AGGACCA CUGAUG ACG-C CAAAG!-C GAA AcAGCAC 2568 UUUGACA CtG-AUGAGGC CGA-AAGGC CCGAA ACUTUCAC 2579 CAGGCCA UCGAUC-ACGrCCC-GAAGGC C GA AACTUAU 2585 AGAGA-AC CUG-AUGAG .CCCGAAA-GC -GAA AUGCCAG 2588 AriMG-AG CUGAUGAGGCCGAV.Gc-CCG-AA ACA!AUC 2591 AGCAGCA CjGAUCAGGCCGAA.AGGCCCGAA AGAACCA 2593 GCAGAG.CC CUG-AtGAGGCCGAA.AGC-CCGAA AAAC-AG 2596 CAtJUGGG JUGAUGAGGCCGAAGGCCGAA ACAAAAG 2601 AAACGZAA CUGAUC-AC7GC CGAAA GGC CGAA ACACGGU 2602 GGCGAUG-G CUtGAUGAGGCCCGAAAGGCCGAA AGCUGTGA 2607 CCACGGJA CUC- AtGAC-GC C GA A.:GGC C GAA AUCCGAG 2608 CACAC-0,G CUC-AUG AG GCC GAAAC-GC CGAA ACrwGCUG 2609 LUCC-UGGU CUG-AUGAGGCCGAAAC-GCClGA ACATJtCC 2620 GCAGC-GU CUCAUGAGGCCGAAAGGCCGAA AC-GtJCCtJ 2626 GCUGGAA CUGAUGAGCGCCGAAA-GCCGAA AL'CCAAA 2628 Ac-GCt3AC CUC-AUC-AGGCCGAAGGCCGAA AGUGUGC 2635 AGGACCG CU'GAUGAGGC CGA-AAGGC CGWAA ACCuA 2640 GGCAGGA CUGAUGAGGCCG-AAAGGCCGAA ACAGGCC 2641 CJUCA CUGAUGAGGCCCGAAA2GGCCGAA AGCUGGG 2642 GAGCAG CUCAtJGAGGCCGAAAGGCCGAA AAACAGG 2653 GCAUCGUJ CCUGAUGAGGCCGAAAG-GCCGAA ACCAGUA 2659 CtJUGC-AC CUGAUG-AGGCCGAAGGCCGA?. ACCC 2689 CCUjCGGA CUC-AUr-AC-GCCGAMAGGCCGAA ACAkUUAG 2691 C-GCCUCIG CUG-AUGAC-GCCCGAAC-GCCGA?. AGACAUU 2700 GGGCAC-G CUGAU GAGGC C CuXMC-Gf-CGA A AGGCUTJC 2704 AG=CGG CUGAUGAGGCCCGAAAG-GCCGAA AGAGGUC 2711 CtJGCUG-A CUC-AUGAGGCCGAAAGCGCCGAA AGC-UGGG 2712 CCCUUCC CUGAUGAGGCCGAAAGGCCGAA AGACCtJC 2721 CrJUGCAC CUGAUGAGGCCGAAAC-GCCGAA ACCCUUC 2724 GCAC.'A.CG CUGAUGAGGCCG-AAACGCCGAA AUGtJACe, 2744 CUGCACG CUGAUGAGGCCGAAAGGCCGAA ACCCACC 2750 GGUACUC CUGAUGAGGCCGAAAG-GCCGAA AUAAAUA 2759 AGAUCGA CUGAUGAGGCCGAAA.GGCCGAA AGUCCGG 2761 CrGC-GGU CtJGAUGAGGCCG-AAA-GCCGAA AGGUCCU 2765 ACGGCA CUGAUG-AGGCCGAA.AGGCGAA AGrCAAAA 2769 CCLTGtJ CUG-AUGACGCCGA.AAC-GCCGAA ACAGACU 2797 C-C-ACV.AU CUG-AUG-AGGCCG-A.AAC-GCCGAA AUUjUCAU 2803 CC-CCUC-G CUJGAUGAGCCCAAAC-C-CCGAA ACCAUGA 2804 CUGCACG CUGAUGAG7GCCGAAAGGCCGAA ACCCACC 2813 G=GCAG CUGAUGAGGCCG-AAAGGCCGAA ACCGGAG 2815 A.2GMUG CUGA;UC-Ac-GCCG-A.C-GCCGAA AGACUGU 2821 CUCC-AG CUCAUCGA:C-GCCG ZLC-GCC-GA-A ACCGUCAG 2823 C-C- UA CCC 3GCGA. .2.C-A SUBSTITUTE SHEET (RULE 26) WO 95123225 1-97 2829 AUGAUUA CUGAtJGAGGCC-GAAGGCCGA-A

AGUCCAG

2837 UCAGAAG CUGAUGAGGCCG-AAAGGCCGAA

ACCACCU

2840 CkGGCG CtJGAUGAGGCCGAAAGGCCGAA

AGUCUCA

2847 GGUGGCtJ CUGAUGAGGCCGuAAGGCCGAA

ACAUUGG

2853 AACAtJAA CUGAL'GAGGCCGAAAGGCC-GAA

AGCUGC

2860 UCACAGU CUGAUGAG-GCCGAAAGGCCGAA ACtJUGGC 2 8-'2 CrJUGGCU C'JGAUGAGGCCGAA.GGCCGAA kAG%-,CC 2877 GUGAUGG CUGAUGAGGCCGAAAGGCCGAA

ZAGCGAA

2899 AAGAUCG CUGAUGAGGCCGAAAGGCCG?%- AAGUCCG-l 2900 AAAACU CUGAUGAGGC CGAAAGGC CGAA AAAUUAk 2904 AAUAGAG CUGAUG-AGGC CGAAAGGC C GAA AUGAAGU 2905 CAAUAGA CUJGAUGAGGCCGAAAGGCCGAA

AAUGAAG

2906 tJAAUAAA CUGAUGAGGCCGAAAGGCCG-AA

ACAUCAA

2907 AAAUtJAA CEJGAUGAGGCCGAAAGGCCGAA

AAAUACA

2908 AGC.AAAA CtJGAUGAGGCCGAAAGGCCGAA

AAGCUUC

2909 AG-AGCAA CtJGAUGAGGCCGAAAGGCCGAA

AGAAGCU

2910 AAAUUA.A CUGAUGAGGCCGAAAGGCCGAA AAAUACA 2911 AAAU- L 3AUGAGGCC-GAAAGGCCGAA AAAUACA 2912 GACAUUA CUGAUGAGGCCGAkAAGGCCGAA AGAACAA 2913 UGACCAG CUG-AUGAGGCCGAAAGGCCGAA

AGAGAAA

2914 CUUJAUGA CUG-AUGAGGCCGAAGGCCGA AAAAGC-k 2915 UCUA.U CUGAUGAGGCCAGGCCGA AAUAAT 2916 CtJCCGCGA CtJGAUGAGGCCGAAAGGCCG-AA ACGAtJA 2917 UCUCCGG CUGAUGAGGCCGAAAGGCCGAA AACGAAU 2918 CUCUCCG CUGAUGAGGCCGAAAGGCCGAA

AAACGAA

2919 CGACCCtJ CUGAUGAGGCCGAAAGGCCGAA

AUGAGAA

2931 CUUCCGA CUJGAUGAGGCCGAAAGGCCGAA ACCUCCAk 2933 CCCUUCC CUGAUJGAGGCCGAAAGGCCGAA

AGACCUTC

2941 UGGGGAC CUGAUGAGGCCGWAGGCCGAA AUIGUCUC 2951 GGAGAGG CUGAUGAGGCCGAAAGGCCGAA AGCGUGG 2952 CAkCAGCG CUGAUGAGGCCGAA.GGCCGAA ACUGCtJG 2955 UGACACA CUGAUGAGGCCGAAAGGCCGAA AGUCACU 2956 UUGAUUC tJGAUGAGGCCGAAAGGCCGAA AAGGAAA 2961 AGUGGCU CUGAUGAGGCCGAAAGGCCGAA ACACAGA 2962 AAUUAAU CUGAUGAGGCCGAAAGGCCGAA AAUACAU 2965 CUUUAUU CUGAUJGAGGCCGAAAGGCCGAA AtJUCAA 2966 CCUCUGC CUGAUGAGGCCGAAAGGCCGAA AGCCAGC 2969 AAAACUU CUGAUGAGGCCG-AAAGGCCGAA AUUGAUU 2975 GCUTGGUA CUGAUGAGGCCGAGGCCGA.

AACUCUA

2976 AGtJAGAG CUGAUGAGGCCGAUAGGCCGAA AACCCUC 2977 CAGCUCA CUGAtUGAGGCCGAAAGGCCG-A-A ACAGCUU 2979 GGC.AAUA CUGATUGAGGCCG-AAAGGCCGAA AGAAtJGA K1,1/l1195/00156 SUBSTITUTE SHEET (RULE 26) Table 6 1 lUman ICAM nt.

Position 86 34'3 635 653 782 920 1301 1373 1521 1594 2008 2034 2125 2132 2276 2810 Hairpin Ribozyme/Suibstrate Sequences Hairpin Ribozyme Sequence Substrate

GGGCCGGG

(GA(;LJGCG

CC2CAUCAG

GCCCUUGG

UGUUCUCA

AGACUGGG

CUGCACAC

ACAUUGGA

CCCCGAUG

AUGACUGC

CUGUUGUJA

ACCCAAUA

UUCLJGUAA

GGUCAGUA

GGGUUGGG

ACCUGUAC

AAGGUCAA

AGAA GCUG ACCAQAGAAAC-ACACGUUGUCGUACAUUACCUGGLJA AGAA CG(C ACCAGAGAAACACACGJtJGIJ;GII ACAIJIIACC(J(GGUIA AGAA GUUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAA GCAG ACCAQAGAAACACACGUUGUGGUACAU3UACCIJGGIJA AGAA GCUC ACCAGAGAAACACACGUUGUGGUACIAUUACCUGGIJA AGAA GCCC ACCAGAGAAACACACGUUGUIGGUACAUUACCUGGLA AGAA OCCO ACCAGAGAAACACACGUUGUGGUACALJUACCUGGUA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGJA AGAA GCUA ACCAGAGAAACACACGEJUGUGGIJACAUUACCUGGUA AG-AA GEJAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAA GCAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUJUACCUGGUA AGAA GCAG ACCAGAGPAACACACGUUGUGGUACAIJUACCUGGUA AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUIJACCUJGGUA AGAA GIJAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUIA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUJACCUGGJA.

CAGGA GCC CCGCU CC AAACU CC CUGCG GCC GAGC(J GULJ GGGCLJ GUU CGGCU GAC CAGCA GAC CCACU GCC UAGCA 0CC AUACA GAC UUGCU GCC CCACA GAC CUGCI3 GUC CUACU GAC GUACA GUU CUGCA GUC

C('CGG(CC

CGCAC[J(C

CUGAUGGG

CCAAGGGC:

UGAGAACA

CCCACUCU

GUGUGCAG

UCCAAUGU

CAUCCGGM

GCAGIJCAU

UACAACAG

UAUUGGGU

UTJACAGAA

UACUGACC

CCCPACCC

GLJACAGGU

UUGACCUrJ Table 7 Mouse ICAM nt.

Position 76 164 252 284 318 447 804 847 913 946 1234 1275 1325 1350 1534 1851 1880 Hairpin R ibozyme/Subsi rate Sequences Hairpin Ribozyme Sequence

GGGAUCAC

UGJAGGAAG

UCACCUCA

GCACAGCG

AAGCGGAC

AGAGCUGG

UCUCCUGG

UCUACCAA

AGGAUCUG

AAGUUGUA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AAA

AGAA

GUGA ACCAGAGAAACACACGEJUGUGGUACAUUACCJGGUA GUUC ACCAGAGAAACACACGUUGUGGUACAUIUACCUGGUA GCUU ACCAGAG-AAACACACGUUGUGGfJACAUUACCUGGUA OCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGrJA GCAC ACCAGAGAAACACACGUUGUGGtJACAUUACCUGGL~A GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGG!JA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGIJA GUUA ACCAGAGAAACACACGUUJGUGGUACAUUACCUGGUA GUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAG AiCCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCtJGGUA GUAG ACCAGAGAAACACACGUUGUGGUAC-AUUACCUGG(JA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA Substrate UCACC GUU GUGAUCCC GAACU GUU CUUCCUCA AAGCIJ GUll UGAGCUGA CAGCA GUC CGCUGUGC GUCCA GUC GUCCGCULJ CCGCG GAG CCAGCUCU AUGCC GAC CCAGGAGA CCACLJ GCC UUGGEJAGA UJAGCG GAG GIAGAUCCU UAACA GUC UACAACUU AGACG GAC UGCUUGGG CAGCA GAC UCUGAAAU CUGCA GAC GGAAGGCA CUGCU GCC CAUCGGGGG UGGCA GCC UCUUJAUGU CUACA CC CGGUGGAC ACGCU GAC UUCAUUCU CCCAAGCA AGAA AUUUCAGA AGAA UGCCUUCC AGAA CCCCGAUG AGAA, ACAUAAGA AGAAL GUCCACCG AGAA AGAAUGAA AGAA Table 8 Rat ICAM Hairpin Ribozyme/Substrate Sequences nt. Hairpin Ribozyme Sequence Position Substrate 59 84 295 329 433 626 806 849 915 1182 1307 1357 1382 1858 1887 2012 2303 2539 AAAGUGCA AGAA OCAG ACCAGAGAAACACACGUUGUGGUAC-AUUACCUGGIJA GGAGCAGA AGAA GCAU ACCAGAGAAACACACGtJUGUGGUACAtJUACCUGGUA GGGAUCAC AGAA GCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACAGUG AGAA GCUG ACCAGAGAAACACACGUUGEJGGUACAUUACCUGGUA AAGCCGAG AGAA GCGtJ ACCAGAGAAACACACGUUGrJGGUACAUUACCUGGUA UUCCACCA AGAA GCGC ACCAGAGAAACACACGUUGUGGtJACAUUACCUGGUA CAUUCUUG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCUCCAGG AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUAL UCCACUGA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACUUACCUGGUA AGGGUCUG AGAA OCCA ACCALGAGAAACACACGUUGUGGUAC-AUUACCUGGtJA ACCUCCAA AGAA OCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGUAAGAL AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGCUUUCC AGAA GCAG ACCAGAGAAACACACGUUGUGGUAC-AUUACCUGGEJA UCCCGAUA AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCCACCA AGAA GUAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGIJA AGPAGGAA AGAA GCCEJ ACCAiGAGAAACACACGUUGEJGGUACAUUACCUGGUA GAGUUGGG AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAULJA.CCUGGEJA AGACUCCA AGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCUCCCAC AGAA GCUT) ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCU CC AUGCU CC UCGCC GUIJ CAGGA GAC ACGCA GUC GCGCU CC UCACU GUU AUGCU GAC CCACU CC UGGCG GAC CUGCG CC CAGCA GAC CUGCA CC CCGCJ CC CUACA CC AGGCU GAC ACACU GUC CCACA CC AAGCIJ GUi)

UGCACUUU

UCUGCLICC

GUGAUCCC

CACUGUGC

CUCGGCULT

UGGUGGAA

CAAGAAUG

CCUGGAGA

UCAGUGGA

CAGACCCUJ

UUGGAGGU

UCUUACAU

GGAAAGOA

UAUCGGGA

UGGUGGGC

UUCCUUCIJ

CCCAACUC

UGGAGUCU

GUGGGAGG

WO 95/23225 201 Table 9: Rat I0AIM HH Ribozyine Target Sequence P11 1395/0() nt Position U1 23 26 31 34 48 54 58 64 96 1 02 108 119 120 146 2.52 i58 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386 IM Target sequence

GAUCCALAU

GCUGACUU

GAACUGCU

CCUCtTGI-tJ Ct3GAAGCU

CUCAAGGU

GAGAACCTJ

CCCCGCCTJ

CCGTJGCCtJ CAAUGGCrJ CCUCtJGCU

CUJCCUGGTJ

GACUGCU

UCCtJACCU GACACUGtJ

GUTIGUG-AU

CCAGACCtT ACCCGG-tJ AUUTJCUUtJ

GAAGCCUU

GGGTJGGAU

CAGCCCCU

GACCAAGTJ

CAAGCUGTJ

CUGAAGCtI

GGCCCCCLI

CACtGCCTJ

GAGCCAAU

G-AAGCCUU

GAAGCUCTJ

CGGAGGAU

ACUGUGCU

UGUGCtJAU AAGCUCUUt CAkCGCAGU C.AAtJGGCU

UUACCCCU

AGAAGCCU1

ACCCACCU

CGCEJGtTGtI

C.ACACUGA

CULUCtCUA tUUCCUCtU

CUGGEJCCU

AGAUATJAC

CAAGCCCC

GGCCUGGG

CCUGAGCC

L7AGCUTCCC

CAACCCG;U

CtJGGtJCCU

CUGGUCGC

GGGGAACU

tICUtJCCCA

CCCAACUC

CCCGGGCC

GGAACUCC

CACCUCAA

ACGAGUCA

ctUccccc CtJGCCUCG

CGUGCAGG

ATJUCGACC

ACUGUGAA

GUGGGAGG

GACACCCC

CCUUAGGA

AGUGGAGG

UCtJCATGC CtGC-CUCG

CAAGCUGA

ACAAACG-A

UGAGAACt3 UGGUCCtJC

AAGCUGAG

CtJCGGCUU

CAACCCGU

ACCCACCU

CCUGCCUC

ACAGGGtJA

UUGGAGCU

nt Position 394 420 425 427 450 451 456 495 510 564 592 607 608 609 611 656 657 668 677 684 692 693 696 709 720 723 735 738 765 769 770 785 786 792 794 807 833 846 851 863 866 HE Target Sequence G13GGC-U Gcr=CCCCU tCCCGUU

?.AGAACCU

GGGUJACUU

CUCGCIU

GCCACCAU

GUGCUGCU

GuAAALGU G -JAU

GAGCC.AAU

AGCCAAUU

GC-C..AUUU

CAAUUUCU

GUCAkCUGU UCAkCU~tyU

GACUGCC

GC'ACCCCtJ AGGrC-m-tJ

CCAGACCU

CGGACrUEY GCCUGTj-U CAkGCAT.7EJ

CUACAACIJ

CAACUULJtJ CtJCCUGGU UCCtJGCCtJ ACrJGJGCU 1 UCUUGUGU I

CUGUGUU

AGGCCUGUI

GGCCUGUUTI

CUJCCUGGU

TJCCtJGCCtJ GCtJCAGAUJ

CCUGGC-G-UT

CUG-ACAGUT

GCLTCACCU I

CAAUJGGCUJ

CCAIUGC

4

'TJ

*CtJG-AACAG ,Ca'-GCGCAk

='GCCACC

AkAAAACCA-- AUCC7UGC:G

C-CCAGC-C

UGCCA:CZ%

ACJGtGTJA

CGUGGGAA

CCAACCAC

ACCAGGGA

tCjCAUC-C UC.:kJGCr)U

AUC-CWJC-A

C.

2

LAGAAUG

AAC-AAUGUJ

UJUCCt7CtUt

CCAGCGCA

cCGACLj

-CAACUCC

G-ALCUUCC

CUGCCUCU

CCCCtJCAC

UUCAGCUC

AC-CUCCCA*

C-GGGtGGA

UGAGAACU

CCU=-G AA.G

UCCUGCCU

CC7JGCCUC CtJC-GtJCGC

UGAAGCUC

LCCUC-GA

C-GAGACUA

AUMUUUG

UAC-CAGCtJ

C?-A.CCCGU

C=CUGC-A

SUBSTITUTE SHEET (RULE 26) P1CIA/B95/OO 156 WO 95/23225 202 867 869 881 885 933 936 978 980 986 987 988 1005 1006 1023 1025 i066 1092 2.093 1125 1163 1164 1166 11.72 1200 1201 1203 1227 1228 1233 1238 1264 1267 1294 1295 1306 1321 -,334 1344 1351 1353 1366 1367 1363 1380 1388 1398 1402 1408 GACCACCU C CCCACWA Ct7CUrUCCU C tJTGCGAAG AAUGGCUEJ C AACCCGUG CACCAAGt A ACUJIMAA UGUGUATJ C GEJUCCCAG GCAGAGAU U UUGTJGUCA UUGAGAAtJ C UACAACUU G-AGAAUCtJ A CAACUTJtJJ CtIACAACTJ U 'UrtCAGCtJC UJACAACUUT U UCAG-CCC ACAACUUU U CAkGCUCCC UtJGUGAU C GUTGGCGUC GUGGGAGU A TUCACC.A CCGGAGGL7 C UCAGAAGG GGAGGUCTJ C AAAGGGG CCtJACCU U GUEJCCCAA AGAGGGGU C UCAGCAGA AGGGGAAU C CAGCCCCU CCCCAACtJ C UUGUTUGAU ACGACGCU U CUUUUTGCU CGACGCtJU C DTJOUGCLUC ACG2CUUCUJ U UUGCCUG CULTUUGCU C UGCGGCCU AUCCAAUU C ACACUGAA.

UUGGGCUU C UCCACAG GGGCUCU C CACAGGUC UUGGAACtJ C CAUGUGCUJ GCGGGCUU C GUGAUCGU CUCCUGGU C CUJGUtCGC UGUGCUAU A UGGUCCUC GGAAAGAU C ATJACGGGU GUCACt3GU U CAAGAAUG CAGAGAUJU U UGUGUCAG AGAGGGGU C UCAGCAGA AGCAGACU C UUtACAUGC AACAGAGU C UGGGG-AAA GUAUUCG U CCCAGAGC CJCGGUGCU C AGGUAUCC UCAGGCCU A AGAGGACU UAGCAG2CU C AACAAUGG AGGGUACU U CCCCCAGG GGGUACUU C CCCCAGGC G-AUGGUGU C CCGCUGCC CUGCCTJAU C GGGAUGGU UGGAGACU A ACUGGAUG CUGGCUGU C ACAGGACA CUGUGCtJU U GAGAACUG UTJCGUGAU C GtJGGCGUC CCGAACUAU C GAGUGGAC 1421 1425 1429 1444 1455 1482 1484 1493 1500 1503 1506 1509 1518 1530 1533 1551 1559 1563 1565 1567 1584 1592 1599 1651 1661 1663 1678 1680 1681 1684 1690 1691 1696 1698 1737 1750 1756 1787 1790 1793 1797 1802 1812 1813 1825 1837 1845 1856 1861 GGGUA7[CUU C CC-CCAGGC ACCCACCU C CUCUGGCU AUACUUGU A GCCUCAGG AGAAGGCU C AGGAGGAG GGGAGUAU C ACCAGGGA AGGGUAMCU U CCCCCAGG ACtC-UC'U U CCUCUUGC C'-U G GGG U U CGACUA CGtUGAAATJ U AUGGUCAA GAAAAUGU U CCAACCAC UGC-GUC;W A AUUGUL'C-G GCCACCAU C ACU=tGUA GUCCUGGU C GCC-GUJUGU ACCUGGGU C AUAAUUGUJ CUGAUCAU U GCGGGC= GUGGCCCtJ C UGCUCGUA UGGGAAGU C CCUGUUUA UCCUACCU U tJGUUCCCA UUACAkCCU A UrUACCGCC ACAkCCUAU U ACCGCCAG AGGAAGAU C AGGAUAUA C-ACeGAUJAU A CAkGUUAC UACAAGUU A CAGAAGGC CCCCGCCU C CCtJGAGCC CUGCACU U GCCCUGG-U GAACAGAU C AAUGGACA GAGAACCU C GGCCUGGG GG-CUUCU C CACAGGtJC GGCCUGUU U CCUGCCtJC CUGCUCGU A GACCUCUC CCCCACCU A CAUACAUU CCGGACUJU U CGAUCUUC CUCCUGGU C CUGGUCGC UCAGAUAU A CCUGGAGA GAUCACAU U CAC%,GUGC GUCCAUUU A CACCUATU CCUC'UGCTJ C CUGGUCCU GAGAACCU C GGCCUGGG GACACUGU C CCCALACtJC AUGGUCCtJ C ACCTJGGAC UCCCUGUU U AAAAACCA GCUCAGAU A UACCUGGA AACAGAGEJ C UGGGGAAA GCGGGCU C GUGAUCGU GCCACCAU C ACUGUGUA ACCCACCU C ACAGGGUA AGAGGACU C GGAGGGGC CCCCUAAU C UGACCUGC CAUGUC-CU A UAUC-GUCC SUBSTITUTE SHEET (RULE 26) PCIAB95/00150 WO 95/23225 203 1865 1868 2.877 1902.

1912 1922 1923 1928 1930 1.9 64 1983 :-996 2005 2013 2015 2020 2039 2040 2057 2061 2071 2076 2097 2098 2'-2.

2128 2130 2145 2152 2156 2158 2159 2160 2162 2163 2166 2167 2170 2171 2173 2174 2175 2176 21.83 22.85 2186 2187 2189 2196 tJAUCCGGU

UCACG-AZJ

ACAGM=C

CUAAAACJ

GAACAGAU

AUGU?.AGEJ

UGGACGcUj '-c'cAGA UGGAW-CtJ AGAG-A7L70 t;GGAAC-CUJ AtUGUAAGTJ C2CU=GCCUJ CUGCCf2Au

UAUUG-AGU

CGGAGCAt7

CCUGACCU

CUC-GLTC-=

GCGUCC=t

Z:GUAGCCU

CCAACUCI

CC-UAC-;c' juccGAClj

AGULGCL'GU

GC:CUGTjtJ CCAACUcJ

UUGCAGA;AU

ucAcAkmu t.7GAUGtIA

U-.UGUTJU

AUGUATJUU

A CAUJUCCU TJAtJUUAUU.

UGATJGUAU

C-AUGUJATiU GUAtJUUAU CAkG-UJAUU I

UGUGCEJAUJ

t7CUCUAUTJ AUuCUUU=

C-AAAALGUI

UGACAGrjU

.ACAGUTJAUI

UUAULUUAUT

CUGACAGUT

GACAC-AAG

AUAUTAAA U

CCCCCAGG

AAGUaMC-A AAtJGGACA

AUUGCCUA

ACCUUMJG

t7ACCUC-G-A ACU GGAUG GTGtJ-CA-C

C-GCCGC-G

UUtCAAGC'J AkTJGCC'UA UCG=- UG

GCGYGAUGG'U

CCCUGtJAC

ACAAACGA

CUGC-AC-GUt

CAAUGGCUJ

UACACCUA

GCCUCAGG

AC-GCCUAA

GULIGAUGU

CTJGGAG7U CGGjUCCUG CrCAUTC ClUGCCUotJ GUUGCtJUtJ

UACAACUJE

~UTUTJA

tJALTUA=MU AL71AAUt2C UUAUtUCA Cr-tUUUGt7J'

AUUCAGAG

Ull.UUAAUU AUUiAAUUC

AAUUCAGA

AUUGAGUA

UGGUCCUC

CCC CUGCU.

ACGAGUCA

CCAACCAC

UUJUAUUG-A

tJA~UAGU A~jU-GUA

UUGAGUAC

GAGtJACCC

AMUMU

2198 2199 2200 2201 2205 2210 2220 2224 2226 2233 2242 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2321 2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2413.

2417 2418 2425 2426 2433 2434 2448 2449 2451 2452 GAAUG;UC' C A G AC UCUU P GGGtJAt C GGGC'-UUCU C TUMUXGCTJ C UGGAGACU A GAGAACCJ C AC LAACAU u CUGGACCU C UCAt:GCluU C XCACAC-cuC CUCCUC-GUJ C AUCCAAkuu C GAUCACAU U AUCACAkuu c AUCAGCGAU A GAGCALC-GU U GGAAAGAU C ACAGIUAU u GCCC'JCUd C CAGGAUAU A GGAAAGAU C TGCGG=JU C GG7,JACU C GC-GfCCUGU C CZUGCCGU A CCCuC-CC' C CCZAUCCAU C CUUGUGUtU C GAACUCU C G-ACuuCC-U U GCAUUUi C CUGc-rUCUU C UGAUUUCu U AkUUUCUUEJu C UAuCCC-GU A UAAAUACU A UG;UGCUAU A C2, Wuutcu C AUCAGGAU A U'CAUGCU C UUAUU7,A.AU U CCUC-GGT u UCAkGAG'uUr C CGC-AGG'7AU C CCGAACAkG- A GAIAGCCU C C-GCCU7ulU u GCCUG-UUU7- C

CGAC-GUCA

CA'UGCCAG

CC-CAGCC

CACA-G,UC

AGCCACUG

*ACUGGAUG

GGCUC-G-

ACArGAAC'J UiCAGUAGU

ACAICC'A

CACG-GUC-C

ACGGUGC-U

UACAAGUU

AACAtIGUA AkUACGGG-U uAtj~uajGU

CUCCA.AUG

CA-AGUUAC

AUACGGGU

CCCAG-

GGL'C-UCA

-ACucuc

CUCCCACA

CCACAGAA

CCUGGAAG

uuCcuC~u

C'JCUAUUA

UUUCACG-A

CUCUGCG

UCACGAGU.

ACGAGUCA

GACACAAG

tJGUGGACOG

UGGUCCUC

AUGCUUCA

UACAAGUU

AC-AGAACtJ

CAGAGUUC

GGAGACUA

UGACA.GUU

A'CAACGA

CUUCCCClC

CUGCCUCG

CCUC-ccuc CUC-cCuclu SUBSITUTE SHEET (RULE 26) PIAB''/l95/OO 156 WO 9)5123225 204 2455 2459 2460 2479 2480 2483 2484 2492 2504 2508 2509 2510 2520 2522.

2533 2540 2545 2568 2579 2585 2588 2591 2593 2596 2602.

2602 2607 2608 2609 2620 2626 2628 2635 2640 2642.

2642 2653 2659 2689 2692.

2700 2704 2712.

2712 2722.

2724 2744 2750 2759 ACAtJUCCIU A CC=TGtU CCCt7GCCt7 C CUCCCACA C=UCCUU U GtJUCCCAA UUACACCtJ A UUACCGCC CGUCGCCG U GUGAUCCC ACCUUUGU U CCCAkAUGU CCUU'UGUU C CCAAUGTJC G-ACCACCTJ '2 CCCACCUA ACCU~ .2 A'UUCCUA ACAU~~ZrJCAC=U CAUACAMUU ~2CUACCUjU GUCCAUU A C.ACCOTU ACC-U7tM'GU U CCCAtIGU CCUUGUU C CCAAtTGUC ACAGCAUUT U ACCCCtJCA UCGGUGCU C ?AGGUAUCC .ALCAGCU C CGGACUU CAkGAGAUU U UGUGUCAG ccuGCACU U UGCCCUGG CUGCtU A GACCUCUC UGCCUCCU C CCACAGCC cujcutccu c UCUCUAUtJ A CJCCUGGU C UGUCUAUm A GUCCtJGGU C GUGGGAG;U A CUUUAGCU C UGGAXGACU A UCAGAGUU C CUCEJCACU A UACAACUU U UCACAGAU C GCUCAGG-U A CCCCACCU A GC"CUGUU C CCACAGGU C AGAAGGGU C ACUAGGGU C UCAGGCCU A AGGGUJACt U GACCACCU C CCCUACCU U CCUACCUU A C-G-AAAG-AU C AAGAUCAU A GGGUGGAU C

UUGCGAAG

CCCCUGCU

CUGGUJCGC

UGGUCCUC

GCCGUUGU

UCACCAGG

CCGUGGGA

ACUGGAUG

UG7ACAGUU

GUGCUGCU

UCAGCUCC

CAAUUCAC

UCCAUCCA

CAUACAUU

CUGCCUCU

AGGGUGCU

CUCCAAGC

CUGAAGCU

AGAGGACU

CCCCCAGG

CCCACCUA

AGGAAGGU

GGAAGGUG

AUACGGGU

CGGGUUUG

CGUGCAGG

2761 2765 2769 2797 2803 2804 2813 2815 2821 2822 2823 2829 2837 2840 2847 2853 2860 2872 2877 2899 2900 2904 2905 2906 2907 2908 2909 2910 2911 2912 2913 2914 2915 2916 2917 2918 2919 2931 2933 2941 2951 2952 2955 2956 2961 2962 2965 2966 2969 UUCuCuAu CG:uGvAAUt

CUCAUGCU

tTCAXIGC-UtJ

GCUCCCAU

CGGACUuu

CCUJGACCU

UAC-ACU

CAACUtj'UtJ

UCGGUGCU

CACAGC-GU

GC-ACCC-CU

UUACCCCUr

UUCGAUCU

UCUUGUGtJ

GGGCCUGU

UGGAGUCU

AGGCCAGCU

GGCUGACU

GAACUGCU

G-GCUGACU

GUUG-AUGU

CUclCUCUU UGAUGt2AU

G-AACUGCU

ACUUCCUU

UUCCUiUCU

AUGUAUUU.

TUGUGUAUU

GUAtlUUAU

UAUUUAUU.

cucuuccu CUUCCUCU1

ALMUCUUU

UUUUGUGU

GAUGGUGU

UJGGAGUCU

CAGUACTU

ACCAUGCU

CCGGACuu

UGCUUWCCU

cuuuccuu Ut.j=GUGUC

UGL'GUAUUC

CU=tGAAU C UGGAAGCU C GAAT-CAAU CGGACUUU C GAUCUUCC CtUUUGCU C UGCGGCCUJ U ACCCCUGC u AUGGJUCAA U CACAGAAC C AC-AGAACU C CUCACCCU C GAL'CTUCC C CUiGGAGG,-U u uck=GCCC C AGCUCCCA C AGGUAUCC A CULYCCCCC C CCAGCGCAk C ACCCACCU U C=-ACUAG U CCCUGGAA C GGUGCUCA C CCAGCACC C CGCGACUUU o CCUUCtJCU C UUCCUCUU u ccuucucu A MUMUA C CUCUJUGCG U UAUUAATJU C UUCCUCUUJ C UCUAUMJC C UAUUJACCC A UUAAUUCA C GUTCCCAG UJ AAUUCAGA A AUCAGAG

CUUGCGAAG

U GCGAAGAC C ACGAGTJCA C AGCCACUG 2 CCGCUGCC C CCAGCACC

CCCCAGGO

J CCUCUGAC J CGAUCUTJC

UGACAUGG

IGAAUCAAU

AGCCACUG

GUJUCCCAG

AAUAXAGTJ

UUCAAGCU

AAGUUUUA

GUCCCUGU U UAAAAACC G-ACGAACU A UCG-AGUGG SUBSTITUTE SHEET (RULE 26) WO 95/23225 WO 95/2225 I'111195/O() I56 205 2975 tGCAAG%-tI C UUJCAAGCU 2976 t3AUAUGGt3 C CUCACCt3G 2977 G-AAGCtJCt U CAAGCt7GA SUBSTITUTE SHEET (RULE 26)

I

WO 95/23225 206 Table 10: Rat 10AM1 HR Ribozyme Sequences PCT/1195/00156 nt pOSitiofl ii1 23 26 3i 34 48 54 58 64 96 102 108 115 119 120 146 152 158 165 168 185 209 227 230 237 248 253 263 267 293 319 335 337 338 359 367 374 375 378 386 Rat HH Ribozyme sequence TJCAGUGt'G CUGATUGAGGCCGAAAGCCGAA ALUGGAUC UAGAGAAG CUGAUGAGGCC-A.AAGGC=-2A AAQIJCAGC AAGAGGAA CUGALUGAGGCCGAAGGCC-GAA AC-CAGtC AGGACCAkG CTJGAUGAGGCCGAAIAGGCCGAA AGCAGAGG GUAUJAUCU CUGATUGAGGCCG-AAAGCTr-CG A %GCUL'CAG GGGGCUUJG CUGAUGAGGCCGAAAGGCCGAA ACC!uUGAG CCCAGGCC CUGAUGAGGCCG-AAAGG-CCG-AA AGG=UC GGCtICAGG CUGAUGAGGC-CGAAAGGCCGAA AC-GCGGGG GGGAGCUA CtJGAUGAGGCCGAAAGGCCGAA zkGGCACGG ACGGGUU=G Ct7GAUGAGGCCGAAAGGCCGAA ACCkUtJG AGGACCAG CUGAUGAGGCCGAAAGGCCGA.A AGCAGCAGG GCGACC-AG CUGAUGAGGCCG-AAAGCCGAA ACCAGGAG AGUUCCCC CtJGAUG-AGGCCGAAAGGCCGAA AGCAGUCC UGGGA.ACA CUGAUGAGGCCGAAAGGCCG-AA kAGGUAGGA GAGUrUGGG CUGAUGAGGCCrGAAAGGCGCAA ACAGIJGUC GGCCCGGG CtJGAtJGAGGCCGAAAGGCGCx AUCACAAC GGAGUUCC CtJGAUGAGGCCGAAGrGCCGAA AGGt7CUCG UUGAGGTJG CUGAIJGAGGCCG-AAAGGCCGA'-A AGCCGGGTIU tJGACUCGU CUGAUGAGGCCGAAAG.A AAAG-AAAU GGGGGAAG CUGAUGAGGCCGAAAGGCCwAA A;tGztrcxC CGAGGCAG CUGAUGAGGCCCGAAAGGCCGAA AAGGCXUC CCUGCACG CtJGAUGAGGCCGAAAGGCCGAA AucCaCCC GGUCAGAU CtJGAUGAGGCCGAAAGGCCcAA AGGGGCLG UUCACAGU CUGAt7GAGGCCGAAAGGCCCGAA ACUGGGUC CCUCCCAC Ct7GAUJGAGGCCGAAAGGCCG?.A ACACtIG GGGGUGUC CtIGAtJGAGGCCGAAAGGCCGAA

AGCUUCAG

UJCCUAAGG CUGAUGAGGCCGAAAGGCCG-AA, kGGGGGCC CCUCCACtJ CUGAUGAGGCCG-AAAGGCCGALA

AGGCAGUG

GCAUGAGA CtJGAUGAGGCCGAAAGGCCGAA kUUGCUC CGAGGZAG CUGAUGAGGCCGAAGGCCG-AA AAGGCtJUC UCAGCUUG CUGAUGAGGCCGAAAGGCCG-AA. AGAGCUUtC UCGUUUC;U CtJGAUGAGGCCGAAAGGCCGAA ALTCCTJCCG AGUtJCUCA CUJGAUGAGGCCGAAAGGCCGAA AGCACAkGU GAGGACCA CUGAUGAGGCCGAAAGGCCGAA

AUAGCACAN

CUJCAGCUU CUGAUJGAGGCCGAAAGGcAA AAGAGCjuU AAGCCGAG CUGAUGAGGCCGAAAGGCcGAA AcCtG~u ACGGGUUG Ct7GATJCAGGCCCAAAGGCCCAA AGCCAt7-UG AGGUC-GGJ CtJGAUGAGGCCGAAAGGCCGAA

AGGG,-,UAA

GAGGCAGG CUG-AUGAGGCCG-AAkAGGCCGAA kGGCUUICU UACCCTGt CUGAUGAGGCCG-AAAGGCCCGALA A.Guc-G,-U AGCUCCAA CUGAUGAGGCCGAAGGCC.AA A'CACAkGCG SUBSTITUTE SHEET (RULE 26) WO 95/23225 207 394 C=GtCAG Ct7GAUGAGGCCGAAAGGCCGAA AGCACCAXC 420 UGCGCUGG CUGAXJGAGGCCGAAAGGCCGAA AGGGGUGC 425 GGUGGCAG CUGAUGAGGCCGAAAGGCCGAA AGCCGAGG 427 UC-GtUEJUU Ct7GAUGAGGCCGAAAGGCCG-AA AACAGC-GA 450 CGCAGGAU CUGAUJGAGGCCGAAAflGCCGAA AGGUEJUU 451 GCCt3GGGG CUGAUGAGGCCGAAAGGCCGA.A AAGUACCC 456 UG-3UGGCAL CUGAUGAGGCCGAGGCCA AAGCCGAG 495 TJACACAGU CUGAUGAGGCCGAAAGGCCGAA AUGGtJCGC 51.0 UUtCCCACG CUJGAUGAGTGCCGAAAGGCCGAA AGCAGCAC 564 GtJG=UGG CUGAt7GAGGCCGAAAGGCCGAA ACAUtJUUC 592 UCCCUGCGU CUCGAUGAGGCCGAAAC-GCCGAA AUACUCZ-C 607 GCAtGA'GA CUGAUGAGGCCGAAAGC-CCGAA, AULGGC-UC 608 AGCAUGAG CUGALC4ZGCCGAAAGGCCGAA AAUUGGCU 609 AAGCAUG-A CUGAUGAflGCCGAAAGGCCGAA AAAUUGGC 61.1 UGAAGCAXU Ct7GAUGAGGCCGAAAGGCCGAA AGAAAUUG 656 CAUUCUUJG CUGAUGAGGCCGAAAGGCCCAA ACAGGAC 657 ACAUJUCUJU CtJGAUGAGGCCGAAAGGCCGAA AACAGUGA 66a AAGAGGAA CUGAUGAGGCCGAAAGGCCGAA AG-CAGUUC 677 UGCGCUGG CUGAUGAGGCCGAAAGGCCGAA AGGCG-tIGC 684 AAAGUCCG CUGAUGAGGCCGAAAGGCCGAA A GCCEJ 692 GGAGUUCC CUGAUGAGGCCGAAGGCCGAA AGGUCUGG 693 GGAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAAGtJCCG 696 AGAGGCAG CUGATJGAGGCCGAAAGGCCGAA AAACAGGC 709 GUGAGGGG CUGAE7GAGGCCGAAAGGCCGAA AAALTGCUTG 720 GAGC UGAA CL"AUGAGGCGAAAGGCCGAA AGtJUGUAG 723 UGGGAG-CU CUGAUGAGGCCGAAAGGCCGAA, AAAAGUUG 735 GCG-ACCAG CUGAUGAGGCCGAAAGGCCGAA ACCAGGAG 738 UCCACCCC CUGAUGAGGCCGAAAGGCCGAA AGGCAGGA 765 AGUUCUCA CtJGAUGAGGCCGAAAGGCCGAA AGCACAGU 769 UtJCCAGGG CUGAUGAGGCCGAAAGGC-CGAA ACACAAGA 770 CUUCCAGG CUGAUGAGGCCGAAAGGCCGAA AACACAAG 785 AGGCAGGA CtJGAUGAGGCCGAAAGGCCGAA ACAGGCCtJ 786 GAGGCAGG CUJGATJGAGGCCGAAAGGCCGAA AACAGGCc 792 GCGACCAG CtJGAUGAGGCCGAAAGGCCGAA ACCAGGAG 794 G-AGCUUJCA CUGAUGAGGCCGAAAGGCCGAA AGGCAGGA 807 UCC-AGGtJA CUGA!JGAGGCCGAAAGGCCGAA AUCUGAGC 833 UAGEJCUCC CTJGAUGAGGCCGAAAGGC-CGAA ACCCCAGG 846 CAAUAAAU CtIGAUGAGGCCGAAAGGCCGAA ACUGUCAG 851. AGCtJGCUA CUGAUGAGGCCGAAAGGCCGAA AGGUGAGO 863 ACGGG0UG CUGAUJGAGGCCGAAAGGCCGAA AGCCAUtJG 866 tJGUCAGAG CUGAUGAGGCCGAAAGGCCG-AA AAGCAUGG 867 UAGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGGUC 869 CUUCGCAA CUGAUGAGGCC(7.iAAGGCCGAA AGWIGA AG 881 CACGGGOU CUGAUGAGGCCGAAAGGCCGAA AAGCc-Auu 885 UUC-ACAkGU CUGAUGAGGCCGAAAGGCCGAA ACUUGGUC 933 CUGGGAAC CUGAUGAGGCCGAAAGGCCGAA AAUACACA 936 UG-ACACAA CUG-AUGAGGCCGAAAGGCCGAA AUCTCLGC 978 AAGUUGUA CUGAUGAGGCCGAAAGGCCGAA AUtJCUCAA 980 AAAPAGUtJG CUGAUGAGGCCGAAAGGCCGAA AGAUtJCUC PCT/1195/0()156 SUBSTITUTE SHEET (RULE 26) WO 95123225 208 986 C-ACUGAA Ct3GAt7GAGGCC:GAAAGC-CCGAA AGG 987 GGAGCUGA CUGATJGAGGC CGAAArC-ccGA A AA~uuGUjA 988 GGGAGCUG CUGAT3GAGGCCGAAAGGCCCGAA AAAGUtJGt 1005 G-ACGCCA.C CUGAUGAGGCCGAAGGCCG-AA AUCACGA 1006 CCUGGUGA CUGAUG-AGGCCGAAGGCCGAA ACUCCCAC 1023 CCUUCTUGA Ct7GAUGAGGCCAAAGGCCGA %CCUCCGG 1025 CCCCJUCU CUGAUC-AGGCCGAAGCCGAA AGACCtJCC 1066 UUGGCAAC CUGAT)GAGGCCGAAAGGCCG N AAGGTjUAGG 2.09 2 LCtJG%-tGA, CUGAUGAGGCCC-AG-G-C-AA ACCCtJ 1.093 %GGGGCL.UG CUGACAGA-CCAAGCCGA, AUUJCCC=J 2125 AUCAACAA CUGAUC-AGGCC-AGrCCGA AGUTGGG 11263 AC-CAAAAG CUGAUGAGGCCGAAGGCCA AGCGUCGU 11.64 CAGCAA CUGAUGAGGCCGAAGCCGAA, AGCGCG 1266 C.ACAGCU~ CUGAUGAGGCCCAAAGGrCCLA AGCA.GCGU 11.72 AGGCC%-zrA CUGAUGAGGCCGAGCCA AGCAAG 1.200 UtCAGUGU CUGAUGAGGCCAAGGCCG- AAUTUGGAU 1201 CCUGUGGA CUGAUGGGCCAAAGGCCGA AAGCCCAA 1203 GACCUGUrjG CUGAUGAGGCCAG-GCG6 AGAAGCCC 1227 AGCZACAUG CUGAUG-AGGCCGAAGf-CCAA AMUUCCA 2228 ACr-AUCAC CUCAUGAGGCCGAAAC-GCCGAA AAGCCCG- 1233 GCG-ACCAG CUGAUGAGGCCGAAGG-CCAA ACCAGGAG 1238 GAGGACCA CUGAUGAGGCCGAAAGGCCGAA AUAGCACAk 1264 ACCCGUAU CUGAUGAGGCCGA-GCCG.-I- AtUUuCC 1267 CAT-JCUJUG CUGAUGAGGCCGAAAGGCCG. AC-AGTjGAC 1294 CUGC-ACA CUGAUGAGGCCGAAAGC-CCGA -AAUC7,CUG 1295 UCGCUGCA CUGAUJGAGGCCGA.CGCCCGA ACCCCUCU 1306 C-CAUGUMA CUGAUGAGGCCGA-GCCGAA AG;UCtGCu 1321 TjUUCCCr-% CUGAUGAGGCCAA.GG3CG ACUCUGUTJ 1334 GC-UCUJGGG CUGAUGAGGCCGAAAGGCCGAA ACGAAUAC 1344 GC-ATJCCt3 Ct7GAtGAGGCCG-AAGGCCGAA AGrCACCGA 1351 AGUCCUCU CtJGAUGAGGCCGA-AAGGCCGAA AGGCCUGA 1353 CCA6UUGUjU CUGAUGAGGCCGA.AGGCCAA ACJGCtJA 1366 CCUGGGGG Ct3GAUGAGGCCGAAGGCCG-A AGUACCCtI 1367 GCCUGGGG CUGAUJGAGGCCGAAAGGCCCGAA AAGtJACCC 1368 GGCAGCGG CUC-AUGAGGCCGAAC-GCCCAA ACACCAUC 1380 ACCAUCCC CUGAUGAGGCCGAAGGCCGA ALVLCGCAG 1388 CAUCCAGU CUGAUGAGGCCGA;IC-GCCG-AA AG-UCUCC-A 1398 UGUJCCUGU CUGAUGAGGCCGAAAGGCCGAA ACAGCCAG 1402 CAGUtJCUC CtJGAUGAGGCCGAAAGGC:CGAA AAGCACAG 1408 GACGCCAkC CtJGAUGAGGCCGAGGCCGA AUCACGAA 1410 Gt3CCACUC CUGAUGAGCCGAGGCCGA AtJAGUUCG 1421 GCCtJGGGG CUGAtJGAGGCCGAA~rGCCGAA AAUAccc 1425 AG-CCAGAG CUGAGAGGCCGAGGCCGAA A-GUGG-GU 1429 CCUGAGGC CUGAUGAGGCCCAAAC-GCCGu4 ACAAauAu 1444 CUCCtJCCT CEJGAUGAGGCCGAAAG-GCCCA.A AC-cctut)cu 145 5 UCCCUC-GJ CUGAUGAGGCCA.GCGCCG-AA AUACUCCC 1482 CCUGGGGG CUGAUGAGGCCGAAGGCCG-A AGUAcccu 1484 GCA6.CAGG CUGAUGAGGCCGCA..-GCCGA-A AGA G CAGU 1493 TAGUCUCC CtJGAUGAGGCCGA;,AAC-CCC-AA ACCCCAGG PCT/1395/()0156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 209 1500 UUGACCAU CUGAUGAGGCCGAAAGGCCGAA AUtJUCACG 1503 GUiGGuuGG CUGAUGAGG;CCGAAAGGCCCGAA ACAUUtUtC 1506 CCAACAAU CUGAUGAGGCCGAAAGGCCGAA AUGACCCA 1509 UACACAGU CU-GAUGALGGCCGAAAGCCGAA AUGUGGC 1518 AcAACGGC CUGADJGAGGCCGAAAGGCCGAA ACCAGGAC 1530 ACAAUUAU CUGAUGAGGCCG-AAAGGCCGAA ACCCAGGU 1533 AAG:CCCGC CUGAUGAGGCCAAGGCCGAA AUGAUCAG i551 tTACGAGcA CUGAUGAGGCCGA AGCCGAA AGGGCCAkC i559 uAAAc-AGG CUGAUGAGGCCGAAAGGCCGAA ACUUCCC-A 1563 UGGGAACA CUGAUGAGGCCAGC--CGAA AGGUAGC-A 1565 -GCGGUAA CUGAUGAGGCCC-AAAGGCCGAA AGGUGJA 1567 CUGGCGGU CUGAUGAGGCCG-AAAGG-CCG-AA AUAGGUGU 1584 tJAtAUCCU CUGAUC-AGGCCGAAAGGCCGAA AUCUtICCU 1592 Gu.AActuuG CUGAUGAGGCCCAAAGGCCGA6A AUAtJCCUG 1599 GCCUtJCUG CUGAUGAGGCCGAAASGCCGAA AACUUJGUA 1651 cGCuCA CUG-AtGAGGCCGAAAGGCCGAA AGGCGGGG 1661 ACCAGGGC Ct3GAtJGAGGCCGAAAGGCCGAA AAGUGCAG 1663 UtJGUCCAUUt CUGAUGAGGCCGAAAGGCCGAA AUCUGUUC 1678 cccAGGCC CUGAUGAGGCCGAAAGCCGAA AGGUUC 1680 G-ACcUGUG CUGAUGAGGCCGAAAGGCCG-AA AG-AAGCCC 1681 GAGGCAGG CUGAUGAG7GCCGAAAGGCCGA AACAGGCC 1684 GACGAGGUC CUGAUJGAGGCCGAAAGGCCGAA ACGAGCAG 1690 AATJGUAUG CUGAUGAGGCCGAAAGGCCGAA AGGUGGGG 1691 GAAGAUCG CUGAtJGAGGCCGAAGGCCGAA AAGUCCGG 1696 GCGACCAG CUGAtJGAGGCCGAAGGCCGA ACCAGGAG 1698 TJCUCCAGG CUGAUGAGGCCGAAAGTGCCGAA AUAUCUG-A 1737 C-CAkCCGUG CUGAUGAGGCCGAAAGGCCGAA AtJGZGAUC 1750 AAUAGGUG CUGAUGAGGCCGAAAGGCCGAA AAAUGGAC 1756 AGGACCAG CUGAUGAGGCCGAAAGGCCGAA AGCAGAGG 1787 CCCAGGCC CUGAUGAGGrCCAAAGGCCGAA AGGUUCUC 1790 GAGUUGGG CUGAUGAGGCCGAAAGGCCGAA ACAGUGUC 1793 GUCCAGTGU CUG-AUGAGGCCGAAAGGCCGAA AGGACCAU 1797 UGGUjIUUUU CUGAUGAGGCCGAAAGGCCGAA AACAGGGA 1802 tJCCAGGUA CUGAUJGAGGCCGAAAGGCCGAA AtUCUGAGC 1812 UUUCCCCA CUGAUGAGGCCGAAAGGCCG-AA ACUCUGUU 1813 ACGAUCAC CUGAUGAGGCCGAAAGGCCGAA AAGCCCGC 1825 UACACAGU CUGAUGAGGCCGAAAGGCCGAA AUGGUGGC 1837 UACCCUGU CUGAUGAGGCCGAAAGGCCGAA AGGUGGGtJ 1845 GCCCCUCC CtJGAt7GAGGCCGAAAGGCCGAA AGUCCUCtJ 1856 GCAGGUCA CUGAUGAGGCCGAAAGGCCGAA AUUAGGGG 1861 GGACCAUA Ct7GAUGAGGCCGAAAGGCCGAA AGCACAUG 1865 CUUGUGUC CUGAUGAGGCCG-AAAGGCCGAA ACCGGATJA 1868 AUUTUAUAU CUGAUGAGGCC-A.AAGGCCGAA ACUCGUGA 1877 CCUGGGGG CUG-AUGAGGCCGAAGC'CGAA AGUACUGU 1901 UGUACCUU CUGAUGAGGCCG?-kAGGCCG-AA AGUUUAG 1912 UGUCCAUU CUGAUGAGGCCG-AAAGGCCGAA AL'CUGUUC 1922 UAGGCAAU CUGAUGAGGCCGAAAGGC-CGAA ACUUACAtJ 1923 CUAAAGGU CUGAUGAGGCCGXA.AGGCCGAA AGCGUCCA 1928 UCCAGGUA CUGAUGAGGCCGAAAGGCCGAA AUCUGAGC PCT/1B95/00150 SUBSTITUTE SHEET (RULE 26) WO 95123225 210 2.930 CAUCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUCUCCA 1964 GCUC-ACAC CUJGAUGAGGCCGAAAGGCCGAA AAAUCUCU 1983 CCCAGGCC CUGAUGAGGCCGAAAGGCCGAA AGGUJCtIC i996 A GC7AA CUAUCAGGCCGAAAGGCCGAA AGCUUCCA 2005 UAGGCAAU CUGAJGAGGCCGAAAGGCCGAA ACUUACAU 2013 C-.tJCCCGA CUGAIlGAGGCCGAAAGGCCGAA AGGCAGCG 2 015 ACCAUCCC CUGAflGAGGCCGAAAGGCCGA AUAGGCAG 2020 GTJACA:GC-G CUtGAUGAZGCCGAAAGGCCGAA ACUCAAtJA 2039 !JCGUUGU CUGAt1GAGGCCCGAAAflG-CCGAA AUCCXCCG 2040 ACCUJCCAkG CUC-AUC-AC-GCCCGAACfGCAA AGGtJCAG 2057 ACCAUUG CUCALGAGGCCCAAAGGCCGAA AflGACCAG 2061 UAGGUGUA CUGAUGAAGGCCGAAAGGCCGAA AUGGACGC 2071 CCUGAGGC CUGAUG-AGGCCGAAAGGCCGAA ACAAGUAU 2076 UUAGGCCtJ CUJGAUGAGGCCGAAAGGCCGAA AGGCUACAX 2097 ACAUCAAC CUiGAUGAGGCCGAAAGGCCGAA AGAGUUGG 2098 ACCUCCAG CUGAUGAGCCGAAAGGCCGAA AGGUCAGG 2115 CAGGACCC CUGAUJGAGGCCGAGGCCGA AGEICGGAA 2128 GAtJCAUGG CUC-AUC-AGGCCGAAAGGCCGAA ACAGC-kCU 2:30 AGAGGCAG CUGAUJGAGGCCG-AAAGGCCGAA AAACAGGC 2:45 ACAUC.A. CUGAUGAIGGCCGAAAGGCCGA AGAGtJUGCO 2152 AAUUGT3A CUGAUGAGGCCGAAGGCCGAA AUUCUCAA 2156 UCLAAA CUGAUGAGGCCGAAGCCGAA AACUGUCA 2158 AA~jUAAUA CUGATJC-AGGCCGAAAGGCCGLAA AUACAUCA 2159 GAAUUAAU CUGAUCAGGCCAAAGGCGAA AAUJACAUC 2160 UC-ALUAA CUG-AUGAGGCCGAA.GGCCGAA AMUACAU 2162 AACAA.GG CUGAUGAGGCGAGGCCGAA AGGAAUGU 21653 CTj'CtjGAAU CUGAUGAGGCCAPAAGGCCGAA AAUAAAUA 2166 AAUUAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUCA 2167 GAAUUAU CUC-AUGAGGCCGAAGGCCGAA AAUACAUC 2170 UCUCAATJU CUGAUJGAGGCCGAAAGGCCGALA AUAAAUAC 2171 UACUCAAU CUGAUGAGGCCGAGGCCGAA AUAAC3G 2173 GAGGACCA CUG-AUGAGGCCGAAASGCCGAA AUAGCACA 2174 AGCAGGGG CUG-AUGAGGCGAGGCCGA AAUAGAGA 2175 UG-ACUCGU GUGAUGAGGCCGAAAGGCCGAA AAAGAAAU 2176 GUC7GUUGG C!JGAUGAGGCCGAAAGGCCGAA ACAUUtUUC 2183 UCAAUAAA CUGAUGAGGCCGAAAGGCCGAA ACUGUCA 2185 ACUCAAUA CUGAUGAGGCCGAAAGGCCGAA AtUhAUGU 2186 tJACUCA.U Ct3GAUGAGGCCGAAGGCCGA AUAACUG 2187 GUACUCAA CUJGAUJGAGGCCGAGGCCGAA AAAUAACtI 2189 GGGUACUC CUGAUGAGGCCGAAACGCCGAA AUAAUAA 2196 CAAUAU CUGAUGAGGCCGAAAGGCCGAA ACUGUCAG 2198 UGACCUCG CUGAUGAGGCCGAAAGGCCGAA AGACAUUC 2199 CUGGCAUG CUGAUGAGGCCGAAAGGCCGAA AACAGUCtJ 2200 GCCtJGGGG CUJGAUGAGCGCCGAAA=GCGAA AAGUAccc 2201 GACCUGUG CUGAUG-AGGCCG-AAAGGCCGAA AGAAGCcc 2205 CAGUGGCU CUGAUG-AGGCCGAAAGGCCGAA ACACAAA.A 2210 CAtJCCAGtJ CUGAUGACGCCGAAAGGCCGAA AGucurCCA 2220 CCCAGGCC CUG-AUGAGGCCG-AAAGGCCGAA AGGUUCUC 2224 A.AC-GtjAGG CUG-AUGAGGCCG-AAAC-GCCG-AA AUGUAUGU PCT/1195/0O 156 SUBSTITUTE SHErT (RUILE 26) WO 95/23225 IICT/IB95/00156 211 2226 2233 2242 2248 2254 2259 2260 2266 2274 2279 2282 2288 2291 2322.

2338 2339 2341 2344 2358 2359 2360 2376 2377 2378 2379 2380 2382 2384 2399 2401 2411 2417 2418 2425 2426 2433 2434 2448 2449 2451 2452 2455 2459 2460 2479 2480 2483 2484 2492

TUCUGGCLI

AGUUC7GI

ACE)ACUG

GCGACCAC

UUCAGUGI

GCACCGUC

AGCACCGT

AACUUM2

UACAUGR~

ACCCGT

ACUCAAUJ7

CAUEJGGAC

ACCCGUAL

CCUGUGG;~

GCCTUGGGC

t7GAGCACC

GAGAGGUC

UGUGGGACG

TJUCUGUGG

CUJCCAGG

TJAATJAGAG

UCGUGmAAA

CGCAAGAG

ACt7CGUJGA UGcAcrJCGU

CUGUGTJC

CGtICCACA

GAGGACCA

tJGAAGCAU AACUrtJGUA AGtIUCUGU

GAACUCUG

UAGUCt7CC

ACUGUCA

tJCGUUUGU

GGGGGAAG

CGAGGCAG

GAGGCAGG

AGAGGCAG

AACAAAGG

tJGUGGGAG UtJC-GGAAC

G-GCC-GUAA

C-GGAUCAC

ACAUUGGG

GACAUUGG

UAGGUGGG

JCUGAUGAGGCCG,-AAACGCCG-AA

SCUGAUGAGGCCGAAAGC-CCGAA

SCUGAUGAGGCCGAAAGCGAA

3 CGAUG2A=GCGAAA=~CGAA JCUGAtJGAGGCCGAAAGGCCGAA 3CUGAUGAGGCCGAAAGGCCGAA 3CUGAUGAGGCCGAAAGGCCGAA

.CUGAUJGAGGCCG-AAAGG,-CCGAA

3CUGAUJGAGGCCGAAAGCAA C= CATJGAGGCC13AA.AZGGCCGAA

SCCGAUGAGGCCGA;AAGGCCGAA

CUGAt7GAGGCCGAAAGC-CCGAA

CUGATJGAGGCCGAAAGGCCG-AA

ICUGAUGAGGCCG-AAAGGC-CAA

CUGAUGAGGCCGAAAGGCCGAA

CE3GAJGAGGCCGAAAGGCCGAA

CUGAUGAGGCCGAAAGGCCGA

CUGATUGAGGCCGAA.AGGCCG-AA

CUCGAUGAC-GCCGAAAGGCCGA

CUGAUGAGGCCG-AAAGGC-CGAA

CU3GAUGACGGCCG-AAGGCCGAA

*CUG-AUGAGGCCGAAAGGCCGAA

CtTGAt7GAGG~CCG-AAAGGCCGAA.

*CUG-AUGAGGCC-AAAGGCCCGAA.

CUGAUGAGGCCG-AAAGGC-CGAA.

Ct3GAtIGAGGCCGAAAGGCCGAA CrJGAUG-AGGCCGAAAGGCCG-AA

CUGAUGAGGCCGAAAGGCCGAA

CUGALTGAGGCCGAAAGGCCGAA

CTJGATJGAGGCCGAAAGGCCGAA

CtJGAUGAGGCCGAAAGGCCGAA CtIGAUGAGGCCGAAAGGCCGAA CtJGAUGAGGCCGAAAGGCCGAA

CUG-AUGAGGCCGAAAGGCCG-AA

CUGAUGAGGCCGAAAGGCCGAA

CUG-AUGAGGCCC-AAAGGCCG-AA

CUG-AUGAGGCCGAAGGCCGA.

CUGAUGAGGCCG-AAAGGCCGAA

CUGAUGAGGCCG-AAAGGCCGAA CtTGAUG-AGGCCGAAAGGCCGAA CUGAUGAGGCCGAAAGGCCGAA CUGAUGAGGCCGu'AAGGCCGA A CtJGAUJGAGGCCGAAAGGCCGA A

CUGAUJGAC-GCCGAAAGGCCG-AAA

CUGAUGAC-GCCGAAAPGCCCGAAA

CUG-AUGAGGCCG-AAAC-GCCG-AA A CUGAUGAGGCCGAAAGGCCGAA A CUGAUGAGGCCGAAAGGCCGAp.A CUGakUGAGGCCGA.A-AGGCCG-A-AA A~GC7%Cr-G

AAGCAUGA

AGCUGL'GU

ACCAGGAG

AAtJUGGA U

ALGUGAUC

AAUGrJG-AU

AUCCUGAU

ACCUGCUC

:XCUU=CC

AEL-ACUGU

AkUAUC',=G AUCUfUTCC

AAGCCCAA

AAGTJACCC

:kCAGGCCC- ACGArG

AGGAGGG

AtIGGAUCG AAC CAAG AGCAGtJUC AC-GAAGtC

A.AAUCAGC

A.ACA

AGAAAUCA

kAAGAAAU kCC%-GAUA kC-AAAUUC- LkGCAtJGA

MUAAUAA

~a=CUGA

~LCCUCCG

LCUGTJUCA

~AGCVUC

AOL=GC

AACAXGGC

.GGZAAUGU

-C-GCAGGG

AC-UAGG

G G UGUA A

CC-C-CGAC

MCArAGU ACAAkAGG

C-GUC-GTJC

SUBSTITUTE SHEET (RULE 26) m WO 95/23225 212 2504 UAGGAAUG CUGAUGAGGCCGAAAGGCCG-AA AUGtJAGGU 2508 AAGGUAGG CUGAUGAGGCCGAAAGGCCGAA AUGEJAUGtJ 2509 AAAGGUAG CtIGAUGAGGCCGAAAGGCCGXAA AAUGUAUG 2510 AAUAGGUG CU.GAUGAGGCCGAAAGGCCGAA AAAUGGAC 2520 ACAUUtGGG CTJGAUGAGGCCGAAAGGCCGAA ACAAAGGU 2521 GACAUUGG GTJGAtJGAGGCCGAAAGGCCGAA AACAAAGG 2533 UGAGGGGU CLUGAUGAGGCCGAAAGGCCGAA .AAUGC UGU 2540 GGAUACCU CUCGAtGAGGCCGAAAGGCCC-AA AGCACCGA 2545 AAAGUCCG CUGAUGA.GGCCGAAAflGCCGAA *kG%-UGCCU 2568 CUGACACA CUGAUCAGGCCGAAAGGCCGAA AAUCUCtJG 2579 CCAGGGCA CUGAUGAGGCCGAAAGGCCGAA AGtJGCAGG 2585 GAGAGGUC CUGAUGAGGCCGAAAGGCCGtA ACGAGCAG 2588 GGCUGUGG CUGAUGAGGCCGAAAGGCCGAA AGGAGGCA 2591 CUUCGCAA CUGAUGAGGCCGAAAGGCCGAA AGGAAGAG 2593 AGCAGGGG CUGAUGAXGGCCGAAAGGCCGAA "kAUAGAGA 2596 GCGACCAG CUJGAUJGAGGCCGAAAGGCCGAA ACCAGGAG 2601 GAGGACCA CtJGAUJGAGGCCGAAAGGCCGAA AUAGCACA 2602 ACAACGGC CUGAUGAGGCCGAAAGGCCGAA ACCAGGAC 2607 CCUGGUGA CUGAUGAGGCCGAAAGGCCGAA ACUCCCAC 2608 UCCCACGG CUGAUGAGGCCGuAAGGCCGAA AC-CUAAAG 2609 CAUJCCAGU CUGAUGAGGCCGAAAGGCCGAA AGUCUCC.A 2620 AACUGUCA CUGAUGAGGCCGAAAGGCCGAA AACtJCtGA 2626 AGCAGCAC CUGAJIJGAGGCCGAAAGGCCGAA ACUGAGAG 2628 GGAGCtJGA CUGAUIGAGGCCGAAAGGCCG-AA AAGUtJGUA 2635 GUGAAUUG CUGAUGAGGCCGAAAGGCCGAA AUCtJGUGA 2640 UGGAUGGA CUGAUGAGGCCGAAAGGCCG-AA ACCUGAGC 2641 AAUGEJAUG CUGAUGAGGCCGAAAGGCCGAA AGGUGGGG 2642 AGAGGCAG CUGAUGAGGCCGAAAGGCCGAA AAACAGGC 2653 AGCACCCU CUGAUGAGGCCGAAAGGCGA ACCUGUGG 2659 GCUUGCAG CUGAUGAGGCCGAAAGGCCGAA ACCCtJUCtJ 2689 AGCUUCAG CUGAUGAGGCCGAAAGGCCGAA ACCCUAGU 2691 AGUCCUCU CUGAUGAGGCCGAAAGGCCGAA AGGCCUGA 2700 CCUGGGGG CUGAUGAGGCCGAAAGGCCGAA AGUACCCt) 2704 UAGGUGGG CUGAUGAGGCCGAAAGGCCGAA AGGUGGUC 2711 ACCUUCCU CUGAUGAGGCCGAAAGGCCG-AA AGGUAGGG 2712 CAC CUUCC CUGAUGAGGCCGAAAGCCGAA AAGGUAGG 2721 ACGUAU CUGAUGAGGCCGAAAGGCCGAA AUCUUUCC 2724 CAAACCCG CUGAUGAGGCCGAAAGGCCGAA AUGAtJUU 2744 CCUGCACG CUGAUGAGGCCGAAAGGCCGAA AUCCACCC 2750 GGUUUUUA CUGAUGAGGCCGAAAGGCCGAA ACAGGGAC 2759 CCACUCGA CUGAUGAGGCCGAAAGGCCGAA AGUEJCGUC 2761 GGAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAAGuccG 2765 AGGCCGCA GUGAUGAGGCCGAAAGGCCGAA AGCAAG 2769 GCAGGGGtJ CUGAUG-AGGCCGAAAGGCCGAA AUAGAGAA 2797 UUGACCAtJ CUGAUGAGGCCGAAAGGCCGAA AUUUCACG 2803 GUUCUGUG CUGAUGAGGCCGAA.AGGCCGAA AGCAUGAG *2804 AGUUCUGtJ CUG-AUGAGGCCGAAAGGCCGAA AAGCAUGA 28213 AGGGUCAG CUGAUGAGGCCGAGGCCGAA AUGGGAGC 2815 GGAAGAUC CUGAUGAGGCCGAAAGGCCGAA kzLAGuCC-G PCT/IB195/0()156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 213 2821. ACCU7CCAG CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 2822 GG-AGCUGA, CtJGAfGAGGCCGAAAGGCCG-AA AUUGUA 2823 UCGGAGCU CUGAUGAGGCCGAAAGGCCGAA AAAAGtJUG 2829 C-GAUACCtJ CUGAUGAGGCCGAAAGGCCGAA AGCACCG-A 2837 GGGGGAAG CUG-AUGAGGCCG-A.AAGGCCG-AA ACCCtJGUG 2840 tGCG%'t'GG CUGAUGAGGCCGAA?.GGCCG-AA AGGGGUGC 2847 AGGUGG-GU CLGAUGAG-GCCGAA.A-GCCGAA AGC-GGUrAA 2853 CtJAGUCGG CUGCAUGAGGCCGAAA=GCAA %CAUCGAA 2860 UUCCAGGG CUGAUGAGGCCGAAAGGCCGA;lA ACACAAGA 2872 UC-G-GACC CUG-AUGAGGCCGAAACGCCGAA ACAc-GCCC 2877 CGUGC-UGG CUG-AUGAGGCCGA.AAGCC-AA AGAC'CCA 2899 kAAGUJCI-G CUGAUGAGGCCGAAAGGCCG-AA AGCUGCCU 2900 AGAGAAGG CUJGAUGACG-CGAAAGGCGAA.AGUCAGCC 2904 XAZACGGAA CUC-AUGAGGCCrGAAAGG--CC-AA kGCA=UC 2905 AG-ACAAG-G CUGAUGAGGCCGAAAGGCCG-AA AGUCAGCC 2906 UUA.AUAAA CUGAUGAGGCCGAAAGGCCGAA ACAUC-AAC 2907 CGCAAGAG CUGAUGAGGCCGAAAGGCCCGAA AAGAGCAG 2908 AALUAAUA CUGAUGAGGCCGAAAGGCCGAA AUACAUCA 2909 AAGAGGAA CtJGAUGAIGGCCGAAAGGCCG-AA AGCAGUtJC 2910 GUAAUAC-A CUG-AUGAGGCCGAAAGGCCGAA AAGGAAGU 2911 GGGDAUA CUGAJuGGGCCG-AGGCCGAA AGAG~A 2912 UG-AAUE3A CUC-AUGAGGCCAGGCCcGA. AAAUACAU 2913 CUGGGAAC CUG-AUGAGGOC =A~CGAA AAUACACA 2914 UCUiGAAUU CUCAUGAC-GCCGAA.Ac-cCCGuAA AUAAAUAC 2915 CUU? -U CUGAGGCCGCA-GCCGA ?A.UAUA 2916 CUtICGCAA CUG-AUGAGGCCG-AAA-GCCGAA AGGAAGAG 2917 GUCUUCGC CUCAUGAGCCGA.GGCCGA. ACGcGAAG 291.8 UG-ACUCGU CUGAUGAGGCCGAA =CGAA AAGAAAU 2919 CAGUGGCU CUGJJGAGGC-CGAAAGGCCGAA ACACAAAA 2931 GGCAGCGG CUCAUGAGGCCGAAAGGCCGAA ACACC-ATJC 2933 GG*C-UGG CUGAUGAGGCCGAAAGGCCG-AA AGACUCCA 2941 GCCUGGGG CUGAUGAGGCGAGC:CGAA ?AGUACUG 2951 GUCAGGG CrGCtJGAGC=AAAAGGCCGA AGCAUGGU 2952 CAACAUCG CUGAUGAGGCCGAAGGCCGAA AGCCGG 2955 CCAUGUCA CUGAUGAGGCCG A.A-GCCG-A AGGAAGCA 2956 AUUGAUUC CUC-AUGAGGCCGAAGCCGAA AAGGAAAG 2961. C-GUGGCtJ CUC-AUGAGGCCGA-ACGCCGA ACACAAAA 2962 CUGGAAC CUGAUGA-GCCGAAAGGCCGAA AAtJACACA 2965 ACUUUAUtJ CUG-AUGAGGCCGAAAGGCCGLAA AUUCAAAG 2966 AGCUUGAA CUGAUGAGGCCG-AAAGGCCG-AA AGC-UUCCA 2969 UAAAACU CUGAUGAGGCCGAAAG-GCCGAA AUtJGAUUC 2975 AGCUGA CUGAUGAGGCCGAAAGGCCGAA AC-cUUCCA 2976 CAG-GUGAG CUGAUGAGGCCCAA.AGGCCGAA ACCAUAUA 2977 tJCAGCUUG CUG-AUGAGGCCGAAAGGCCGAA ACAGCUUC PCT"I/i 119510()156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 214 Table 11: Human 11-5 HRE Target Sequence Pc'rIB95o()15( nt.

Position 12) 13 36 37 38 56 57 63 64 69 74 78 91 97 104 116 117 130 145 155 156 157 159 162 165 171 179 192 200 201 206 207 212 216 222 EH Target Sequence AUGCACU U UCUUUGC UGCACU U CUUUGCC GrCACUUU C UUUJGCCA ACUUUCtJ U TJGCCAA CUUUCUU U GCCAAAG AGAACGU U UCAGAGC G-AACGTJU U CAGAGCC AACG'tUUJ C AGAGCCA GGAUGCEJ U CUGCAUU GAUGCUUJ C UGCAUTU UCUGCAU U UGAGOTJU CUGCAUU U GAG=UU UUUGAGU U UGCUAGC UUGAGUU U GCUAGCU GUUUGCLU A GCUCUUG CCUtAGCU C UUGGAGC UAGCUCU UJ GGAGCUG GCUGCCU A CGUGUAU UACGUGU A UGCCAUC AUGCCAUJ C CCCACAG CAGAAAU U CCCACALA AGAAAUU C CCACAAG AGUGCAU U GGUGAAA GAGACCU U GGCACUG CACUGCU U UCUACUC ACUG=tJ U CUACUCA CUGCt3UU C UACUCAU GCUUUCU A CUCAUCG UUCUACU C AUCGAAC UACTJCAU C GAACUCU UCGAACU C UGCUGAU UGCUG-AU A GCCAAUG UGAGACU C UGAGGAU UGAGGAU U CCUGUUC GAGGAUU C CUGUUCC UUCCUGU U CCUJGUAC UCCUGUU C CUGUACA UUCCUGU A CAUAAAA UGUACAU A AAAAUCA UAAAAAU C ACCAACU nt.

Position HIH Target Sequence 245 247 248 249 257 273 291 305 307 308 316 319 322 323 326 334 338 380 388 389 392 397 409 410 411 413 419 437 440 447 454 462 463 466 479 480 481 497 498 AAGAAAU C GAAAUCU Z AAAUCUjU *1 AkAUCUTJU r

AGGGAA'

GGAGACU C

AGG=GU

AAAGACU

AGACUJAU L GACUAUU C AAAAACtJ TJ AACtJUGU C UUGUCCU U UGUCCU A CCUUAAU A AAGAAAU A AAUACAU U GGAGAGU A AACCAAU U ACCAAUU C AAUJUCCU A CUAGACU A CAAGAGU U AAGAGUU U AGAGUUU C AGUUUCU U TJUGGUGU A AGUGGAU A GGAUAAU A AGAAAGU U UGAGACU A AACt7GGU U ACUGGUU U GGUUUJGU U CAAAGAU U AAAG-AUU U AAGAUUU U AGGACAU U GGACA:UU U T UCAC-

CC-AA

G~f-"ACAC

-AAACUGU

CUGUGGA

UUCAAA.A

CAAMAC

*A-AAAACUJ

GUCCUU

CUMAA

*AAUAAAG

AUPAAAG A

?AGAAAU

CAUTJGAC

G-ACGC-CC-

AACCAAU

CCUAGAC

CUJAGACTJ

GACUACC

CC-UGCAA

UCUUGGU

CULG

UUGIGUGU

G-GUGUAA

ZAUCAAC%

AUAGAAA

-AAAGUU

GAGACUA

AACUC-GU

UGUUGCA

GUjUGCAG GCAkGCCAk U UGGAGG

UGGAGGMA

GGAGGAG

U-uA2CUGC

UACUGCA

GACAUUU U ACUGC-kG SUBSTITUTE SHEET (RULE 26) PCT/IB95/00156 WO 95/23225 PT135OI5 215 500 532.

538 539 542 543 544 545 549 551 554 555 556 560 561 573 577 579 580 581 588 597 598 611 616 617 619 620 625 627 629 630 631 636 638 644 647 653 655 656 637 658 661 672 676 678 681 682 AcAt=U A AAAGAGU C CAGGCCU U AG7GCCUU A CCtUuAAu U CUUAAUU U UIJAAUUU U UAAEUUU C UtUuCAAu A UCAAXAU A AUALTAAU U

AAUUUUC

AUtJUUCA

UJUCAAUA

UCAAUAUX

CAAUA

AAVALTAA

UAUUUA

AUtJUAAC

UAACUUC

AACUTJCA,

ACUUCAG

tL.UAADU

ALMAUUU

UUAACUU

AGUAAAU

UAAAU

AAAUAU

AAtUAUUU C ACAGG A AAUAIIUU A UUUCAG U UCAGGCA U CAGGCAU C AGGCAUA CAGGCAU A CUGACAC UGACACtI U UGCCAGA G-ACACUE U GCCAGAA AAAGCAU A AAATUU AUAAAAU U CUUAAAA UAAAAUU C UUAAAAU AA.AUUCU U AAAAUAU AAUUC=t A AAAtUA ULTAAAAU A UAUUJUCA AAAAUAU A UtUCGAA AAUATJAU U UCAGAUA AUAUAUU U CAGAUAU.

684 685 686 688 689 691 692 693 697 698 703 704 708 715 719 720 724 725 728 731 733 734 735 745 746 752 753 757 761 762 765 767 768 769 771 772 773 778 779 783 788 789 791 794 805 UACUUUU U AcutUUU U CUUUUUU C UtJUUUCU u UUUUC=t A UUCUUAU U UCUUJAUU U CUUALUTU A UUUAACU U UUVAACUU A

UCUEUAUU

CUUAUUU

UUAUUUA

AUUUAAC

UUUAACU

UAACUtJA

AACUUAA

AACAUUC

AC-Auucu UUAACADU U CUGUJAAA UjAACAXUU C GGUAAAA AUUCLUGU A AAAUGUC AAAAUGU C UGtJUAAC UGUCUGU U AACUUAA GUCUGU A ACUUAAU GUUAACU U AAUAGUJA UUAACUU A AUAGUAU ACUUAAU A GUAUUUA UTAAUAGU A UUtU.UGA AUAGUAU U UAUGAAA UAGUAUU U AUGAAAU AAUUU A UGAAAUG AAAtJGGU U AAG-AAUU AAUGGUU A AGAAUUU UAAGAAU U UGGUAAA AAG-AAUU U GGUAAAU AUUTUGGU A AAUUAGU GG.-UAAAU U AGUAUUU GUAAAUU A GUAUUUA AAUTUAGU A UUEJAUUU UTUAGUAU U UAtJUUAA UAGLMUU U AUUUAAU AGUALUU A UUUAAUG UAUUUAU U MAAU=~t AUUUAUU U AAUGUtJA IJUUAUUU A AUGUUAU UUAAUGU U AUGUUGU UAAUGU A UGUUGUG GUUJAUGU U GUGUUCU GUUGUG,'U U CUAAUTAA UCJGUGUU C UAAUAAA GUGOUUCU A AUAAAAC UUCUAAU A AAACAAA CAAAAAU A GACAACU

UAUJAUU

UtJCAGATJ

CAGAUAU

UCAGAAU

GAAUCAU

TJUGAAGU

GAAGUAU

AAGUAUU

AGUAUUU

GUAUUU

UUUUcCU

C-CAAAAU

AAUUC-AU

UUG-AUAU

AUAUAcu UArUACUU C AGAMUC A UJCAGAAU C AGAAUCA C AUUGAAG U GAAGtJAU A UtUCCU U tiuccucc U UCCUCCA U CCUCCAG C CtTCCAGG C CAGGCAA U GAUAUAC A tU.CUUUU A CUUUUUU U UUUUCUU U UUUCUUA SUBSTITUTE SHEET (RULE 26) WO 95/23225 216 Table 12: Human ILr-5 HE Rib ozymae Sequences nt. HH Ribozyme Sequience Position 8 GCAAAGA CtJGAt7GAGC-CCCAAAGGCAA AGUGCAU 9 GGCAAAG CtGANUGAGGCCCGAAAGGCCGAA AAGtJGCA UGGCAAA CUGAUGAGGCCCAAAGGCC- GAA -AXAGUC-C 1- MILWC CU.GALGAGGC-CAAAGGCCGAA AGAAAGU 13 Ct-UU-GC CUGALGAGGCCCAAAGCO2A AAGAAAG 36 GCUCUGA CUGAUGAGGCCCAAAGG-CCAA ACGU.jCU 37 GGCTJCUJG CUGATJGAC-GCCG-AAAC-GCCCGAA AAC=JUC 38 UGG-CCTZ CUGAUGAGGCI-C-AAAG%--C--AA -AAACGtU 56 AAUGCAG CUGAUGAGGCCG.AA CGAA ACAUCc 57 AAAUGCA CUG-AUCGGCCGAAA GGCCCGA AAG-CAUC 63 AAACUCA CUCAUGAGCC-JA-AGGCCCGAA AL'C-CA-A.

64 CAAACUC CtJGALGAGGCCGAAAGC-CCGA- A AALGCAG 69 GCUAGCA CUGAUG-AGG-CCG-AAAGG- CC-AA ACUCAAA AC-UAGC CUGAL'GAGGCCGAAAGGCCGAA AACtJCAA 74 CAAGAGC CETCGAUGAGGCCC-AAAGGCCC-AA AGC.AAAC 78 GCUCCAA CUGAUGAGGCCG-AAAC-GCC2,AA AGCUC-C CAGCUCC CtJGAUGAGGCCG-AAAGGCCG-AA AGAGCUA 91 AUACACG CUGALGC,'GGCC-A-AAC-GCC2AA, AC-GCAGC 97 GAtJGGCA CUG-ALGAGGCCG-AAAGG-CCG-AA ACACGUA 104 CUGUJGOG CUGAUGAGGCCGAAAGG-CCAA ALGGCAkU 1U6 tJUGUGGG CUGAUC-AGGCCAP)AGCCGA AUUUCtJG 117 CULTGUGG CUGAUGAGGCCGAAAGGCCGAA AAUUjUCU 1.30 UUUCACC CUGAUGAGGCCGAAAGGCCGAA AUGCACU 2.45 CAGUGCC CUG-AUGAGGCCG-AAACGGCCCGAA AGGucuc 1.55 GAGUAGA CUGATUGAGGCCGAAAGGCCGAA AGCAGUG 2.56 UGAGtJAG CtJGAtTGAGGCCG-AAAC-GCCG-AA AAGCAGtJ 157 AUGAGUA CUGAUGAGGCCGAAAGGCGAA AMAG 159 CGAUGAG CUGATJGAGGCCGAAAGC-CCCAA AGAAAGC 2.62 GUjUCGAU CUGAUC-AGGCCG-; AAGGCC-GA A AGUAGAA 165 AGAGUJUC CUGAUGAC-G'CCA.AAGG,-jCCAA; AUGAGtJA 2.72. AUCAGCA CUGAtIGAGGCCGAAAC-GCCG--A. AGuutcG-A 2.79 CAUUGGC CUGAUGAC-GCCGAAAGC-CC-GAA ATCAkGCAk 192 AUCCtJCA CUGAUGAGGCCG-AAAGC-CCGA-,A AGUCUCA 200 GAACAGG CUGAUGAGGCCG-AAAGGCCG-AAz AucctJcA 201 GGAACAG CUGAUG GGCCGA.AAGGCCG-AA AAtICCUC 206 GUACAGG CUGAUGAGGCCG-,AAGCcCGA AAGGAA 207 UGUACAG CUGAEJGAC-GCCG-AAAGGCCG-AA AACAGGA 21.2 UMUJG CUGAUGAGGCCG-AAAGGCCcGA:A ACAGkGzA 22.6 UC-AUTjtJU CUG-AUGAGGCCGAA-:AGC-CCGA AUGUjACA 222 AGUUGGU CUG-AUGAGGCCGA.AAGGCCGA; A AUUUUtJA 245 rCUGAAA CUGAUrGAGGCrC2LAA-GC-GAA; A:UUUcutJ IT/I1195/O() SUBSTITUTE SHEET (RULE 26) WO 95/23225 (7/11195/00156 217 247 tICCCUGA CUGAUGAGGCCGAAAGGCCCGAA AGAUIJUC 248 UTJCCCUG CUGAUGAGGCCGAAAGGCCGA.A AAGAUrU 249 AUUCCCtJ CUGAUGAGGCCGAAAGGCCGAA AAAGAtU 257 GtJGUGCC CUGAUGAGGCCGAAAGGCCGAA AtJucccu 273 AcAGUU cuGAUGAGGCCGAAAGGCCGAA ACUCUCC 291 UCCACAG CUGAUGAGGCCGAAAGGCCGAA Acccccu 305 UUUUGAA CUGAL'GAGGCCGAAAGGCCGAA AGUCUU 307 GUUUUG CUGAUGAGGCCGAAAGGCCGAA AUAGUCU 308 kGUUtUU CUGAUGAGGC'CGAAAGGCCGAA AAUAGUC 32.6 UJAAGGAC CUGAUGAGGCCGAAAGGCCG-AA AGUUUUrJ 319 UAUUAAG CUGATUGAGGCCGAAAGGCCGAA ACAAGUU 322 CUUEYAUU CUGAtIGAGGCCGAAAC-GCCGAA AGGACAA 323 TUCUUUAU CUGAUGAGGCCGuAkAGGCCGAA AAGGACA 326 AUUUCUU CUGAUGAGGCCGAAAGGCCGAA AUUAAGG 334 TJCAATJG CUGAUTGAGGCCGAAAGGCCGAA AUUUCUU 338 GGCCGEJC CtJGAUGAGGCCGAAAGGCCGAA AL'GUAUU 380 AUUGGUU CUGAUGAGGCCGAAAGGCCGAA AcUcucc 388 GUCUAGG CUGAUGaAGGCCGAAAGGCCGAA AUUGGUU 389 AGUtCUAG CUGAUGAGGCCGAAAGGCCGAA AAUUGGU 392 GGUAGUC CUGAUGAGGCCGAAAGGCCGAA AGGAAUU 397 UUGC-AGG CUG-AUGAGGCCG-AAAGGCCGAA AGUCaMG 409 ACCAAGA CUGAUGAGGCCG-AAAGGCCGAA ACUCUUG 410 CACCAAG CUGAUG2AGGCCGAAAGGCCGAA AACUCUU 411 ACACCAA CtJGAUrAGGCCGAAAGGCCGAA AAACUCU 413 UUACACC CUGAUGAGGCCGAAAGGCCGAA AGAAACU 419 UGUUCAU CUJGAUGAGGCCGAAAGGCCGAA ACACCAA 437 UUUCUAU cuGAUGAGGccGAAAGGccGAA AucC-Acu 440 AACUUUC CUGAUGAGGCCGAAGGCCG-AA AUUAUCC 447 UAGUCUC CUGAUGAGGC-CGAAAGGCCGAA ACUtJUCU 454 ACCAGUTJ CUGAUGAGGCCGAA;k GCCGAA AGUCUCA 462 UGCAACA CUrGAUGAGGCCGAAAGGCCGAA ACCAGUU 463 CUGCAiAC CUGAUGAGGCCGAAAGGCCGA.A AAccAGu 466 UGGCUGC CUG-AUGAGGCCGAAAGGCCGAA ACAAACC 479 CCtJCCAA CUGAUGAGGCCGAAAGGCCGAA AUCUUUG 480 UCCUCCA CUGAUGAGGCCGAAAGGCCGAA AAUJCUUU 481 CUCCUCC CUGAUGAGGCCGAAAGGCCGAA AAAUCUU 497 GCAGUAA CUGAUGAGGCCGAAAGGCCGAA AuGucctJ 498 UGCAGUA CUGAUJGAGGCCGAAAGGCCGAA AAUGUCC 499 CUGCAGU CUGAUGAGGCCGAAAGGCCGAA AAAuGuc 500 ACtJGCAG CUGATJGAGGCCGAAAGGCCGAA AAAAUGU 531 AAGGCCU CUGAUGAGGCCGAAAG-GCCGAA ACtJCUUU 538 CAAAAUJU CUGAUGAGGCCGAAAGGCCGAA AGGCuG 539 UGAAAAU CUGAUGAGGCCGAAAGGCCGAA AAGGcctJ 542 UAUUGAA CUGAUGAGGCCGAAAGGCCGAA AUUAAGG 543 AUAUUG-A CUGAUGAGGCCGAAAGGCCGAA AAUUAAG 544 UAUAUUG CUGAUGAGGCCGAAAZGCCGAA AAAUJUAA 545 UTJAUAUU CUGAUGAGGCCGAAAGGCCGAA AAAAUtJA 549 UAAAUUJA CUGAUGAGGCCGAAAGGCCGAA kUUGAAA 551 GtJUAAAU CUGAUJGAGGCC GAAAGCcGAA AUAUtJGA SUBSTITUTE SHEET IRULE ?6) WO( 9513225 218 554 GAAGUUA CLUGAUG-AGGCCGAAAGGCCGAA AtUtAUAU 55:5z UGA.GUU CUGAUGAGGCCGAAAGGCCGAA AAUUAUA 556 CUGAAGTJ CUGA!rzAGGCCGAAAGGCCGAA A.AUUAU 560 CCCtJCUG CIJGALIGAGGCCGAAAGGCCGAA AGtJUAAA 561 UCCCUCtJ CUGALIGAGGCCGAAGCCGAA AGUIAA 573 AAAUAUU CUGAtGAGCCGAGGCCGAA ACUUUCC 577 CCUGAA CUGAUGAGGCCGAAGGCCGAA AUUUACU 579 UGCCLG-A CUGAUGAGGCCGAAGGCCG-AA AUAUUUA 580 ATJGC:tJUG CEJGAJuGGGCCGAAAGGCCGAA AAUAU~Tj- 581 tJAUGCU CGUGAGGCCGAAAGGCCGAA AAUAUU 588 GUGUCAG CUGAUGAGGCCGAAAGGCCCAA ATJGCCUG 597 TJUCGCA CJGAUGAGGCCGAGGCCGA. AGwGUCA% 98 UUCUGGC CUGAUGAGGCCGAAAGGCCGAA AAGUGUC 611 AGAAUUU CUG7AUG AZGCCGCAAAGiGCCGAA AUC-ZMTU 616 UUUUAG CUGAUGAGGCCGAAGGCCGA ALTUUUAU 617 AtUUA CUGAUGAGGCCGAAAGGCCGAuA AUUJUA 619 AUAtUUU CUGALGAGGCCGAC-GCCGA AGAAUUU 620 UAUAUUU CUJGAUGAGGCCGAAAGGCCGAA AAGAAUU 625 UGAAAUA CLTGATGAGGCCGA.AAGGCCGAA AtJUUtJAA 627 UTCUGAAA CUGAUGAGGCCGAAAGGCCGAA AtJAUUUU 629 UAUCUJGA CUGCAUGAGGCCGAAAGGCCGAA AUAtJAUU 630 AUAUCUJG CUGAUGAGGCCGAAA=GCGAA AAUAUAU 631 G-AUAUCU CUGATJGAGGCCGAAAGGCCGAA AAAtJAUA 636 AUUCUG-A CCUGAtJGAGGCCGAAAGGCCGAA AUCUGAA 638 UG-AUUCU CUJGAUGAGGCCGAAA.GGCCG-AA AUAUCUG 644 CUUCAAU CUGAUGuAGGCGAAAGGCCGAA AUUCUGA 647 AUAhCtJUC CUGAUGAGGCCGAAAGGCCGA AUGAUUC 653 AGGAAAA CUGAUGAGGCCGAAGGCCGA ACUtJC 655 GGAGGA. CUGAUGAGGCCGAGGCCCGA AUACUUC 656 UGGAGGA CUGAUGAGGCCGAAAGGCCGAA AAUACLTU 657 CUGGAGG CUGAUGAGGCCGAGGCCGAA AAtJACJ 658 CCUGGAG CUGAUGAGGCCG7 -,CCGAA AAAAAC 661 UUGCCUG CUG-AUGAGGCCGAAAGGCCGAA AGGAA 672 GtJAUAUC CUJGAUGAGGCCGAAAGGCCGAA AUUUUGC 676 AAAAGUJA CUGAUGAGGCCGAAAGGCCGAA AUCAAUU 678 AAAAAG CUGAUG-AGGC-CGAAAGGCCGAA AUAUCAA 681 AACAAA;A CUGAUGAGGCCGAAAGGCCGAA AGtU.UAU 682 UAAGAAA CUGAUGAGGCCG-AAAGGCCGAA AAGUAUA 683 AUAAGAA CUGAUGAGGCCGAAAGGCCGAA AAAGUAU 684 ijAULAAC-A CLGAUGAGGCCGAAAGGCCGAA AAAAGUA 685 AAAIJAAG CUJGAUGAGGCCGAAAGGCCGAA AAAAAGu 686 UAAAUAA CUGAUGAGGCCGAAAGGCCGAA AAAAAAG 688 GUtJAAAU CUGATJGAGGCCGAAAGGCCGAA AGAAAAA 689 AGUUAAA CUGALGAGGCCGAA.AGGCCGAA AAGAAA 691 UAAGUUA CUGAUGAGGCCG-AAAGGCCGAA AUAAGAA 692 tUUAAGUU CUG-ATGAG-GCCGtAAAGGCCGAA AAUAAGA 693 GLUUAAGU CUGAUGAGGCCGAA-AGG-CCAA AAAU-AG 697 G-AAUGUU CUG-AUGAGGCCGAAAGGCCG-AA AGUUAAI\ 698 AGAAUGU CUG-AUGAGCGCCGAAAc-GC.CGAA AAGUUAA Pc~r/iB'5/00 156 SUBSTITUTE SHEET (RULE 26) I 1 0 WO 95/23225 219 703 UUACAG CtJGAUGAGGCC-GAAAGGCCGAA A1Gt3UA 704 =MU~CA CUGAUGAGGCCGAAAGGCCGAA AAt7GUUA 708 G-ACALTUtU CtUGAUGAGGCCGAAAGGCCGA.A ACAGAAU 715 GMt=hCA CUGAt7GAGGC-CGAAAGGCCGAA ACAUUtTE 72.9 UUAAGUtJ CUGAUGAGGCCGAAAGGCCGAA ACAGACAN 720 AUUAAGtJ CUGAUGAGGCCGAAAGGCCGAA AACAGAC 724 UACUAUXU C-UGMLMGGCCGAAAGGCCGAA AGLUMAC 725 AUACUAU CUMUtGAGGCCGAAAGGCCrAA AAGuuAA 728 UAAAt3AC CUGALGAGGCCGAAAGGCCGAA AtJUAAGU 731 tICAUAAA CUGAUGAGGCCGAAAGGCCGAA ACUAtJUA 733 LUUCAtJA CtGAUGAGGCCuAAAGGCCu-AA AUACUAU 734 AUU~CAU CUGAUGIAGGCCAAGGCCGA AAUTACUA 735 LALWCA CLGAUGAGGCCGAAAGGCCGAA AAAUJACU 745 Au.AtU CU'GAUGAGGCCC-AGG2C:GAA ACCAUUU 746 AAATUJCU CUGAUGAGGCCGAAAGG~CCGAA AACCAUU 752 UUUACCA CUGAUGAGGCCGAAAGGCCGAA. ATiuCUUA 753 ATUUACC CUGALtGAGGCCGAAAGGcc:GAA AAuTJCUU 757 ACUAATU CtLGADGAGGCCGAAAGGccGAA kCCAAAU 761 AAATJACUT CtGALGAGGCcGAAAG~ccGAA AuutJACC 762 UAAAUAC CUGAUJGAGGCCGAAAGGCCGAA AAUtJUAC 765 AA.At7AAA CtJGAUGAGGCCGAAAGGCCGAA ACt3AAUtJ 767 L7UAAAUA CUGAUGAGGCCGAAAGGCC-GAA AtJACUAA 768 ALUAAAU CUGAUGAGGCCGAAAr.GCCGA AAUACUA 769 C-AUUA CUCAtGAGGcccAAGGccGAA AAtJAcu 771 AACAUUA CUGAUSAGGCGAGGCCGAA ALAAtA 772 UAACMU5U EJGAUGAGGCCGAGGCCG-A ?AAUU 773 AUACAU CtGAUGAGGCCGAAAGGCC:GA.A AAAUAAA 778 AcA.ACAu ctJGAtJGAGGCCGAAAGCCGAA AC-AuuAA 779 CACAACA CtJGAUGAGGCCGAAAGGCCGAA AACAUUA 783 AGAACAC CUGAUJGAGGCCGAJAAGGCCGAA ACAUAAC 788 UUAUU-AG CtJGAMJAGGCGAAAGGCCGAA ACACAAC 789 UtJUAtJUA CUG-Ai.AGGCCGAAAr.GCCGAA AACACAA 791 GUUtUUAU CUGAruGGGCCGAAAGGCCGA AGAACAC 794 UTJUGUUU CUJGAUAGGCCGAAAGCCGA AUUAGAA 805 AGtJUGtC CUGAT7GAGGCCGAAAGGCCGAA AUUUUUJG rc'111195/001I56 SUBSTITUTE SHEET (RULE 26)

I

WO 95/23225 220 Table 13: Mouse IL-5 HR Rihozyme Target Sequence PCTI95/00156 HK Target sequence 12 36 36 37 43 58 59 59 66 82 91 112 113 141 141 158 167 196 197 197 202 202 206 212 212 218 218 218 232 241 241 241 241 243 243 244 245 CG,-CUUt uCUUCJ C'cCUly G-AAgacU GaAgActJ AAgacPU tcaGaGtl

GC-AUGCU

GAUGCUTJ

gAUGcUtJ CUGCActJ UgAcucU Gct~gUGtJ ugGAgAU gG-AgAUU GAGACCtJ GtgACct7 gtJccgCU cCGArTCU UGAGGcU GAGGcUU gAGGCuU tUtCCtJ(tJ UUC=Ut UGtJCccU tJACtTCAU tUacuCAU UaaqAaat

UAAAAAU

UAAAAAU

uaUGCATJ gAC-AAAU gAgAaAU gagAAAU gAgAaAU ga..AAucU

GAAATJCU'

AAAUCU

AAU=UUjJ CUULG--u UCt-gAAL GcU9AAG CAGAGuC CAgAGtUC

AGAGUCA

AIJGAgaA CLTGCAcU

UGCACUU

uGcActU CGAGugu aGcUGtJG uggGCCA CCCAugA CCAugAG GaCACaG GaCAc~g AcCGAgC tJGuMJAc CCUGL7CC

CEJGUCC

CtJGUCC CCtuacuC CcUAcuc ci.CaUAA aAAaUCa

AAAAUCA

aCcAGCU ACCAgCU acCAgCU C-GaGAA

TJUUCAGG

UUucAGG

UUUCAGG

UUUCAGg UCAGgGg UCAGW3g CAGGGgC AGGGgcU nt Position 253 259 269 269 269 287 301 301 303 303 304 315 318 31.9 322 330 334 334 384 385 393 405 406 409 481 4 82 483 483 495 553 557 564 564 565 565 569 569 613 614 AGCGGgcUJ UagACAU Ga.AGCAat GaAGAa-U G-AAgaAU u~GGGGGIJ AAAugCtJ AAAugCtJ AtGCuAU ugCtlnm AACcUGtJ cUGUCaU Ut;CatUU CaULAPAU

:AAGAAAU

?AUACAU

AAt~aCaU AggCAgu ggCAgtU C7igGAUtJ

CAAGAGY

A.A'GAGUUJ

AGUUcCU UCaCAAU cAcAAUTJ AcAPAUuU AAAUUgtJ GC-UGuuU UuUcCAU I UtauAutJ 1 tJUAuaUUt i uaUAUU7J tJ?.UAuUU

UUUAUGU

ub-UATJGtJ AAAGuGU AAgUGutT'.

GaC.AuAC C rUC a AgA

AAACTJGU

AkAaCugtJ aA~t.Tgt

CT;GUGGA

TJUC XAA uUC%-aa.A CCaAaAc CcAAAAC cAAAACc allLTAAUJA

AATJAAAG

ALLAAAGA

AAGAAAtJ C--,TjuGAC CGACcGCC CkCCgCC CCt~gGAu Ct~gGAuU

CCUGCAA

cCtJUGGTJ

C'JTCGGQUG

C-UGUgA UAAgUUA A.AgEuuaA AgtUuaAa aGUUAAa AAcAgAU Cat~uAU UauaUU atlgUJCCU AugUcCtJ ugOCCuG ugtuccrjg cUGUaGtJ CuGuagu uaaCCtrJI a.ACcUL'U I4H Target Sequence SUBSTITUTE SHEET (RULE 26) WO 95/23225 PCT/IB95/00156 221 620 tJUAACctJ u ut~uGtJAU 1407 cCAgUUU A CUcCAGg 793 caAGgCU u IGuGcAU 1407 ccAgUUU a CUCCAGG 816 CUGag~tTJ a tACUCcc 1410 gt~tJUaCU C CAGGaAA 818 G-AguUAT a cUCCcuC 1434 AUgCULtJ U atlutaAt 825 ACt~cCctJ c CcCECA 1434 allgcUuU U kUJUUAAu 825 aCE~ccCtJ c CcCcEJCa 1434 allgcuUU u AuUUAAUJ 839 AuCcucU U cGTUGCAk 1435 LUgCUUtJU a UuE~aAUU 840 uCcucUU c GUUGCAu 1435 ugcUUUt a utJUAaUt 863 cAkAgUAU U cCAGGCu 1438 UuTjtJAUU U ?AAuUcug 864 AAgUAUU c CAGGCug 1438 urUUUAUU U AAUucUg 864 AAGU-AU c caggO~ig 1439 UUUAUUU A AUucUgU 913 gAaCUCU U GGucCaG 1443 UUTUaAuU c UGuaAGa 917 UctiuggU c CAGAUGG 1447 AUJUCUGU A AgAUGUu 957 uuagcAkU c Cm-tucuc 1458 ugtUcaU a UUJAUUUA 960 GCAucc-U u UcUcCuA 1458 tUgUUCAkU A uUAUUUA 960 GcaUcCU u uCUCcUa 1460 UucAUAU u AtUUAug 962 AUc~uuU c UCcUaGC 1461 UcAT.MuU Ak UUtUAA 975 gcccCt2U u AgAtL.gA 1463 AUAUtJAU U UAUGAug 987 aGaUGAU A cuuAAUG 1475 AuGgAUJU c aGUAAgU 990 UGAuACU u AAugacU 1479 AUUcaGU A AgUUAaU 1000 UGACuCU c UugCuGA 1483 aGuAAGU u AAUAUUU 1027 CgggG-CU U ccugcuc 1483 aGtUhAgU U AaUAUTU 1034 UCCUGcU C CUaUcuA 1484 GUAAgUU A aUlAUUUA 1037 UgcUCcU A UcUAACU' 1487 agUUAAU a UUuAuUA 1039 cUccuAU c UAACUUC 1487 AgUUAaU A UUjUAUUa 1039 cUCcUAU c TAACUUc 1489 tUUau u uAuuc 1041 CcUAUcU A ACUcAa 1489 UUAAuAU u UAUUaCA 1051 UUcAAliU U AAuAccC 1489 UUAaUAU U UAUUacAk 1148 uGAcUJU u cUuaUGU 1490 U?-AUaUU u AuUAc-kc 1213 GCgGaU u UUGGaa 1490 U~aTAUU U kUuAc:Ac 1213 gcUGGAU u uUgGAA 1490 TJAaUATUU U AUUacAc 1214 cugGAUU U UGGAaaA 1491 AAUAUUU a uuaCAcg 1215 ugGAUUJU U GGAaaAG 1491 AAUJAUuU a UuAcAcg 1234 gGGACAU c UccuUGC 1491 AaUAUUU A IUuAcAcG 1236 GACAUcU c cuUGCAG 1491 AaUAUUU A Lrtac:AcG 1275 ugGGCCU U AcUUcUC 1494 AUuUAUU a CAkcgUAU 1276 gGGCCUU A cUUcUCc 1502 cACGUaU A UaauAUu 1280 CUUAcUU c UCcgUgU 1502 cAcgUAU a IJAAUaUU 1298 UgAACUU a AGAaGC-A 1507 AUAUAaU a UUcUaaU 1310 gcAAAGU a a-AuACcA 1509 AUAAuAU U CUaAuAA 1.310 GCAAAgU a aAUAcca 1509 allaaUaU U CUAAUAA 1310 GcaAAgU a AAUAccA 1510 UAAuAUU C UaAuAAa 1350 AAAGCAU A AAAUggU 1510 UAAuAUU c uaauA.A 1358 AAAUGGU U ggGAugU 1510 UAAUAUU c UaaUAAA 1.370 UgUuaUU C AC-gUAUC 1510 UaaUa3U C tJA.AUTAAA z.375 UUCAGgU A UJCAGggU 1512 allaUUCU A kTU-eAAgC 1377 CAGgUAU C AGggUCA 1515 UUCUAAUJ A AAgCAgA 1383 UCAGggU C AcUGgAG 1405 cccCAgU U UACUcCA SUBSTITUTE SHEET (RULE 26)~

I

WO 95/23225 222 Oil 0 Pii PCT/11395/00156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 WO 9523225PCT/1B95/OO 156 223 14 f9 151

H

0 0 P4 oC)tfLnN -4 CM0~ v )C SUBSTITUTE SHEET (RULE 26)

I

PCT/I 195/0(156 WO 95/23225 224 DIH ~I IgiI I I

H

HI

Di uA C C) II Hull 0 0 C) U1 (N L -4 :Ce

C

Po a r rHq SUB STITUTE SHEET (RULE 26) WO 95/23225 PC'T/11395/OO 156 225 Table 17 Mouse re/A HH Target sequence nt. Position HH Target Sequence nt. Position HH Target Sequence i19 AAtJGGCU a caCaGgA 467 cCAGGCU c cuguUCg 22 aGCUJCcU a cGUgGUG 469 ApG~A u AGcCAGC 26 CctjCcatJ u GcGgACa 473 UuE~gAGU C AoauCAg 93 G-AuCUGJ U uCCCCUC 481 kC-C%-7azU C C-AGACCA 94 AuCUGUU U CCCCUC% 501 AACCCCU U UCACGUU 100 UuCCCCJ C AUCJUuC 502 ACCCCUU U CACGUtJC 103 CCCUCAU C TUTuCF-cu. 508 UUCAcGU U CCIAUAG 105 CIJCAUCU U UCCcuCA 50G9 LC-kcGUU C CUAJAC-A 106 UCAUCUU u CCcuCAG 512-' cGUUCCU A UAG-AgGA 129 cC-kr^uU C UGGgC~L1 5i4 UUCCt3AU A G-AgGAGC 138 G-GgCCuU A UGUGGAG 534 GGGGACU A UG-ACUUG 148 UGGAGAU C AUcGAaC 556 UGC-GcCU C UCUUCC 151 AGAUCAU c GAaCAGC 561 CUCUGCU U CCAGGUG 180 ATJGCGaU U CCGCUAu 562 CUtGCLUU C CAGGUGA 181 UGCGaUU C CGCTTAUA 585 aAgCCAU u AGcCAGc 186 UUCCGCU A uAAaUGC 538 GGCC'CU C CuCCUC-a 204 GCGGCGCU C aGCGGG 613 C-CCGG C C'JvuCaC 217 CGuU u C~C-GCG 616 CUGUCCU c uCaCAUC 239 CAkCAGAU A CCAXC~kk 617 gucCCUU C CUCAgCC 262 CCACCAU C AAGAUCA. 620 COUCCJ C AgCCaug 268 UCAAGAU C AALU 623 UCCt~gcU CCAUCUC 276 A-AUGGCU A r-ACAGGA 628 AUCCgAU u UUUGAULA 301 UuCGaAU C UCCCUGG 630 C-,gAZUuU U UGAUAAC 303 CGaAUCU C CCUGGCJC 631. CgAUuUU-r U G-uAC 310 CCCUGGU C ACCAAGG 638 UC-gCcAU u GGuuCC 323 GGcCCCU C CUCcuga 661 CCGAG= C A:-GCAUCU 326 uCCaCCU C ACCGGCC 667 UCAAGAU C UG-CCGAG 335 CCGGCCU C AuCCaCAX 687 Cr-gAACU C UGGgAGC 349 AuGAaCU U GUgGGQA 700 GCULGCC-U C GUGooo 352 AGaUcaU c GaAcAGc 715 AUGAGAU C UjUCUgC 375 C-AUGGCU a CUA.UGAG 72.7 GAGAUCU U C-UgCUG 376 AUGGucU C UccGgaG 718 AGAUCUUJ C UUgCUGU 378 G.2CUaCrU A UGAGGCU 721 UucUCCU c CauUGcG 391 CUGAcCJ C UGCCCaG 751 AaGACAU U GAC-GUGU 409 GCaGuAU C CAuAGcU 759 CAGGJG A UUU")TCACG 416 CCgCAGU a UCC.AuAg 761 GGUGUAU U UCACGGG 417 CAu.AGcU U CCAGAAC 762 GUJGUAUU U CACGGGA 418 AuAGCUU C CAGAACC 763 UGUAUUJ C ACGGGAC 433 UGGGgAU C CAGUGUG 792 CG-AGGCU C CUJUUUCu 795 GG-CUCCU U UUCuCAA 1167 G-ACCAGUJ U UuCCcCC 796 GCUCCUT U UCUCAAG :168 AU -GUGU U uC--cCCA 797 CUCCUUUJ U CuCAAGC 1169 UG-AGUJuU L C-CCCAL 798 UCCVUU C UCAAGCJ 1182 AUGcUGU U aCCaUCa 829 UGG-CCAU U GUGUUCC 1183 UGcUGUJ a CCaUCaG SUBSTITUTE SHEET (RULE 26) PCT1B95/00 150 WO 95/23225 226 834 835 845 849 872 883 885 905 906 919 936 937 942 953 962 965 973 986 996 1005 1006 1015 1028 1031 1032 1033 1058 1064 1072 1082 1083 1092 1097 1098 1102 1125 1127 1131 1132 1133 1137 1140 1153 1158 1680 1681 1683 1686 2.690 AUUGUGU U UUTGUGUU C GACUCCU C CCUCCgU A cCAGGCU C UuCGaGU C CGaGtJCU C GCGGCCU U CGGC=JU C GcGAG%-tJ C AUGGAgU U UGG-AgU C uuCCAGu A GCCucAbU c AGAuGAU C CagUacJ u ACCGGAJ U GAgACcU u AGGA~cU A GAGACCJ U AGACCJU C AGAGuAU C GAAG-AGU C GAGUCCU U AGUJCCJU U GUCCJUUu C CCGGCCU C UacAcctJ u GgCGuAU U UGUGCCU a aaGCCUU C CGaAaCJ C CIJCAaCU U UCAaCUU C CT.UCUGU C CAGCCCU A GCCaUAU a cAUCCCU c AcaCCUJU c UCCaUcU c UUtJACuU u cCagCAU C GCACCAU C AUCAACU u GAAGACU U AAGACUJ C G-ACuutCU C uucUiCCU C CCUCCAU U

*CCGGACU

CGGACUC

CgUACGC

*CGCCGAC

CUGt~uCG

UCCAUGC

CATJGCAG

CuGAuCG

UGAUCGC

AGUGAGC

CCAGUAC

MIUACu

CUUGCCA

CACAuGA

GCA-CCG

gCCaGAc GAaGAGA cAAGagu

UGAGACC

AAGAGuA

AUGAAGIA

CUUJUCAa UCAauGG CAauGGA AauGG-AC CAaCcCG GAucCAa

GCUGUGC

CCCGaAa CCGaAGu AaCtJUCU

CUGUCCC

UGUCCCC

CCCA~.GC

caCCUc gCcUUAC agCacCA cCagCAU CagCuUC AgCgCgc CCUcAGC

AACUUUG

UGAUGAG

ctuccUc

UCCUCCA

CUCCATU

CAUUGCG

GCGGACA

1184 1187 1188 1198 1209 1215 1229 1237 1250 1.268 1279 1281 1286 1309 1315 1318 1331 2.334 1389 1413 1414 1437 1441 1467 1468 1482 1486 1494 1500 1501 1502 1525 1566 1577 1579 1583 1588 1622 1628 1648 1660 1663 16 4 1665 GGccccj C CUcCUGa GUccCuJ c CTUcaGCc UEaCCaU C aGGGCAkG GGgAGuU u AGuCuGa CAkGcCCJ a caCCUUc cuGGCCU U aGCaCCG GGuCCCJ u CCucAGc CCC-AgcU C CUGCCCC CCAGcCU C CAkGgCuC CCCaGCU C CuGCCcc CCAUGG~u c cCuuCcu gUGGgcU C :%GCUqcG AUgAGuU u UccCCCA CuCCUGU u CgAGUCu cCCCAGU u CUJAaCCC- CAGUuCU A aCCCCgG gGGuCCU C CcCAGuC CuuUuCU C AaGCUGa ACGCUGU C gGAaGC-- CUGC-AGU U UGAUGcU UGC-AGU U G-AUGcUG GGGGCCU U GCUUGGC CCUUGCU U GTGCAACA GgaGUGU U CACAGAC gaGUGUU'T C ACAGACC CUGC-CAU C uGUgGAC CuUCgGU a GggAACU GACAACU C aGAGUUJ UCaGAGU U UCAGCAG CaGAGJU U CAGCAGC aGAGUUU C AGCAGCU gGuGCAU c CCUGUGu AUGGAGU A CCCUGAa UGAaGCU A UAACUCG AaGCUAU A ACUCGCC UAUAACU C GCCUgGJ CUJCuCCU A GaGAggG CCCAGCU C CUGCcCC UCCUGCU u CggUaGG CGGGGCU u CCCAAUG cUGaCCU C ugccCAG cuCUgCU U cCAGGuG uCUgCUU c CAGrjuGA CUCgcUU u cGGAGgU SUBSTITUTE SHEET (RULE 26) WO 95123225PC/B/O15 PCT/IB95/00156 227 1704 1705 1707 1721 1726 173 1 1734 1754 AUGGACtJ UGGA=tt

GACUUCU

uuUGAGU

GUCAGAU

AM=GC

AGlCtccu CaGugCtJ u cucuGcu C UCuGCuC C uC-CuCu C AGAUCAG C AGCUCCEJ C CUAAGGu A AC-GGCU C CCaAGAG SUBSTITUTE SHEET (RULE 26) WO 95/23225 P)CTIB95O()156 228 Table 18 Human re/A HH Target Sequences nt. Position HH Target Sequence nt. Position HH Target Sequence 19 AAUJGGCU C GUCUGUJA 467 GC7C--G7J A UCAGUCA 22 CG,-cUCGU C UGUAGUG 469 ACGCUU C AkGTJCAGC 26 CGUCtJGU A GUGCACG 473 UAUr-MGU C AGCGcAU 93 GAACtJGU U CCCCCtJC 481 ACGCAU C CAGACOam 94 AACUGUU C CCCCUC-A 501 AACCC= U CCAAMUTU 100 UCCCCCU C AUJCUUCC 502 A C C C C U C C-AAGUUC 103 CCCUCAU C UUCCCGG 508 UCC-AAGU U CCT)AG 105 CUCAUCU U CCCGGCA 509 CC~AcU C CUUAGA 106 UCAUCUU C CCGCAG 512 AGU-UCVJ A U7GAG 129 CAGGCCU C UJGGCCCC 514 UUCCU A -A-AGAC 138 GGCCCCtJ A UGUGGAG 534 GC-GC-ACU A CGACCUG 148 UGGAGAU C AUUGAGC 556 CGCGGCJ C UG(7UUCC 151 AGAUCAU U GAGCAGC 561 CrjCtjC-Ct U aGU 180 AJGC-GCtJ U CCGLtMhC 562 UCUC-~TJ C GAGGTJGA 181 UJGCGCUUW C CGCEJCA 585 G-ACCCAU C AGGrCA:GG 186 UUCCGC-U A CAAGUGC 598 GCC""u C C--=UG 204 GGGcGCU C CGCGGGC 613 cc-CcUGL c =cuccUC 217 GCAGCAU C CCAGGCG 616 CUGUCCU U CCUCAUC 239 CACAGAU A CCACCAA 617 ucucLIIJ-u c CUCATMcc 262 CCACCAU C AAGAUCA 620 =j LuucCU C AUCCCAU 268 UCAA-AU C AAUGGCU 623 UCCUCAU C CCAUCUtJ 276 AAUGGCU A CACAGGA 628 AUCCCAU C 'JtJUGACA 301 UGCGCAU C UCCCUGG 630 CCCA UCU U UGU-CAA6 303 CGCAUCU C CCtJGGUC 631 CC-A-UCU U G-ACAAUC 310 CCCUGGU C ACCAAGG 638 UC-ACAAU C G3UGCCCC- 323 GGACCCU C CUCAkCCG 661 CCCGAGCU C kAGAUCU 326 CCCUCCU C ACCGGCC 667 UCAAGAU C UGCCGAGk.

335 CCGGCCU C ACCCCCA 687 CGAAACU C CGGCAGC 349 kCGAGCU U GUAGGAA 700 C-CUC-CCU C GGUGGGG 352 AC-C=tGU A GGAAAGG 715 A-UGAGAU C UUCCUAC 375 G-AUG=C U CUAUGAG 717 GAZA=C U CCUACUG- 376 AUGGCU C UJAUGAGG 718 AGAUCUU C CUACtJGU 378 GGCtJUCU A UGAGGCU 721 UCUUCCU A CUGUGUG 391 CUGAGCU C UGCCCGG 751 AC-GACAU U GAc-GUGUJ 409 GCUGCAU C C-ACAGUU 759 C-ACGUGU A TjUU~CACG 416 CCACAGtJ U UCCAGAA 761 GG-UGTJAU U UCACGGG 417 CACAGUU U CCAGAAC 762 GCGUAUtJ U C-ACGGGA 418 ACAGUUEJ C CAGAACC 763 UGUATUU C ACC-GGA--C 433 UGGAAU C CAGUGUG 792 CGAGZGCtJ C CUUUUCG 795 GGCUCCU U UUCGCAA 1167 GAUGAGU7 U CCCCACC 796 GCUC=U U UCGCAAG 1168 AUGAGUU U CCOACr-k 797 CUCCUUU U CGCAAGC 1169 UGAGU-C C COACCAO 798 UCCUUUU C GCAAGCU 1182 AUC-GU U UCC-UUCU 829 UGGCCAU U GUGUUCC 1183 UGGTUUU U CCUUCUG 834 AUUGUGU U CCGGACC 1184 G-rUjw C Cr-uTCuc-G SUBSTITUTE SHEET (RULE 26) WO 95123225 PCTIII95/IOO 156) 229 835 UU.GUGUU C CGGACCC 1187 GULTUCCtJ U CUGGGCA 845 GACCCCU C CCUACGC 1188 UUUCCUU C UGGCAG 849 CCUCCCU A CGCAC 1198 GGCAGAtJ C AGCCAGG 872 GCAGGCU C CtJGUGCG 1209 CAGGCCTJ C GGCCUtJG 883 UGCGUGU C UCCAUGC 1215 UCGGCCU U GGCCCCG 885 CGUGUCU C CAUGCAG 1229 GGCCCCU C CCCAAGU 905 GCGGCCU U CCGACCG 1237 CCCAAGU C CUGCCC 906 CGGC-tJ C CGACCGG 1250 CCAGGCU C CAGCCCC 919 GGG-AGC-U C AG3CAGC 1268 cCtJGCJ C cAkG-CAU 936 AUGGAAU U CCAGUAC 1279 CCAUGrGU A UCAGCUC 937 UGGAAUTJ C CAGLICC 1281 AUC--.UAU C GUU 942 UUCCAGU A CC-UGCCA 1286 AUC-kGCU C UGGCCCA 9 53 GCCAGAtJ A CAGACGA 1309 CCCCUGU C CCAGUCC 962 AGACGAU C GUCACCG 1315 UCCCAGU C CUAGCCC 965 CGAUCGU C ACCGGAU 1318 CAGUCCU A GCCCCAG 973 ACCGGAU U GAGGAGA 1331 AGGCCC-U C CUCAGGC 986 C-AAACGU A AAAGGAC 1334 CCCUCCU C AGGCJGU 996 AGGACAU A UGAGACC 1389 ACGCUGU C AGAGGCC 1005 GAGACCU U CAAGAGC 1413 CUCG U UGAUGAU 1006 AGACCUU C AAGAGCA 1414 UGCAGUU U GAUGAUG 1015 AC-AGCAkU C AUGAAGA 1437 GGGGCCU U GCUU=GCC 1028 GAAGAGU C CUUUCAG 1441 CCUUGCTJU U GGCAACA 1031 GAGUCCU U UCAGCGG 1467 GCtJGUGU U AAA 1032 AGUCCUU U CAGCGGA 1468 CUGUGUU C ACAGACC 1033 GtJCCUUU C AGCGGAC 1482 CUGCA*U C CGUCG-AC 1058 CCGIGCCU C CAkCCUCG 1486 CA7UCCGUT C GACAACU 1.064 UCCACCU C GACGCAt 1494 GACAACU C CGAGUUU 1072 G-ACGCAU U GCUGUGC 1500 UCCGAGU U UCAGCAG 1082 UGUGCCU U CCCGCAG 1501 CCGAGUJU U CAGCAGC 1083 GUGCCUJU C CCGCAGC 1502 CGAGUUU C AGCAGCU 1092 CGCAGC C AGCUUCU 1525 AGGGCAU A CCUGUGG 1097 CUCAGCU U CUGUCCC 1566 AUGGAGU A CCCUGAG 1098 UCAGCU C UGUCCCC 1577 UGAGGCU A UAACUCG 1102 CUUCUGU C CCCAAGC 1579 AGGcUAU A ACUCGCC 1125 CAGCCCU A UCCC=U 1583 UUAAjCrj C GCCUAGU 1127 GCCCUAU C CCUUUAC 1588 CUCGCCU A GUGACAG 1131 UAUCCCU U UACGUCA 1622 CCCAkGcu C CUGCUCC 1132 AUCCCUU U ACGUCAU 1628 UCCUGCU C CAkCUGGG 1133 UCCCUUU A CGUCAUC 1648 CGGG-GCU C CCc-ZAUG 1137 UUUACGU C AUCCCUG 1660 AUGGCCU C CrUUUCAG 1140 ACGUCAU C CCUGAGC 1663 GCTJUCTJ U UCAGGAG 1153 GCACCAU C AACUAUG 1664 CCUCC U CAGGAGA 1158 AUCAACU A UGAUGAG 1665 CUCCUUU C AGGAGAU 1680 GAAGACU U CUCCUCC 1681 AAGACUU C UCCUCCA 1683 G-ACUJUCU C CUCCAUU 1686 UUCUCCU C CAUUGCG 16,90 CCUCCAU U GCGGAcAk 1704 ALUGGACU U CUCAGCC SUBSTITL7C SHEETI (POLE 26) WO 95123225 WO 9523225PC'r/11395/00156 230 1705 1707 1721 1726 1731 1734 1754

'C--GACUU

G-ACUCU

C-CUGAGU

GTJCAGAU

AUCAGCU

AGCUCCTJ

CU-GCCCY

C UCAC-CCC C AGCCCtJG C AGAU.CAG C AGCUCCU C CUAAGGG A AGGGGGU C CCCAGAG SUBSTITUTE SHEET (RULE 26) WO 95/23225 PC'11195/0()156 231 Table 19 Mouse rel A HH .Ribozyme Sequences nt. HH Ribozyme Sequence Sequence 19 tJCCTJGUG CUGAUGAGC-CCGAAAGGCCG-AA AGCC-tktJ 22 CACCACG CUGAUJGAGGCCGAAAGGCCGAA AGGAGCUJ 26 tTGUCCGC CUGAtYGAGGCCCGAAAGGCCGAA AUGGAGG 93 GAGGGGA CUGAUGAGGCCGiAAAGGCCGAA ACAGAUC 94 UGAGGGG CUJGAUGAGGCC-GAAAGGCCGAA AACAGAU 100 GAA.UGAU CUGAUG-AGGCCGAAAGGCCGAA AGGGGAA 103 AGGGAAA CUGALUGAGGCCGAAAGGCCGAA PAUGAGGO 105 tJGAGGGA CUGAUGAGGCCG-AAAGGCCGAA AGAUGAG 106 CtJGAGGG Ct7GAUGAGGCCGAAAC-GCCGAA AAGAUGA 129 AGGCCCA CtJGAUCGAGGCCGAAAGGCCGAA AAGCCUG 138 CLTCCACA CUGAUGAGGCCGAAAGGCCGAA AAGGCCC 148 GJCGAU CrJGAUGAGGCCG-AAAGGC.-GAA AUCUCC-A 151 GCtJGTJUC CUGAUGAGGCCGAAAGGCCGAA AUGAt7Ct 1J100 AUAGCGG CUGAUGAGGCCG-AAAGGCCGAA AUCGCAU 2,81 UAUAGCG CUGAUGAC-GCCGALGGCCCGAA AAUCGr-k 186 GC-AUUUA CtJGAUGAGGCCGAAAGGCCGAA AGCGGA.A 204 GCCCGCU CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 217 CGCCAGG CUGAUGAGGCCGAAAGGCCGAA AUAGUGC 239 tUUGGUGG CUGAUJGAGGCCCGAAAGGCCGAA AUCUGUG 262 UGAUCIJU OUGAUGAGGCCGAAAGGCCGAA AUGGUG 268 AGCCAUU CUGAUGAGGCCGAAAGGCCGAA AUCUUJGA 276 UCCtJGUG CUGAtJGAGGCCGAAAC-GCCGAA, AGCCAUU 301 CCAGGGA CUGAUGAGGCCG.AAAGGCCGAPA AUUCGAA 303 GACCAGG CUGAUGAGGCCGAAAGGCCGAA AGAUUCG 310 CCtJUGGU CUGAUGAGGCCGAAAGGCcGAA kcr-AGGG 323 UCAGGAG CUGAUGAGGCCG-AAAGGCCGAA AGGGCC 326 G-GCCGG,-U CUGAUGAGGCCGAAAGGCCGAA AGGuGGA 335 UGUGGAU CUGAUGAGGCCGAMGGCCGAA AGGCCGG 349 UCCCCAkC CUGAUGAGGCCGAAAGGCCGAA AGuuCAU 352 GCUGUJUC CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 375 CUCAUAG CUGAUJGAGGCCGAAAGGCCG-AA AGCCAUC 376 CUCCGGA CUGAUGAGGCCGAAAGGCCGAA AGAcCATJU 378 AG-CCUCA 3CGAUGAGGCCGAAAGGCCCrA AGUAGCC 391 CUG-GGCA CUGAUG-AGGCCGAAAGGCCGAA AGGUCAG 409 AGCUAUG CUGAtIGAGGCCG-AAAGGCCGAA AUACUGC 416 cuAUGGA CUGAUGAGGCcGAAAGGCCGAA ACUGCGG 417 GUUCUGG CTJGAUGAGGCCG-AAAGGCCGAA AGCUAUG 418 GGUUCUG CUGAUGAGGCCGAAAGGCCGAA AAGCUAU 433 CACACUG CUGAUGAGGCCG-AAAGGCCGAA AUCCCCA 467 CGAACAG CUGAUGAGGCCG-AAAGGf-CCAA AGCCUGG 469 GCUGGCtJ CUGAUGAGGCCAAAG~ACCGAA AuGG=tt.

473 CUGAUCUJ CUGAUG-AGGCCGMAAGGCCGA-A ACUCAAA 481 UGGtJCUG CUGAUGAGGCCG-AAAGGCCGAA AUU~CGCU SUBSTITUTE SHEET (RULE 26) XVO 95/23225 lICT/11195/00156 232 502. AACGUGA CUJGATJGAGGCCG-AAAGGCCGAA AGGGU 502 GAACGUG CUGAUGAGGCCG-AAAGGCCGAA AAGGGG 508 CUAUGG CUGAUGAGGCCGAAAGGCCGAA ACGUGA2 509 UCUAU7AG CUGAt7GAGGCCGAAAGGCCGAA AACGtJGA 512 UCCUCUA CUJGAUGAGGCCGAAAGGCCGA-A AGGAACG 514 GCUCCtJC CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 534 CAAGUCA, CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 556 GGAAGCA Ct3GAUGAGGCCGAAAGGCCGAA AGCGCA 562. CACCUGG CUGAUGAGGCCC?AAGGCCGAA AGCAGAG 562 UCACCUG CUGAUGAGGCCG-AAGGCCGAA AAGC:AGA 585 GCUGG-tJ CUC-AUGAGGCCGAAAGGCCGAA AUGGCU= 598 UCAGGAG CUG-AUGAG3CCGAAAGCCGA AGGG-GCC- 613 GUGAGAG CUGAUCAGGCCGAAAGGCCCAA ACAGGG 616 GAUG3GA CGAUGAGGCCAAGGCCGA AGGACAG 617 GGCUGAG CUGAUGAGGCCGAAAGGCCGA .AGGGA C 6 20 CAUGGCU CUGAUGAGGCCGAAGGCCGAA AGGAAGG 623 GAGAUGG CGAUGAGGCCGAAAGGCCGA XGCAGC-A 628 UAUCAAA CUGAUGAGGCCGAAAGGCCGAA ATJCGGAU 630 GtJUAUC.A CUGAUGAGGCCCAAAGGCCG2LA AAAUCGG 631 GGtJUAUC GUGAU-GPAGGCCCAAAGGCCGAA AAAAUCG 638 GGAACAC CUGAUGAGGCCG-AAAGGCCGAA AtGGCCAk 662. AGAUCtJU CUJGAUGAGGCCGAAAGGjCCGAA AGCUCGG 667 CTUCGGCA CLTGAUGAGGCCG-AAAGGCCGAA AUCUUG-A 687 GCtJCCCA6 CUGAt7GAGGCCGAAAGGCCGAA AGUUtCCG 700 CCCCACC CtJGAUGAGGCCGAAAGGCC3A-A AGCAGC 72.5 GCAAGAA CUGAtIG-AGGCCG-AAAGGCCGA AUCUCAU 72.7 CAGCAAG CUGAt7GAGGCCGAAAGGCCGAA AGAUCtJC 72.8 AC.AGCAA CtTGAtJGAGGCCG-AAAGGCCG-AA AAGAUCU 721 CGCAAtJG CUGAtUGAGGCCGAAAGG7CCGAA AGGAGAA 751 ACACCUC CUGAUGrAGGCCGu'AAGGCCGAA AUGUCLU 7S 9 CGUGAAA CUJGAUGAGGCCGAAAGGCCGAA ACACCUJC 761 CCCGUGA CUGAUGAGGCCGAAAGGCCGAA AUACACC 762 UCCCGUG CEJGAUGAGGCCGAAAGGCCGAA AAJACAC 763 GUCCCGU CtJGAUGAGGCCGAAAGGCCGAA AAAUACA 792 AGAAAAG CUJGAUGAGGCCCAAAGGCCGAA AG-CCUCG 795 UUGAGAA CUGAUGAGGCCGAAAGGCCG-AA AGGAGCC 796 =UGAGA CL7GAUGAGGCCGAAAGGCCGAA AAGGAGC 797 GCUtJGAG CUJGAUGAGGCCG-AAAGGCCGAA AAAGGAG 798 CUGAUGAGGCCGAAAGGCCGAA: AAA.AGGA 829 C-GAACAC Ct2GATJGAGGCCG-AAAGGCCGAA AUGGCCA 834 AGUCCGG CUGAUGAGGCCGAAAGGCCG-AA ACACAAU 835 GAGUCCG CUGAUGAGGCCGAAAGGCCGA.A AACkCAA 845 GCGUACG CUG-AUGAGGCCGAAGGCCGAA AGGAGUC 849 GUCGGCG CUGAUGAGGCCG-AAAGGCCCGAA ACGGAGG 872 CGAACAG CUGAUGAGGCCGAAGGCCCGA AGCCUGG 883 C-CAUGGA CUGAUG-AGGCCG-A-AAGGCCCGAA ACUCCGAA 885 CUGCAtJG CtJGAtJGAGGCCGAAAGGCCG-A.A AGACUCG 905 CGAUCAG CUGAUGAGGCCGAAAGGCCGAA AGGCCGC 906 GCGAUCA CUGAUGAGGCCGAAAGGCCCGAA AAGGC:CG SUBSTITUTE SHEET (RULE 26) WO 95/23225 233 919 GCtJCACU CUGAUGAGGCCGAAAGGCCGAA AGCUCGC 936 GUACUGG CUJGAtJGAGGCCAAGGCCGAA ACUCCAU 937 AGUACUG CUGAUJGAGGCCGAGGCCCAA AACUCCAk 942 tJGGCAAG CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 953 UC-AUGUG CUGAUGAGGCCGAAAGGCCGAA AUGAGGC 962 CGGUGGC CUGAUGAGGCCGAAAGGCCGAA AtTCAUCU 965 GUCtJGGC CUGAUGAGGCCGAAAGGCCGAA AGIJACLG 973 UCtJCUUC CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 986 ACtJCUUG CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 996 G7JCUJC.A CUGAUGAGGCCGAA.GGCCGA6A AGGUCCU 1005 ACUCUUG CUGAUGAGGCCGAAAGGCCC P AGGUCUiC 1006 UACUCUU CUGAUGAGGCCGAACQGCCuGAA AAGGEJCt 1015 LCUUCAU CUGAUGAGGCCGAAGGCCGAA AUACUCU 1028 UUGAAAG CUG-AUGAGGCCG-AAAGGCCGAA ACUCtJUC 1031 CCAUUGA CUGANUGAGGCCGAAAGCGAA AGGACUC 1032 UCCAUUG CUGAUGAGGCCGAAAGGCCGAA AAGGACU 1033 GUCCAUtJ CUGAUGAGGCCGAAGGCCGA AAAGGAC 1058 CGGGUUG CUGAUGAGGCGA.GGCCGAA AGGCCGG 1064 UUGGAUC CUGAUGAGGCCG-AAAGGCCGAA AGGVGUA 1072 GCACAGC CUGAUGAGGCCGAAAGGCCGAA AUACGCC 1082 UUUCGGG CUGAUGAGGCCGAAAGGCCGA.A AGGCACA 1083 ACGG Ct3GAUGAGGCCGAAAGGCCGAA AAGGCUU 1092 AGAAGUU CUGAUGAGGCCGAAAGCCAA AGUUUCc, 1097 GGGACAG CUGAUGAGGCCGAAAGGCCGAA AGUUJGAG 1098 GGGGACA CUG-AUGAGGCCGAAAGGCCGAA AAGUUGA, 1102 CUUGGG CUGAUGAGGCCGAGGCCGAA ACAGAAG 1125 GAAGGUG CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 1127 GUAAGGC CUGAUGAGGCCGAAGGCCGAA AUAUGGC 1131 UGUGCU CUGAUGAGGCCGAAAGGCCGAA AGGGAUG 1132 AUGC-UGG CUGAUGAGGCCGAAGGCCGA AAGGUGU 1133 GAAGCUG CUGAUGAGGCCGAAAGGCCGAA AGAUGGA 1137 GCGCGCU CUGAUJGAGGCCGAAAGGCCGAA AALJAU 1140 GCUGAGG CUG-AIGAGGCCGAAAGGCCGAA AUGCtIGG 1153 CAAAGt3U CtiGAUGAGGCCGAAAGGCCGAA AUGGUGC 1158 CUCAUCA CUG-AUGAGGCCGAJAAGGCCGAA AGuTJGAU 1167 GGGGGAA CUGAUGAGGCCGAAAGGCCGAA ACUCAUC 1168 UGGGGGA CUGAUJGAGGCCGAAAGGCCGA AACUJCAU 11.69 AUGGGGG CUJGAUJGAGGCCGAAAGGCCGAA AAACUCA 1182 UG-AUGGU CUGAUGAGGCCGAGGCCGAA ACAGCAU 1183 CUGAUGG CUGAUGAGGCCGAAAGGCCGAA AAcAGCA 1184 UCAGGAG CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 1187 GGCUGAG CUGAUGAGGCCGAGGCCGA AAGGGAC 1188 CUG-CCCU CUGAUG-AGGCCGAAAGGCCGAA AUGGUA 1198 UCAGACU CUGAUGAGGCGAAGGCCGA ACuccc 1209 GAAGGUG CUGAUGAGGCCG-AAAGGCCGAA AGGOCUG 1215 CGGUGCU CUGAUGAGGCCGAAAGGCCGAA AGGCCAG 1229 GC-UGAGG CUCGAUGAGGCCGAAAGGCcGAA AGGGACC 1237 GGGGCAG CUG-AUCAGGCCGAAAGGCCGAA AGCUGGG 1250 GAGCCUG CUG-AUGAGGCCG-AAAGGC:CGAA AGGCUGG PCT/11395/00156 SUBSTITUTE SHEET (ROLE 261 WO 95/23225 PCT/1B95/0()156 234 1268 GGGGCAG CUGAUJGAGGCCCAAAGGCCGAA AGCUGGG 1279 AGGAAGG CUGAUJGA~fCCGAAAGGCCGAA kCCAUGG 1281 CGOAGCU CtJGATJuGGGCCGAAACGCCGAA AGCCCAC 1286 UJCGGGGA Ct3GAUGAGGCCCAAAGGCCGAA AACtJCAU 1309 AGACUCG CTJGAUGAGGCGCAAAC-GCCGA ACAGGAG 131J5 GGGUTJAG CUJGATJGAGGCCGAAAGGCCGAA ACtJGGC-G 1318 CCGGGGU Ct7GAUGAGGCCCGAAAGGCr-CGAA AGAACUG 1331 GACUGGG CtJGAUGA-GCCGAAAGC-CCGAA AGGACCC 1334 UCAGCUU CUGAUGAGGCCGAAAGcCCG-AA AGAAAAG 1389 GGUUCC CUGAUGA=GCGAAGGCCGAA ACAGCCGU 1413 AG=AUCA CUJGAUGAGGCGC-GCCGA ANCUGC.AG 1414 CAGCAUC CUGAUGAGC-CCGAAGG-CCGAA AACTMG2A 1437 GCCAA-C CUGAUGAGGCCGAAAGGCCG-AA AGGC-CC 1441 UGUUGCC CtYGAIGAGGC -CGAAGCv-,-CAA AGf-'AGG 1467 GUCUGUG CUGAUGAGGC-CGAAAGC-CCGAA aACAct'C 1468 GGUCUGtT CUGAUGAGGCCGAAAGGC,-GAA AACACfJC 1482 GUCCACA CtJGAUGAGC-=CGAAAGCYr-CGAA AUGCCAG 1486 AGUUCCC CUGAUGAGGCCGAAAGGCCGAA ACCCGAAG 1494 AAA=Ut CUGAUGAGGCCG-AAAGG-r-CCAA AGUJUGUC 1500 CUGCUGA CUGAUGAGGCCGAAGGcG-,.z AGucuGA 1501 GCUGC:UG CUGATJGAGGCCGAA.QCGCCGAA AACJCLG 1502 AGCUGCU CUGAUG-AGGCCG-AAAGGCC,-GAA A.ACUCU 1525 ACACAGG CUJGAUGAGC-CCGAAAG-CCGAA AUGCACc 1566 UUCAGGG CUGAXTJGAGGCCG-AAAC7%rGCC-AA ACUCCAU 1577 CGAGUUA CEJGAUGAGGCCGAAAGGCCGAA :kC=C-k 1579 GGCGAGU CtGAUGAGGCCG-AAAGCCrGAA AUAGCtU 1583 ACCAGGC CUGAUGAGGCCGAAGCCG- AGUUAtJA 1588 CCCUCUC CU7GAUGAGGCCGAAAGGCCG-AA AGGAGAG 1622 GGGGCAG CUGAUGAGGCCG-AAAGGCCGAA AGCUGGG 1628 CCtJACCG CUJGAtJGAGGCCGv-AAGGCCGAA AGCAGGA 1648 CAUUGGG CUGAUGAGGCCGAAAGGCCG-AA AGCCCCG 1660 CUGGGCA CUGAUGAGGCCG-AAAGCCC-AAL AGGUCAG 1663 CACCUGG CUGAUGAGGCCG-AAAGGCGCAA AGCAGAG 1664 UCACCUG CUGAUGAGGCCG-AAAGGCCCGAA AZGCAGA 1665 ACCUJCCG CUGAUGAGGCCGAAA.GGCCGAA AAGcGAG 1680 GGAGGAG CUGAUJGAGGCCGAAAGGCCAA AGUC 1681 UJGGAGGA CUGAUCGAGGCCG-AAG(GCCGAA AAGUCUU 1683 AAUGGAG CUGAUGAGGCCGAAAGGCCG-AA AGAAGUC 1686 CGCAAUG CUGAUC-AGGCCGAACGCCGA. AGGAGA.

1690 UJGUCCGC CUGAUGAGGCCGAAAGC-CCG-AA AUGGAGG 1704 AGCAGAG CUGAUGAGGCCGAA.AGGCCGAA AGUCC-AU 1705 GAGCAGA CUGAUGAGGCCG-AAAGGCCGAA AAGtJCCA 1707 AAGAGCA CUGAUGAGGCCG-AAAG3CCGAA AGAAGUC 1721 CUGAUCUJ CUGAUGAGGCCGAAAGGCCGCA.A ACEJCAAA 1726 AGGAGCU CUGAUGAGGCCCGAAAGGCCG-AA AUCUG-AC 1731 ACCUUAG CTUGAUGAGGCCGAAAC-GCCCGAA AGCUGAU 1734 AGCACCU CUGAUGAGGCCGAAAGCCGAA AGGAGcu !754 CUCUTUGG CUGAUGAGGCCGAAAGGCCCGAA AGCACTTG SUBSTITUTE SHEET (RULE 26) WO 95/23225 P1C'!'!95/0(1156 235 Table Human re/A HH Ribozyme Sequences nt. Position HH Ribozyme Sequences 19 UACAGAC CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 22 CACUACA CUGAtJGAGGCCG-AAAC-GCCGAA ACGAGCC- 26 CGtJGCAC CJGAUGAGGCCGAAAGGCCG-AA AC-AGACG 93 CAGGGGG CtJGATJGAGGCCGAAAGGCCAA ACAGUUC 94 UGAGGGG CUGAUGAGGCCGAAAGGC-CGAA AAC-AGUU 100 GGAAU CUGAUJGAGGCCGAAGGCCG-A AGc-GGGA 103 CCGGG-AA Ct7GAUGAGGCCGAAAGGCCG-AA AUGCAGG 105 UGCCGC-G CtJGAEJGAGGCCCGAAAC-GCCGAA AGAIJGAG 106 CUGCOG CUJGAUGAGGrCCGAAAC-GCCGAA AAGAUGA 129 GGGGCCA CUGAUGAGGCCG AGGCCGAA AGGCCtJG 138 CUCCACA CUGAUG-AGGCCGAAAGGCCGAA AGGGGCC 148 GCUC-AAU CUGAUJGAGGCCGAAAGGCCGAA AUCUCCA 151 GCUGCUC CUGATJGAGGCCGAAAGGCCGAA AtIGAUCtJ 180 GUJAGCGG CUGAUGAGGCCGAAAGGCCGAA AGC-GCAU 181 UGUJAGCG CUGAUGAGGCCGAAAGGCCGAA AAGCGrCA 186 GCACUUG CUGAUGAGGCGAAAGGCCGAA AGCGGAA 204 GCCCGCG CtJGAUGAGGCCGAAAGGCCGAA AGCGCCC 217 CGCCUJGG CtJGAUJGAGGCCGAAAGGCCGAA AUGCUGC 239 UUGGUGG CUGAUGAGGCCGAAAGGCCGAA AtJCUGtJG 262 UGAUCUU CUGAUGAGGCCGAAAGGCCGAA AUGGUC-G 268 AGCCAUU CUGAtJGAGGCCGAAAGGCeCGA6A AUCUUGA 276 UCCUGUG CUGAUGAGGCCGAAAUGGCCGAA AGCCAUU 301 CCAGGGA CUGAUGAGGCCGAAAGGCCGAA AUGCGCA 303 G-ACCAGG CUGAUGAGGCCGAAAG-GCCGAA AGAUGCG 310 CCUtJGGU CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 323 CGGUGAG CUGAUG-AGGCCGAAAGGCCGAA AGGGUCC 326 GGCCGGU CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 335 UGGGGGtJ CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 349 UUCCUAC CUGAUG.AGGCCGAAAGGCCGAA AGCUCGU 352 CCUUtJCC CUGAUGAGGCCGAAAGGCCGAA ACAAC-CtJ 375 CIJCAt7AG CUGAUGAGGCCGAAAGGCCGAA AGCCAUC 376 CCUCAUA CUGAUGAGGCCGAA.AGGCCGAA AAGCCAU 378 AGCCUCA CUGAUGAGGCCGAAAGGCCGAA AGAAGCc 391 CCGGGCA CUGAUGAGGCCGAAAGGCCGAA AGCUCAG 409 AACUGIJG CUGAUGAGGCCGAAAGGCCGAA AUGCAGC 416 UJUCUGGA CUGAtJGAGGCCGAAAGGCCGAA ACUGUGG 417 GtJUCUGG CUGAt7GAGGCCG-AAAGGCCGAA AACUGUG 418 GGUUCUG CUGAUGAGGCCGAAAGGCCGAA ALAACUGU 433 CAC.ACUG CtIGAUGAGGCCGAAzAGGCCGA1A AUUCCCA 467 UGACUGA CUGAtTGAGGCCGAAAGGCCGAA AGCCUGC 469 GCUG-ACU CUGAtYGAGGCCG-AAAGGCCGAA AUAG-CCU 473 AUGCGCU CUGAUGAGGCCGAAAGGCCGAA ACUGAUA 481 UGGUCUG CUGAUGAGGCCGAAAGGCCGAA AUjGCGCEJ 501 AACUUGG CUGAUG-AGGCCGAAAGGCCGAA AGGGtjU SUBSTITUTE SHEET (RULE 26) WO 95123225 PCVIB95/00156 236 502 GAACt3UG CUGAL'GAGGCCCAAAG-CCGA.A AAGGGGU 508 CUATAGG CUGAUGAGGCCCGAAAGGCCGAA ACUUGGA 509 tJCTAG CUJGAUGAGGCCGAAAGGCCGAA AACUUGG 512 UCUUCLUL CUGAUJGAGGCCGAAAGGCCGAA AGGAACtJ 514 GCUCUUC CtJGAUGAGiGCCGAAA-GCCGAA AUAGGAA 534 CAGGUCG CTUGAU-GGCCGAAGGCCGAA AGUCCCC 55;6 GGAAGCA cUGATJGAGGCCGAAAC-GCCG-AA AGCCGC-T 561 CACCUGG CtJGAUGAGGCCGAAAGGCCGAA AGCAGAG 562 UCACCUG CTJGAXUGAGGCCGAAAflGCCGAA AAGCAGA s UGCCtJ CUG-AUGAGGCCGAAGGCCG. AUJGGGUC 598 GCAGGCG Ct3G AUGAGGC CAAG-C C GAA AGGGGCC 613 CAGGAAG CUGAL"GA-GCCGAAAGGCCGrAA ACAkGGCG 616 GALGAGG CUGAGAC-GCCGAGCGCCCA2 kCGACAG 617 GGAUGAG CUG-AUGCAGCCGAAGGTCCAA AGGACA 620 AL'GGGAU CUCGALGAGGCCGAAAGGCCCGAA AGGAAGG 623 AAGAUGG CtJGAU-AC-G7CCGAAAGGCCGAA AUGAGGA 628 UGUCAAA Ct7GAUGAC-GCCG-AAAGGCCGAA AUGiGGAU 630 AUUGUCA CUGAUGAGGCCGAAAGGCCGAA AGAIJGG 631 GAUUJG3C CUGAUG-AGGCCGAAAGGCCGAA AAGAUGG 638 GGGG06C CUGAL'GAGGCCGAAAGGCCGAA AUGUCA 661 AGAUCUU CUGAUGAGGCCCGt-AGGCCGAA AGCUCGG 667 CUCGGCA CUGAUGAGGCCGAAAGGCCGAA AUCU=JGA 687 GCUJGCCA CtJGAUGAGGCCGAAAGGCCGAA AGUUTJCG 700 CCCCACC CUGAUGAGGCC.AGCGCCGAA AGGCAGC 71.5 Gt3AGGAA CUGAUGAGGCCGAAAGGCCGA AUCtJCAU 717 CAGUAGG CUGAtJGAG-GCCGAAAGGCCGAA AGAUCUC 718 ACAGUAG CJGAUGAGGCCGAXAGGCCGAA AAGAUCtJ 721 CACA CA G CUGAUGAGGCCGAAAGGCCGA.ZA kGGAAGA 751 ACACCUC CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 759 CGUGAAA CUG-AUGAGGCCGAAAGGCCGAA ACACCUC 761 CCCGUG-A CtJGAUG-AGGCCG-AAAGGCCGAA AUACACC 762 UCCCGTJG CUC-AUC-AGG-,-CCAAAGGCCGAA AAUACAC 763 GUCCCGU CUGAUGAGGCCGAAAGGCCGAA AAAUACA 792 CGAA.G CUGAUG-AGGCCGAGAGCCGAA AGCCUCG 795 UUGCGAA CUGAt7G-AGGCCGAAAGGCCGAA AGGAGCC 796 CUUJGCGA CUGAUJGAGGCCGAAAGGCCG-AA AAGGAGC 797 GCUJUGCG CUGAT-TCAGGCCCGAAAGGCCGAA AAAGGAG 798 AGCtJUGC CUGAUG-AGGCCGAAAGGCCG-AA AAAAGGA 829 C-GAACAC CtJGAtJAGGCCGAAAGGCCGAA AUGGCC-A 834 GGUCCGG CUG-AUG-AGGCCG-AAAGGCCGAA ACACAAU 835 G=GCCG CUGAUG-AGC-CCGAAAGGCCGAA AACACAA 845 GCGUAGG CUGAUGAGGCCGAAAGGCCCGAA AGGGGTJC 849 GUCUGCG CUGAUGAGGCCGAAAGGCCGAA AGGGAGG 872 CC-CACAG CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 883 GCAUGGA CtJGAUG-AGGCCGAAAGGCCGAA ACACGCA 885 CUGCAUG CUG-AUG-AGGCCGAAAGGCCG-AA AGACACG 905 CCGGUCGG CUGAUG-AGGCCGAAAGGCCGAA AGGCCGC 906 CCC-GUCG CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 919 GCUCACU CUG-AUGAGGCGAAAGGCCGAA kGcuccc SUBSTITUTE SHEET (RULE 26) WO 95/23225 icriB95/00156 237 936 GUACUGG CUG-AUGAGGCCCGA-AAGGCCGA:A AUTCA 937 GGUACUG Ct7GAUGAGGCCGAAAGGClCGAA AAtUCCA 942 tTGGCAGG CUGAUGAGGCCGAAAGGCCGAA ACU;GGAA: 953 tJCCUCUG CtJGAUGAGGCCGAAAGGCCG-AA kUTCUjC-CC 962 CGG-UGAC CUGAUGAGGCCGAAGGCCGA AUCGUCEJ 965 AUCCGGU CUGAUGAGGCCC-AA.GG-CC-AA ACGAUCG 973 tJCECCUC C!JGAUGAGGCCGAAAGGCCGAA AUCCCG%-u 986 GUCCUUU CUGAUGAGGCCGAAZAGGCCC-AA -CGU tC 996 GGUCUCA CUGAUGAGGCCGAAAGGCAA ALGUCC

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1005 GCUCUUG CUGAUGAGCCAAAGCCA AC7-UCUC 1006 UGGCTJU= CUG-AUGAGGCCGAAAGGCC-GAA 1015 UCtUUCA:U CUGAUGAGGCCGAAGGCCGAA AUGCU 1028 CUCAAAG CUGAUGAGGCCGAAAGGC-,CAA kCU=tjUC 1031 CCGCUGA CUGAUC-AGGCCCGAAAGGCCG-AA AC-CACUC 1032 UCCGCUG CU GA UG AG G CC GA A A CTG C G AA AAGGACU 1033 GUCCGCU CUG A UG AG G CC G AAGGC-C G~ A A..G.AC 1058 CGAGGUG CUGAUC-GGCCGAA-ACGCCCGA-A AGGCCGG 1064 AUGCGUC CUGAUGAGGCCG-AAAGc-CCGAA AGGUGGA 1072 GC-ACAGC CUGAUGAGGCCGAAGGCCCAA AtIGCGtJC 1082 CUGCGGG CUGAUGAGGCCG-AAGGCCGAA AGGCACA 1083 GCTJGCGG CUC-AUGAGGCCCGAAA-GCC-A. AAC-G!c.Ac 1092 AGAAGC-U CUGAUGAGGCCGAAGGCCGAA ACCQGC-, 1097 GGGACAG CUGAUGAGGCCGAAAGGCCG-AA AC- GAG 1098 CGGGACA CUGAUGAGGCCCGAAAGCCC-AA AAC-CtJGA 1102 GCUUGGG CUGAUGAGGCCG-AAAGGCCGAA Ar-NGAAG 1125 AAAGGGA CUGAUGAGGCC-GAAAGGCCC-AAk AGCGCUG 1127 GUAAAGG CUG-AUGAGGCCGAAAGGCC-AA AUAGGC-C 1131 UGACGUA CUGAUGAGGCCGAAAGGCCGAA AGGGAMA 1132 AUGACGU CUGAUG-AGGCCG-AAAGGCC-GAA ?A.GGGA.U 1133 GAUGACG CUGAUGAGGCCG-AAAGGCCGAA AAAGGGA 1137 CAGGGAUJ CUGAUGAGGCCGAAAGGCCCAA ACGUAAA 1140 GCUCAGG CUGAUJGAGGCCGAAAGGCCG-AA AUGACGtJ 1153 CAUAGUU CUGAUGAGGCCGAAGGCCGA AUGGUC 1158 CtJCATUCA CUGAUGAGc-CCCGAAAGC-CCGA AGUMUu 1167 GGUGGGA CUGAUGAGGCCGAGGCCG-A ACUCAUC 1168 UGGUGGG CUGAUGAGGCCGAAAGGCCGAA AACUCAU 1 169 AUGGUGG CUGAUGAGGCCGAAGGCCGAA A-AACUCA 1182 AGAAGGA CU7GAUGAGGCCGAAAGCGCCAA ACACCAU 1183 CAGAAGG CUGAtJGAGGCCGAAAGGCCCAA AACACC-A 1184 CCAGA.AG CUGAUGAGGCCG-AAAGGCCCGAA AAACACC 1187 UGCCCAG CUGAUGAGGCCGAGGCCG-AA AGGAAAC 1188 CUGCCC-k CCGAUGAGGCCGAAAGGCCG-AA AAGCZA 1198 CCUGGCtJ CUJGAUGAGGCCG-AAAGGC:CGAA AUCUJGCC 1209 CAAGGCC CUGAUGAGGCCGAAAGGCCG-A. AG-GCCUG 1215 CGGGGCC CUGAUGAGGCCGAAAGGCCG-AA AGfCCGA 1229 ACUUJGGG CUGAUGAGGCCGAAAGGCCGAZLA AGGGGCC 1237 GGGGCAG CUG-AUG-AGGCCC-AAGCGCCGAA:k ACZJGGG 1250 GGGGCUG CUGAUGAGGCCGAAAGGCCG-AA ACZUG 1268 AUGGCUG CUGAUGAGGCCG-AAAGGCCGAA AC-C-AGGG SUBSTITUTE SHEET (RULE 26) WO 95123225 PCIAB95/00156 238 1279 GAGCtJGA CUGAUGAGGCCGAAAGGCCGAA AkCCAUJGG 1281 C-AGAGCtJ CUGAUGAGGCCGAAAGGCCGAA AUACCAU 1286 UGGGCCA CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 1309 GGACUGG CTJGAE7GAGGCCGAAAGGCCGAA ACAGGGG 1315 GGGCUAG CUGALUGAGGCCGAAAGGCCGAA ACtJGGGA 1318 CtJGGGGC CUGAUGAGGCCGAAAGGC-CGAA AQGACUG 1331 GCCUGAG CUGAt7GAGGCCGAAAGGCCGAA AGGGCC

T

J

1334 ACAGCCU CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 1389 GGCCUCtJ CUGAUGAGGCCGAAAGGCCGAA AC.AGCGU 1413 AUCAUCA CUGAUGAGGCCG-AAAGGCCGAA ACUGCAG 1414 CATJCAUC CUGAUGAGGCCGAAAGG7CCGA ACUGC.N 1437 GCCA.GC- CUG-AUGAGGCCGAGCCGAA AGGCCCC 1441 UG=JGCC CUGAUGAGGCCGAAAGGC"CAA AGCA.AGG 1467 GUCUGUG CUGAUGAGGCCGAAAGGCCGAA ACACAGC 1468 GGUCUGtJ CUGAUGAGGCCGAAAGGCCG-AA AACACAG 1482 GUCGACG CtJGAUGAGGCCGAAAGGCCGAA AUGCCAG 1486 AGUUGUC CUGAUGAGGCCGAAAGGCCGAA ACGGAUG 1494 AAACUCG CUGAUGAGGCCGAAAGGCCGAA AGUUGUC 1500 CUGCUGA CtJGAUGAGGCCGAAAGGCCGAA ACUCGCA 1501 GCUGCUG Ct3GAUJGAGGCCGAAAGGCCCGAA AAGUCGG 1502 kGCUGCU CUGAUGAGGCCGAAAGGCCGAA AAACtJC- 1525 CCACAGG CUGAUGAGGCCGAAAGGCCGAA AUGCCCU 1566 CUCAGGG CUGAU-GAGGCCGAAAGGCCG-AA ACUCCAU 1577 CGAGUUA CUGAUGAGGCCGAAAGGCCGAA AGCCUCA 1579 GGCG-AGU CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 1583 ACUAGGC CUGAUGA-GCCGAAAGGCCG-AA AGUUAUA 1588 CUGTJCAC CUGAUJGAGGCCGAAGGCCGAA AGGCGAG 1622 GGAGCAG CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 1628 CCCAGUG CUGAUGAGGCCG-AAAGGCCGAA AGCAGGA 1648 CAUUGGG CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 1660 CUGAAAG CUGAUGAGGCCGAAAGGCCGAA AGGCCAU 1663 CUCCUGA CUGAUGAGGCCGAAAGGCCG-AA AGGAGGC 1664 UCUCCUG CUGAUGAGGCCGAAAGGCCGAA AAGGAGG 1665 AUCtJCCU CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 1680 GGAGGAG CUGAUGAGGCCGAAAGGCCGAA AGUCUUC 1681 tJGGAGGA CUGAUGAGGCCGAAAGGCCG-AA AAGUCUU 1683 A.UGGAG CUGAUGAGGCCGAAAGGCCGAA AGAAGtJC 1686 CGCAATUG CUGAUGAGGCCGAAAGGCCG-AA AGGAGAA 1690 UGUCCGC CCUGAUGAGGCCGAAAGGCCGAlA ATJGGAGG 1704 GGCUGAG CUGAUGAGGCCGAAAGGCCG-AA AGUCCAU 1705 GGGCUGA CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 1707 CAGGGCU CUGAUGAGGCCGAA.GGCCGAA AGAAGUC 1721 CUGAUCtJ CUGAUJGAGGCCGAAAGGCCG-AA ACUCAGC 1726 AGGAGCU CUGAUGAGGCCG UXGGCCGAA AUCUGAC 1731 CC CW AG CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 1734 ACCCCCU CUGAUG-AGGCCGAAGGCCG-A AGGAGCU 754 CUCUGGG CUGAUGAGGCCG-AAAGGCCGAA AGGGCAG SUBSTITUTE SHEET (RULE 26)

I

Table 21 Hu~man rel A nt. Position Hairpin Ribozyme/Target Sequences Hairpin Ribozyme sequence Substrate UGAGGGO AGAA GUUC ACCAGAGAAACACACGUUGUGGLJACAUUACCUGGUA GAACU GUtJ CCCCCUCA

GCUGCUUG

GCCAUCCC

GUIJCUGGR

GAAGGACA

UUGAGCUC

CCCACCGA

AGGCUGGG

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

AGAA

GCUC

GUCC

GUGG

GCAG

GUGU

GCUG

GCGEJ

ACCAGAGAAACACACGUUrGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUEJACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACGUIUGUGGUACAUUACCUGGUA

ACCAGAGAAACACACG!JUG[JGGUACAUUACCUGGUA

ACCAGAGAAACACACGIJUGUGGUJACAUJUACCUGGUA

ACCAGAGAAACACACGUUGUGGUACAUUACCUGGtJA

ACCAGAGAAACACACGUUGUGGUACAUJACCUGGJA

GACCA CC GGACJ CC CCACA GUU CUGCC CC ACACJ CC CAGCU GCC ACGCA GAC CGGCG CC AUACA GAC CACCG CAC CCACC GAC

CAAGCAGC

GGGAUGGC

UCCAGAAC

UGUCCUUC

GAGCLJCAA

UCGGUCGG

CCCAGC:CU

UUCCGACC

GAUCGUCA

CCACCGAC

CCCCGCCC

900 955 1037 1045 1410 1453 1471 GGUCCGAA AGAA GCCG UGACCAUC AGAA GLJAU GUCGGUGG AGAA GCUG GCCCGCG AGAA GUGO CAUCAUCA AGAA GCAG ACAGCUGG AGAA GUC CUCCA GU UGAUGAUG GCACA CAC CCAGCUC3U UCACA GAC CUG7GCAUC GAUGCCAG AGAA GUCA ACCAGACAAACACACGUUGUGGUACAUUACCUGCLJA -rable 22 Mouse rel A nt. Position Hairpin Ribozyme/Target Sequences Hlairpin Ribozyme sequence Substrate 137 273 343 366 633 676 834 881 1100 1205 1361 1385 1431 1449 1802 2009 2124 2233 2354 GUUGCUUC AGAA GAGAUUCG AGAIA GCCAUCCC AGAA GGGCAGAG AGAA UTUGAGCUC AGAA CCCACCGA AGAA AGGCUGGG AGAA GAUCAGAA AGAA AGGUGUAG AGAA GOCCAGAG AGIA GGGCUUCC AGAA CAGGAUCA AGAA ACUCCUGG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGrJA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGIJ ACCAGAGAAACACACGUUGUGGUACAUTJACCUGGUA GCUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGEJ ACC-AGAGAAACACACGUUGUGGUACAUUACCUGGIJA GCCG ACC-AGAGAkAACAC-ACGEJUGUGGUAC-AUUACCUGGUA GCGG ACCAGAGAkAACACACGUUGUGGUACAUUACCUGGU.A GUGC ACCAGAGAAACACACGUIUGUGGtJACAUUACCUGGUA GCGrJ ACCAGAGAAACACACGEJUGUGGUACAUUACCUGGUA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGA ACCAGAGAAACACACGUUGrJGGUACAUUACCUGG(JA OCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA OCAC ACCAGAGAAACACACGUUGUGGUACAUUACCJGGrJA GCCA ACCAGAQXAAACACACGUUGUGGUACAUUACCUGGUA GEJCI ACCAGAGAAADCACACGEJUGUGGUACAUUACCUGGUA GAACA CC GAACA CUU GGACU GCC AGCL GAC ACACJ CC GAGCU CC ACGCC GAC CGGCG GCC CCGCA GCC GCACC GEJC ACGCU GUC CUCCA GUU GCACA GAC UCACA GAC CAGCU CC GGACA GAC GUGCU CC UGGCC CC

GAAGCAAC

CGALAUCUC

GGGAUGGC

CUCUGCCC

GAGCUCAA

UCGGUGGG

CCCAGCCU

UUCUGAUC

CUACACCLJ

CUCUGCCC

GGAAGCCC

UGAUGCUG

CCAGGAGU

CUGGCAUC

CCCCACUU

UGGAGCC-A

CGACACCA

UUCAGAAU

GAUGCCAG

AAGUCGGG

UGGCUCCA

UGGUGUCG

AUUCUGAAL

UCAGUAAA

AGAA

AGAA

AGAA

ACAA

AGAA

AGAA AGACA CCC UUUACUGA WO 95/23225 .PCT/IB95/00156 241 Table 23: Human TN-F-a HH Ribozynae Target Sequence nt. HE~ Target Sequence nt HH Target Sequence Position Position 28 GGCAGUt U CtICUUCC 29 GfcAG=tt C UCUCCtJ 321 GU'JC.ACAU C ALICULUCU 31 -AGGUMUCU C tJUCCUCU 324 AGAUCXU C jucucGA 33 GJt5CtCU U CCUCUCA 326 AUCAUCU U CUCGAAC 34 UJUCUCUU C CUCUCAC 327 UCAUC'JU C UCGAALCC 37 Utjtuccu C UCACAUA 329 AUCUUCU C -AAcCC 39 UtJCCUCU C ACAUACtJ 352 AGCCUJGU A GCCCAUG 44 CUCACAU A CUGACCC 361 CCCAUGU U G!ThQCAA 58 CAC=GC C CACCCUC 364 AUGUUGU A GCAAACC 63 CCACCCtJ C UTJCCCC 374 AAACCCtJ C AG,-UGA 67 ACCCUCU C UCCCCtJG 391 -GCACtJ C C-AGtJGGC 69 CCUCUGU C CCCUJGGA 421 AUGCCCU c crrGGccA 106 -CAUGAU C CGTGGACG 449 GAGAGAU A ACCAGCU 136 AG-GCGCU C CCCAAG-A 468 GUGCCAU C AGAGGC 165 CAGGGCU C C.A=GGG 480 GGCCtJGt AL C--tiAUC 177 CG-GU-CU U GUJUCCUC 484 TJGUACCU C ;,UCtJACU 180 UGCUUGU U CCUCA-C 487 ACCUCAku C muAJccc 181 GC-UUGUU C CUCAGCC 489 CUCAUCU A CtJCCCAG 184 UGUtJCCU C AGCCUJCU 492 AUCUACU C ccAGuc 190 UCAGCCU C UUCUCCU 499 CCCACGU C CUCUUCA 192 AGCCUCU U CUC=tJC 502 AGGUCCU C UUCAAGG 193 GCCUCU C UCCOUCC 504 GUCCtJCt U CAAGGGC 195 CtJCUUCU C CUUCCUG 505 IJCCtJCtU C P.AGGGCC 198 UUCUCCU U CCUGAUC 525 UGCCCtJ C CAcccAu 199 UCUCCU C CUGAUCG 538 AUGUGCU C CUCACCC 205 UCCUGAU C GUGGAG 541 UGCUCCU c ACCC-ACA 226 CCACGCTJ C UUCUGCC 957 ACACCAU C AGCCGCA 228 ACGCUCU U CUGC-CUG GCCGCAU C GCCGUCU 229 CGCCUU C UGCCUGC 8UCGCCGU C uCCUjACC 243 CUGCA-CU U UGGAGUG 570 GCCGUCU C CUACCAG 244 UGCACtJU U GGAGUGA 573 GUCtJCCU A CCAGACC 253 GAGUGAU C GGCCCCC 386 CCAAGGU C AACCUCC 273 GAAGAGU C CCCCAGG 592 UCAAkCCU C CUCUCUG 286 GGGACCU C UCUCUAA 595 ACCUCCU c uCUC-CCA 288 GACCUCU C UCUAAUC 597 cuccucu c uGCCAUC 290 CCUCUCU C UAAUCAkG 604 CCGCCAU c AAG= 292 UCUCUCU A AUCAGCC 657 CCCUG-U A UGAGCCC 295 CtJCUAAU C AC-CCCUC 667 AGCCCAU c uAuctJGr 302 CAc-CCCU C UGGCCCA 669 CCCAUCU A UCUGGGA SUBSTITUTE SHEET (RULE 26) WO 95/23225 PCTAB95/00156 242 671 CAUCUAU C UGGGAGG 960 UC--C-AtU C AGGAAUG- 682 GAGGGGU C UUCCAGC- 2.00 AACCA7CU A AG-AAUC 684 GGGUCU U CCAGC-UG 2.007 UiAAG-AAU U CAAACUG 685 GGGUCUU C CAGCUGG 1008 ?-AGAAtJU C AAACUG 709 ACCGACU C AGCGCtJG 1021 GGC-GCCU C CAGAACU 721 CtJGAGAU C AAUCGGC 1029 CAGAACU C ACTJGGGG 725 G-AtI-AAU C GGCCCGA 1040 GGC-GCCU A c-kGCUu 735 CCCGACU A UCUCGAC 1046 UACA=C U UGAUCOC'- 737 CGACT3AU C UCG-ACt-JtJ 2047 AC.AGCUU u GAUCCCtJ 739 ACUAUCU C GACEJUUG .052. CUUUGAU C CO-UGACA: 744 CtJCGACtJ U UGCCGAG 1.060 CUGALCAU C UGGAAUC 745 UCGACUU U GCCGAGU 2.067 CUcGAAU C UGGAGAC 753 C-CCGA.GU C UGGGCAG lo085 -G A c -71 u UGGTUCU 763 GGCAGGU C UJACUUUG 1086 CGAGCCUU U C-GuUCUG 765 CAGGUCU A CUUJUGGG 1090 CUUUGGU U CUGGCC-3 768 GTJCUACU U UGGGAUC 109i UUUC-=UTJ C UG-GCCA:G 769 UCUACUU U GGGAU-A 1113 C-G-ACU U GAGAAGA 775 UUGGGAU C AU.UGCCC 1124 AAG-ACCU C ACCEJAGA 778 GGATJCAU U GCCCUGU 1129 CUJCACCtJ A G-AAUUG 801 CGAACAU C CAACCUJ 2.135 UAGAAAU U -ACAC.

808 CCAACCU U CCCAAAC 1151 UGGACCU U :AGGCCU 809 CAACCUU C CCAAACG 1152 GC-ACCUJU A GGCCUUC 820 AACGCCtJ C CCCUGCC 2.158 UAGGCCtJ U CCUCUCU 833 CCCCAAU C CCUULtAU- 1159 AGGCC=t C CUTCUCUC 837 AUCCCU U UAUUACC 1162 CC-uUCCUJ C UCUCCAG 838 AUCCCUU U AtUtACCC 1164 tUUCCUCUL" C UCCAGAU 839 UCCCUTUU A UUACCCC 1166 CCUCUCtJ C CAGAUGU 841 CCtJUUAU U ACCCCCU 2.174 CAGAtJGO U UCCkGAC 842 CUUUAUU A CCCCCUC 1175 AGA=Gt U C C.%AACU 849 ACCCCCU C CUUCAGA 1176 C-AUGUUU C CAGACUU 852 CCCUCCtJ U CAGACAC 1183 CCAGACU U CCUU UAG 853 CCUCCtJU C AGACACC 1184 CAGCACUW C CUGAG-A 863 ACACCCEJ C AACCUCU 2.187 ACUJUCCU u G-AGAC-AC 869 UCAACCU C UCCUGGC 1208 C-cCCCU C CCCAUCG 872. AACCUCUT U CTJGGC-UC 1224 C-CCZAGCU C CCUCtJAU 872 ACCUCUU C UGGC-UCA 2.228 GC-UCCCU C U-AIMU 878 UCUGGCU C AAAAAGA 1230 UCCCUiCU A UUTUAUGTJ 890 AGAGAAU U GGGGGC-U 1232 CCU=tJU U tJAUGTJU 898 GGGGGCU U AGGGUCG 1233 CUCUTAUU U Z.UGUUUJG 899 G-GGGCUU A CGGUCGG 2.234 UCriAUUU A ZUGUUUC-C 904 UUAGGGU C GGAACCC 1238 UJUAUGU U UGC.ACUU 917 CCAAGCU U AGZAACUU 1239 UUAUGUUJ U C-CACUG 918 CAAGCUU A GAACUUU 1245 jU~C-CACtJ U GUGAUUA 924 UAGAACU U UAAGCAA 1251 =UUGAU U AUJUUAUU 925 AGAACUU U AAGCAAC 1252 UC-UG-AUU A UUUAUUA 926 GAACUUU A AGCAACA 1254 UG-AUUA-U u UAk~uAUU 945 CACCACU U CGAAACC 2.255 GAUUAUU U AZUUU 946 ACCACUU C GAAACCU 1256 AUUAUrUU A UUAUUUA 959 CUGGGAU U CAGGAAU 1258 uAuuuAu U AUMuuAU SUBSTITUTE SHEET (RULE 26) WO 95/23225 PTI19/( PCIAB95/00156 243 1259 1261 1262 1263 1265 1266 1267 1269 1270 1272 1273 1274 1276 1277 1278 1280 1281 1282 1294 1296 1.297 1298 1300 1301 1315 1317 1334 1345 1350 1359 1360 1361 1362 1386 1393 1394 1401 1414 1422 1423 1425 1426 1427 1431 14 32 1436 1,437 1438 AUUUAU A UUUAT-)UU UUAUUAU U UAU~TMU UAUUAUU U AUUUAUU- AUUAIJUU A UUUTJOUA UAUUMU U UAUUAUtJ AtJUUAUtJ U AUUAUUU UUUAUU~rU A UUAUUUA tJAULUAU U kUJAjU KTUUAUTU A UUUALUU UUAUUAU U UAUUUAU UAUUAUJU U AM=AU AMUA=U A UUJUAjUU UAUtJTAU U UAtUUAC .kUUUAUU U ALUJLCA UUUAUUU A UUUACAG UAUUUAU U UACAGAU AUUUAU7U U ACAGAUG UUTAUW A CAGADGA UGAAUGU A UrJLAUUU AAUGUAU U UAUUUGG AUGUAUU U ATj'UUC-G UJGUAUUU A UUUGGG-A UALTUAU U UGGGACA AUrUUAUU U GGGAGAC CCC-GGG--U A UCCUjGC-G GGGGUAU C CUGG CCAUGU A GGAGCUG GC-UGC.-r U GGCUCAG CUUG=C C AGACAUG GACAUGU U UUCCGG ACAUGUU U UCCGUGA CAUGUUJU U CCGU7GAA AUJGULTUU C CGUGAAA GAACAAU A GGCUGUU AGGCUGU U CCCAUGU G=CGUU C CCAUGUA CCCAUGU A GCCCCCtJ CUGGCCU C UGUGCCU uGUC-ccTJ U CUUUUG-A GUC-CCUTU C UUUUGAU GCCUCU U TJUGAUTUA CCUCUU U UGAUUAU CUUCUUU U GAUTUAUG TUUUUGAU U AUGULIUU 1440 1442.

1446 1448 1449 1451 1456 1457 1461 1464 1466 1479 1480 1494 1498 1501 1512 1517 1528 1533 1537 1540 1546 1549 1551 1552 1566 1572 1576 1577 UG"UUUU U GMUUUUU A UUAA.AA:U A AAAAUAU U AAAkUJAW A AUATUAU C AUCUGAU U U;CuGA-juU A AL)UAAGUJ U AAGUCGU C GUjUGUCj A% utr3_CAU u (icujGArU u CAACjGtj C UGUCACU C CACUCAU U G-AC-GCCUj C CuCGCU C AGGGAGU U GUJUGUGTJ C uGucUGUc- A CUGUAA7U C UCGC-CCU A GCCTJACU A CUACtJAU u UACUAUEJAJ C GAGAAAU A UAAAGGU U GGU'UGCU U GUUGCUU A .AAAUa~u

AAA:UATUUT

TUAUCrUG

AUCUGAU

UCLAUU

UCGAUUAA

A"AGUUGU

A;GUUGUC

GUCUJAAA

UJAAC-A A

AAC-AAUG

UC-GUGUCAC

.GGACC

~-CAcuEJ

A~TXGCUG

GCUGAGG

uGcUCCC

CCCAGGG

GUGCCU.G

UGUAA:UC

AUCC-CCC

C-CCU71C CL7AUULCA2 TjUCAGUG

CAGU-'GGC

AGUGC-CG

.AAC-GUJUG

GCUUAGG

AC-GAAAG

GGAAAGA

UUUGAUU

AUUAUGU

UUAUGLuU UAUG~jU-L

UGUUUUUT

TjUUUAAAA

UUAAAA

SUBSTITUTE SHEET (RULE 26) WO 95/23225 PCT/IB95/00150 244 Table 24: Human TNF-a Hammerhead Ribozyme Sequences nt. 11H Ribozyme sequence Position 28 C-GAAGAG CUG-AIGAGGCCGAAAGGCCGAA ACCrUGCC 29 AGGAAGA CUGAUGAC-GCCG-AAkAC-GCCGAA AACCUC 31. AGAGGAA CUjGUGAGGCCGAAAGGC-CAA AGAACCU 33 UGAGAGG CUGAUG-AGGCCGAAAGGCCGAA AGAGAAC 34 GUCAGAG CTGAUCAGGCCGAAAGGCCCAA AAGAGAA 37 UALTGUGA CUGAL'GUCAGGC.-CGAAAGGCCG-AA AGGAAGA 39 AGUJAUGU CEGAUG-AGGCCG-AAAGGCCGAA AGAGGAA 44 C-GGUCAG Ct7GAtGAGGCCGAAACGCCGAA AUGUG.G 58 GAGGGUG CUGAUGAGGCCGAAAGGCCG-AA AGCCGJG GGGGAGA CUC-AUCAGC-CCGAAAGCCG;LA AGGGUGG 67 CAGGGGA CUG-AtGAGGCCG-AAAGGCCGAA kACTA-GU 69 UCCAGGG CUGAUGAGGCCGAAGGCCG-A- AGAGAGG 106 CGUCCCG CUjGAUGAGGCCGAAGGCCCGA AUCAUGC 136 UCUUGCG CUGAUG-AGGCCGAGGCCGAA AGCGCCU 165 CCGCCUG CUGAUG-AGGCCG-AAAGGCCGAA AGCCC7JG 177 GAGGAAC CUGAUGAGGCCGAAGGCCGAA AGCAzCCG 180 GCJGAGG CUGAUGAGGCCGAGGCCGAA ACA.GCA 181 GGCUGAG CUGAtJGAGGCCGAAAGGCCGAA AACAAGC 184 ACAGGC-U CUiGAUGAG~CCCAAAGGCCCA AGGAACA 190 AGGAGAA CUGAUG-AGGCCGAAAGGCCGAA AGGC-UGA 192 GAAGGAG CtJGATGAG-GCCG-AAAGGCCG-AA AGAGGCU 193 GGAAGG-A CUGAtJGAGGCCGAAAGGCCGAu'A AAGAGGC 195 CAGGAAG CUGAUGAGGCCGAAAGGCCGAA ACAAGAG 198 GAtJCAC-G CUGAUGAGGCCGAAAGGCCG-AA AGGAGAA 199 CGAUCAG CtJGAUGAGGCCGAAAGGCCGAA AAGGAGA 205 CUGCCAC CUGAUGAGGCCGAAAGGCCrGAA AUCAGGA 226 C-GCAGAA CUG-AUGZAGGCCGAAA.GGCCGA-A AGCGUGG 228 CAGGCAG CUGAUGAC-GCCGAAAGGCCGAA AGAGcGtJ 229 GCAGGCAk CUGAUGAGGCCGAAAGGCCCGAA AAGAGCG 243 CACUCCA CUG-AUG-AGG-CCAAAGCCCGAz, AGUCCAG 244 UCACUCC CUG-ATJGAGGCCGAAAGGCCGAA AAGUGCA 253 GGGGGCC CUGAUGAC-GCCGAAAGGCCGAA AUCACUC 273 CCUGGGG CEJGAUG-AC-GCCG-AAAGGCC-A-A ACIJCUUC 286 UUAGAGA CUG-AUG-AGGCCGAAAGGCCGAA AGGuccc 288 GAUUTAGA CUGAUG-AGGCCG-AAAGGCCCGPA AGA GGUC 290 CUGAUUA CUG-AUG-AC-GCCGAAAG-GCCGA-A AGAGAGG 292 C-CUGAU CUG-AUGA;C-GCCGAAAGGCCGA-A

AGA-GAGA

295 CAGGGCU CUGAUGAC-GCCGAAA C-GCcc;AA: Auu-AGAG 302 UGGGCCA CUCGAUG-AGGCCGAAAGGCCGA.A AGGGCUG SUBSTITUTE SHEET (RULE 26)

M

95/23225 rc("r/1139510()156 245 3 2: AG-AAGAu cGAUGAGGCCG-AA.GGCCGAA AUCUGAC 322 UCGAGA cUGAUGAGGCCGAAAGGC-CAA AtJGACt' 326 -,UUCGAG CUG-A!AGc-CCGAAAGCCGAA AGAUGAU 327 G tJUCGA cUGAUGAGGCCGAAGCCG-AA AAGAUGA 329 c:G mc CLGAUGAGGCCGAAAG-GCCGAA

AGAAGAU

352 CAUGGGC c!JGAUGAGGCCG-AAAGGCCGAA

ACAGGCEJ

361 UULGCUAC CUGALGAGGCCGAAAGGCCGrAA ACAUGGG 364 GGUuUjGC CUGAUGAGGCCCGAAAGGCCG-AA ACAACATJ 374 G=ACU CGUAGGA CCGAA GGGtUt 39: GCCACUG CUGAUGAGGCCGAACGrCCG- AC-CUGCC- 42: UG-GCCAG CJG-AUGANGGCCGAACGCCGAA

AGGWCA;

44129 AGGt7CGAUGAGCCGAAA-CGAA UCtA'cC 468 GC-CCt CUC-AUGMG -CC-AA.AGGCCGA-% ALkUCMC 480 CAUGAGG CUiGAUGAGGCCGAAAGGCCCAA ACAGGCC 484 AGUAGAtJ CtJGAUGAGGCCGCAAAGGCCGAA AGtjUA-A.

487 cGGAGUA CUGAUGAGGCCGA.AAGGCCG-AA AUGAGGU 489 CUGGGAG CUGAUGAGGCCGAAGGCCG-AA AGAUGAG 492 GACCUGG CUGAUGAGGCCGAAAGGCCCGAA AGUAGAU 499 UGAAGAG CJGAUGAGGCCGAAAGGCCGAA ACCUGGG 502 caCUUGAA CU-A:UGAGGC,-CAAAGG-,-C-GAA AGGACCtJ 504 C-C-CUG CUGAUGAGGCCG-AAGG--C GAA AGAGGAC ;Cr 5 GGCCCLTU CUGAUGAGGCCGAAAGGCCGP.A AAGAGGA 525 AUG,-UG CtJGAUGAGGCCGAGGCCGA. AGGGG:M*A 538 GGGTJGAG CtJGAUGAGGCCGAAAGGCCAA AGCACAU 54: tIGUGGGU CUG-AUGAGGCCGuAAGGCCGAA AGGAGCA 553 UC-CGC-CU CUGAUGAGGCCGAAAGGCCGA-A AUGGGUt 562 AG-ACGGC CUJGAUGAGGCCGAAAGGCCGAA AUGCGCC 568 C-GUAGCGA CUG-AUGAGGCCG-;AAGGCCGAA ACGGCGA 570 CUGGUAG CUGALGAGGCCGAAAGGCCGAA AGACGGC 573 -GUCUGG CUGCAUGAGGCCGAAAGGCCGAA AGGAGAC 586 GGACGWU CUGAUGAGGCCGAAAGGCCGA.A ACCUUGG 592 CAGAGAG CUGAUGAGGCCGAAAGGCCGAA AGGtJUGA 59Q5 L'GGCAGA CUCAUGAGGCCGAAAGGCCGAA AGGAGGU 597 GAUGGCA CUGAUGAGGCCGAAAGGCCGAA AGAGGAG 604 GC,7-UCUtJ CUGAUGAGGCCGAAAGGCCG-AA AUG-GCAbG 657 CGCGCUC% CTIGAUGAG-GCCGAAAGGCCGALA ACCAGG 667 CCAGAtJA CUGAUGAGGCGAAGGCCG-A AUGGGCU 669 UCCCAGA CUGAUGAGGCCGAAAGGCCG-AA AGAUGGG 67.- CCUCCCA CUC-AUC-AGGCCCG GCGCCG-AA AUJAGAUG 682 GCUGW.A CUGAUGAGGCCGAAAGGCCGLAA ACCCCUC 684 CAGCUGG CUGAUGAGGCCGAAAGGCCGA AGACCCC 685 CCAGCUG CtJGAUGAGGCCGAAAGGCCG-AA AAGACCC 709 CAGCGCU CUGAUGAGGCCGAAAGGCCG.A AGUCGGU 72:- GCCGAUU CUGAUGAGGCCGAAAGGCCCGAA AUCUCAG 725 UCGGGCC CCGAUGAGGCCGC-GCA UGU 735 GUCG-AGA CUJGAUGAGGCCCG:AAGGCCGA.A %,,UCGGG 737 A.AGUCGA CUG-AtGAGGCCGAAA-GCCGAA AUAGUCG 739 CAAAGUC CUG-AUGAGGCCGA.AGGCCG-AA AGAUAGU 744 CUCCGCA CUGAUGAGGCCG-AAAGGCCCGAA AGUCGAG SUBSTITUTE SHEET (RULE 26) WO 95123225 246 745 ACTJCGGC CUGAUGAGG-CCGAAAGGC-CGAA AAGtCGA 753 Ct.GCCCP. CUGAUGAGGCCGAAAGGCCSAA ACtJ XGGC 763 CAAAGt7A C UG A UGA G GCC GA A A G CC G AA ACCL'GCC 765 CCCAAAG CT GAUGAGGCCGAAACGCCGAA A G AC C', 768 GAUICCCk CUG A TJG AG GC CG A AA ,G CC GA A AGtJAGAC 769 tJGAtJCCC C U ~G A lG G CC GA AA GG C GAA AAGUAGA 775 GGGCAAU CrJGAUGAGGCCGAAAGGCCGAA AWCCCAA 778 ACAGGGC CUGAUGAGGCCGAAAGGCCGAAM AUGAtICC 801 AAGGUtJG C U G A UGN=CG A AAG G C C GA AtATJCCG 808 GUUUEGGG CUGLVGAGGCCGAAAC-GCCaAA AGGUYULGG 809 cGUUUGG CUGAUGAGGCCGAAAGGCCCAA AAGGG 820 GGCAGGG CUGAUGAGGCCGAAAGGCCGAA AGGC',",U 833 At3AAGG CUGAUGAGGCCGAAA.GGCCGAA ALtUCGGG 837 GGUAAUT-A CU GA UG A G G CCGAA G GCC GAA Ac-cGAUUJ 838 GGGUAAU CUGATJGAGGCCGAAAGGCC-GAA AAGGGAU 839 GGGGUAA CUGAUGAGGCCGAAAC-GCCGAA AAAGGC'A 841 AGGGGCU CUGAUGAGGCCGAAAQGCCGAAz AL'AMW~ 842 CAGGGGG CtJGAUGAGGCCGAAAGGCC,-AA AAUAAAG 849 UCtJGAAG CUGAUGAGGCCGAAAGGCCGAA AGC-WGUt 852 GUGUCUG CTJGAtJGAGGCCGAAAGGCCGAA AGGAGG 853 C7 GUCtJ Ct3GAtGAGGCCGAAAGGCCGAA AAGGAGG 863 AGAGGTUt UaMUGAGGCCCGAA.AC-GCCCGAA AGGa'taG.Jj 869 GCCAGAA. CUGAUGAGGCCGAAAGGCCCGAA AGGUUGA 871 GAGCCAG CUGAUGAGGCCGAAAGGCCGAA AGAGGCU 872 UGAGCCA CUGAUGAGGCCG-AAAGGCCGAA AAC-AGCtJ 878 t7CM=JUt CUGAUGAGGCCGAAAGGCCGAAM AGCCAGA- 890 AGCCCCC CUGAUGAGGCCGAAAGGCCGA.A ATJUCUCtI 898 CGACCCtJ CUGAUGAGGCCGAAAGGCCGAA AGCCCCC 899 CCGACCC CtJGAUJGAGGCCGAAAGGCCGCAA AAC-CCCC 904 GGGUUCC CtJGAUGAGGCCGAAAGGCCGAA ACCCtJAA 917 AAGTJUCU CUGAUGA.GGCCGAAAGGCCGAA AGCJUGG 918 AAAGt3UC CUGAUGAGGCCGAAAGGCCGMAAAGCtUG 924 UUCT=U CUGAUGAGGCCGAAAGGCCCGAA AGtJUCUA 925 GtJUGCTUt CUGATJGAGGCCGAAAGGCCG-AA AAGtMCt 926 T7GtJUGCU CLGAtMGGGCCGAAAGGCCGAA A.A; CC 945 GGUUCG CtJGATJGAGGCCGAAAGGCCGAA AGLGGLG 946 AGUUUC CUGAUGAGGCCGAAAGGCCGAA AAGUGGU 959 AMtCCUG CUGAUGAGGCCGAAAXGGCCGAA AUCCCAG 960 CAtUCC3 CtJGAUGAGGCCGAAACGGCCGAA AATUCCCA 1001 GAAUUCU CUGAUGAGGCCGAAAGGCCGAA AGX:GGUU 1007 CAGUUUG CUGAUGAGGCCGAAAGGCCG-AA AUUCUUA 1008 CCAGUUU CUGAUGAGGCCGAAAGGCCGAA ?A.UUCUU 1021 AGUUCUG CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 1029 CCCCAGU CUGAUGAGGCCGAAAGGCCG-AA AMTJCtG 1040 AAAGCUG CUGAUGAGGCCGAAAGGCCG-a.A AGGCCCC 1046 GGGAUCA CTUGAUGAGGCCGviAAAGGCCGAA AG=UA 1047 AGGGAUC CUJGAUGAGGCCGAAAGGC-CGAA AAGCtUGU 1051 UGUCAGG CUGAtJGAGGCCGAAAGGCC-A-A AUCAAAG 1060 G-AUUCCA CUGAUGAGGCCGAAAGCCCGAA AZGUCAG P(TII 51016 SUBSTITUTE SHEET (RULE '26) m WO 95/23225 247 1067, GUCUCCA CCGAUGAGCGAAAGGCCGAjl AUCCCAG 1085 AGA~cA cuGAtLGAGGCCGAAAGGCC%.GAA ACGG=tCC 1086 cAGAcc cuGAUGAGGCCGAAAGGCC A AAGGCC 1090 UGGCCAG CCGAUGAGGCCGvAiAGGCCGAA ACCAAAG 1091 CCC-A cuGAUGAGGCCGAtUW-CCCGAA AACCAAN 11-3 UC'XCUC CUGAGAGGCCGAAAGcGCCGuAA AGUCCUG 1124 UCUAGu CGaUGAGGCGA.'%GGCCG-AA AGGUCUUV 1129 cA.ut3c cUGAUGAGGCc"GAAAGGCCGAA AGGUGAG 1135 tJUGtJGuc CUGAUCAGGCGAAAGGCCGAA AUUUCUA 1151 AAGGCCU CUGAETGGCCGAGGCCGA A AGGSUCCA 1152 GAA.GCC cUGALuGAGGCGAAAGGC CGAA AAGGUCC 1158 AGAGAGG cUGAtmGCCAAAGGc"GAA AGCLtA 1159 GAGAGAG Ct7GAUGAGWGCCGAAAGGCC GAA AAGGCCU 1U62 cpUGGAGrA cCJAUG.ACGCCGAAAGGCCGAA AGGAAGG 16164 AUCUGGA CUGAtrAGGCCGAAAGGCCGAA AGAGGAA 1166 AcAt7cuG CCVGAUGAGGCc"GAAAGGCCGAA AGAGAGG 1174 GUCUGGA CUGAtGAGCCAAAGGC'CGAA ACAUCUG 1175 AGUCU;GG CUGAUGAGGCCGAAAGGCCG-AA AACAUCI3 1176 AAGUCUG CtJGAUCGAGGCCGAAAGG;CCGAA AAACAUC 1183 CUCAACG CUGAUGAGGCCGAAAGGCCGAA AGUJCUGG 1184 tUCUCAAG Ct7GAUGAGGCCGAAAGGCCGA,% AAGtJCUG 1187 GUGUCtJC CtJATGAGGCCGAAAGGCCGAA AGG-AAGU 1208 CCAUGGG CUGAruNAGGCCGAAAGGCCCAA AGG 1224 ALTAGAGG CUGAUGAGGCCGAAAGGCCGAA AGCUGGC 1228 AtJAAAUA CtJGAtMAGGCCGAAAGrCCGAA AGGGAGC 1230 ACAuAAA CUGAUGAGGCCGAAAGGCCGAA AGAGGGA 1232 AAACAUA CtIGAUC-AGGCCGAAAGGCCGAA AUAGAGG 1233 CAAACAU CUGAUGAGGCCGAAAGGCCGAA AAUAGAG 1234 GCAAACA CUGAUGAGGCCGuAAGGCCGAA AAAUAGA 1238 AAGt!GCA CUGAtJGAGGCCGAAAGGCCGAA ACAUAAA 1239 CAAG;UGC CUGCAUGAGGCCGAAAGGCCGAA AACAtJAA 1245 UAAUC.AC CTJGAUGAGGCCGAAkAGGCCGAkA AGtJGCAA 1251 AAUAAAU CT.GAUGAGGCCGAAAGCCGAuA AUCACAA 1252 UAAUAAA CUGAUGAGGCCG-uAAGGCCGAA AAUCACA 1254 AAUAAUJA CtJGAUGAGGCCGAAAGGCCGAA AtIAAUCA 1255 AAAUAAU CUGAUGAGGCCGAAAGGCCCGAA tAUAAUC 1256 UAAAUAA CUGAUGAGGCCG?)AGGCCGAA AAALTAAU 1258 AAtUAAU CtlGAUGAGGCCCGAAAC-GCCCGA AUAAAUA 1259 AAUAAA CUGAU;GAGGCCGAAAGGCGAA AAUAAAtJ 1261 AtJAAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA 1262 AAUAAATJ7 CUGAUGA=GCGAAAGGCCCGAA AAUYAAUA 1263 tJAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU 1265 AAUAAUA CU=AGAGGCCGAAAGGCCCGAA ATUAAAUA 1266 AAAUAAU CUC-AUGAGGCCGJAAGGCCGAA AAtYAAAU 126"1 UAAAtJAA CUGAUGAGGCCGAAAGGCCG-AA AAAUAAA% 1269 AAUAAAU CUCAUGAGGCCGAMGGCC-GAA AUAAAUA 1270 AAAt;AAA CtJGAUGAGGCCGAGGCCGA AAUAAAUJ 1272 AUAAATJA CUGAUGAGGCCGAGGCCGM 1273 AAUAAAU CUGAUGAGGCCGAAAGGCCCGAA AAUAAUA rC'ii 119SAM(11$6 SUBSTITUTE SHEET (RULE 26) WO 95123225 248 I& 4 AAAtYAAA CTUGAt;GAGGCCM4AAAGGCC A WUAAtI' i.276 GTJAAAtTA CUGAT.GAGGCCGAAAGGCC*G-AA AUAAATJA 2.277 UTJGAAAY CUGAUGAGGCCGAAAGGCCGAA AAtIAAAtJ -27 8 C't.GAAA CUGAUG-AGGCC~AAGGCCGAA AA~tAAA 2280 AUCUGUA CUGATJGAGGCCGAAAGGCCGAA AUAAAUA 1281 CAUCUGU CUGAt7,GAGGCCGAAAGGCCGAA AAUTAAAtI 1282 UCAUCUG C UGkUGAGGCCGAAAGGCC=A AAAUAAA 1294 AAUA UAGGCNAAAGGCC3AA ACU~ 1296 C-MAAU CUGAUGA=GCGAAAGCGCCGAA AM3CAbVU 1297 CCCAAMt Ct;GAUGAGGCCGAMAGGCCGAA AAtrACAtJ 1298 UCCCAAA CUGAt;GAGGCCGAAAGGCCGAA AAAUACA 1300 %OCUCCCA CUGAUGAGGCCGAAAGGCCGAA AUAAAt;A 1302. GUCIUCCC Ct7GAUGAC-GCCGAAAGGCCGAA AAUAAAUJ 1315s CCCAGGA CUGAUGAGGCCGAAAGGCCGXA ACCCCGG 1317 CC"CCAG CUGAT.GAGGCCGAAAGGCCGAA AUACCCC 1334 CAGCC CUGAUGAGGCCGAAAGGCC.GAA ACAUUGG 1345 CLuAGCC- CUGAUGAvGCCGAAZGCCGAA AGGCAGC 1350 CAUGUCO CUGAUGAGGCC-GAAAGGCCGAA AGCCAAG 2359 CACGGAA CTGAUjGAGG3CCGAAAGGCCGMA ACAUGtX 1360 UCACGGA CUIGAUGCAGGCCGAAAGGCCGAA AACALGU 1361 tUtCACGG CUGAUGAGGCCGAA.AGGCCGAA AAACALTG 1362 ULUCACG CU.GAUCGAGGCCG-AAAGGCCGAA AAAACAU 1386 AACAGCC CCGAUGAGGCCGAAAGGCCGAkA AtJtJGUC 1393 ACAUGGG CUGAUGAGGCCGAAAGGCCGAA AC.7GCCU 1394 UACAUGG CVGAUGAGGCCGAAAGGCCCGAA ?.ACAGCC 1401 AGGG=G CUGAUGAGGCCGAAAGGCCGAA ACAtJGGG 1414 AGGCACA CUGALGAGGC%-GAAAC-GCCGAA AGGCCAG 1422 UCAAAAG CUGALGAGGCGAAAGGCCGAA AGGCAC.A 1423 AUCAAA?. Ct7GAtJGAGGCCGAAAGGCCGAAk AAGGCAC 1425 t3AAUCAA CUGAUGAGGCCGAAAGG;CCGAA AGAAGGC 1426 At7AAUCA CUGAUGAGGCCGAAAGGCCGAA AAGAAGG 1427 CAtAAUC CtJGAUGAGGCCGAAAGGCCGAA AAAGAAG 1431 AAAACAU CTGAUGAGGCCGAAAGGCCCAA At3CAAAA 1432 AAAAACA CUGAUGAGGCCGAAACGCCGAA AAUCAAA 1436 UUUAAAA CUGAUGAGGCCGAAA-GCCGAA ACAUAAU 1437 UUUt3A CUGAUGAGGC~ruuAGGCCGAA AACAUAA 1438 AUUtJUAA CUGAUGACG CCCAAAGGC-CAA AAACAUA 1439 UAUt3UUA CUGAUGAGGCCGAAAGGCCGAA AAAACAU 1440 AM=~2U CCGAUGAGGCCGAAAGGCCGAA AAAAACA 1441 AATJAlUUU C UGAUGAGGCCGAAAGGCCGAA AAAAAAC 1446 CAGAUAA CUGAUGAGGCCGAAAGGCCGAA ALTUtUUAA 1448 AUCAGAU CLTGAUGAGGCCGAAAGGCCGAA AUAUUMJ 1449 AAUCAGA CUGAUGAGGCCGAAAGGCCGAA AAtTAUUtJ 1451 UUAAUCA CUJGATUGAGGCCGAAAGGCCGtAA ATJAAUAU 1456 ACAACUtI CUGATUGAGGCCGAAC-GCCGAA AUCAGAtI 1457 GArnAcu cuGAuc-AGcccGA.A.AGct GAA AAucAGA, 1461 UUUAGAC CUGAUGAGGCCG-AAGGCCCGAA ACUUAAU 1464 tjttA CUGAUCGCGCCCAAAGGCCGAA ACAACUU 1.466 CAUUGUU CUGAUGAGGCCGAAAkGCCCGAA AGACAAC lICT/11195/00016 SUBSTITUTE SHEET (RULE 26)

I

WO 95/23225 249 1479 GUCACCA, CUGAUGAG-GCCGAAAGGCCGA AUCAGCA 1480 GGUCACC CUGAtGAC7GCCGAAACGAA AALCAGC 1494 AAUGAGTJ CUGAUGAGGCCGAAAGGCCGAA ACAGtJUG 1498 CAGCAAU CtGAUGAGGCCGAAAGGCCCAA =CuAA 1501, CcU-AGC CtJGAt7GAGGCCGAAAGGCCGAA AUGAGtG i52.2 GGGAGCA CUGAUGAGGCCGAAAGGCC-G.M AGGCCtC 1517 CCCt3GG-7 CUGAUGAGCCGAAAGGCCGAA AGCAGAG 1528 CAGACAC CUGAUGGGCCGAAAGGCCGAA ACtJCCCYJ 1533 GAtJUCA CtJGAUGAGGCCGAAAC-GCCGAA ACACAAC i537 GGCGAU CUGAUGAGGCCGAAGCCGAA ACMGACA 1540 GUA.GGCC C3GAUGAZGCCGAAAGGCCGAA AUtTACAG 1546 IJGAAUAG CUGAUGAGGCCGAAAGGCCGAA AGGCCG,% 1549 CACUGAA CTJGAUGAGGCC-GAAAGGCCGAA AGUAGOC 1s51 GCCAC!G CLMGAuGGGCC-GAAAGGCCGAA AUAGUAG 1552 CGCCACU CLTGAtJGAGGCCGAAAGGCrCGAA .AAUAGOA 1566 CAACCTJU CUGAt7GAGGCCGAAAGGCCGAA ALTJUCUC 1572 CCUAAGC CUGAUGAGGCGAAAGGCCGAA ACCtJUA 1576 CUTCCU CtJGAUGAz=CGAAAGGCCGAA AGCAACC 1577 UCLJUCC CUGAEUGAGGCCGAAAGGCCGAA AAGCAAC PC19510MM156 SUBSTITUTE SHEET (RULE 26) WO 95123225 250 Table "25: Mouse TN-f-a HE- Taxget Sequences PC('T/1139S1()()150 mit.

Positionl 101 101 102 i02 106 '110 U16 137 i39 177 207 228 228 236 236 249 249 261 261 263 263 264 264 266 269 270 276 297 299 300 304 306 3 14 315 315 324 HH Target Sequence UgGAAAU

GGCAGGU

GGCACgL

GCAGGTU

9CAGgUU GUtJCLgt tuguccct gCZCcCUtJ guCCCutl ticCCOuu tUuUCACtJ GCCaCAU caCAuCU

GCAUGAU

AC-GCaCtJ GGGCutJ

GGGGCU

CAGaaCT CAGaACU GGUgCCtJ GGuGCCtJ UCAGCCt7 UCAgCCtJ

AGCCUCU

AgCCUTJ

GCCUCUU

gCCtCUU CCLITJCUj~t TJUCtJCaU t3CrJCaUtJ tJCCEJGCU CCAkCGCtI

ACGCEJCU

CGCOCUU

CtiuCtugu UCUGtUCt CUGaACY tJGaACUU uGaaCU gGGtJGaU GcucCCA CtgUcCC CUGUcCC UgUcCCtJ UgUCCCtJ CCtULUCA

UCACUCA

CaCtJCAC CACuCAc ACucACtJ Act7Ggcc uCctCcuc CCUJCcAg

CGCGACG

CCCcAaA

CAGAACU

CAGaact7

CAGCGG

cA(3gcGg tugL7CcA UGucUCa UUCUCaU UCCUcau CUCaUtC CLTcauUC UCaUUCC tjcautjCc atllJCCUG CCUGcUu CUGcUuG

GUGGCA.G

tJUCUGuC CUGuCUa UGuCUaC uActJGaa ctgAAcU cG~gGtJG GGgGUGA GGGguGa GgtJCCcC at.

Positioni 324 347 364 3 660 366 369 376 390 396 401 404 406 406 407 409 409 409 432 444 501 560 560 564 567 569 572 572 572 579 580 580 582 582 584 585 608 6i5 615 618 HH Target Seq~uence GgCGAU GAGAagtJ

CC

t JCcCU tJ~UCU UcCCUC= CUCjcAIJk CAGuuCUJ A9ACCCU ucaCAcU cUCAGAU AGAUCAtJ

AUCAUCTU

AUcAtUcU

UCAUCIU

AUCUUCJ

AuCuUCU allcUt~cU

AGCCUGU

AcGUcGU AcGCCCtJ gGgUUjGU GG9UtJGt

UGUACCU

ACCUugtl cuugEJCU gUCtJACtJ GUCUaCtJ GuCUactJ

CCCAGGU

CCAGguU CCaGGutJ

AGGUUCU

AGGUuCU GUuCt3CU UuCtJCUU CcCGaCtJ aCgUGcU AcGUGCtJ

UGCUCCU

GGuCCC cCCAaaU Uci.UCAG AUCAGUu auCAGut) AGuu~ta

U=GCCA

AcaCUcA

AGATJCAU

AUCtUtCU UUCCaiA CUCaAAa c t ~caAAM tJCaAAau aA~auuC Aa.AAtC AAAauUc GCCCAcG

GCAAACC

CJGGCCA

CCTuguC CCUugUC gUCUACU

UACU.CCC

CUCCCANG

CCAGGUu CCAGguu CCAgG~u

CUCUUCA

UCUUcAa UCuUcaa UUtCaagg

UUCAAGG

CAAGGGa AAGGGaC CgugCUC CUCAcCC

CUCACCC

ACCCACA

SUBSTITUTE SHEET (RULE 26) WO 95/23225 PC'r/Iu9/o(J1 56 251 AcACCgUi C AGCau ACACcgtJ C AgCCqatJ agcCgAU u uGCJaUc alUUGctU a UC t JCAuA UuGCUaU C t7CaUACC GcuaTjC3 C allACCAG agAAaGU C A7ACCtJCC UC.AcCCr1 C Ct3CtCUG UcAAcctJ c ct~ct7C'G AcctJCCt C UCUGCI-g 7c-Uczu C UjGCCguc ctjGCCgt3 C AagaGcC- CUGCCgtJ C AAGAGCC CUGcCgU C aaGAgcC CCCUGGU A UGAGCCC Ccc7GGU a ugaGCCc AGCCCAU a UAcCUGG CCCAUaU A cCUGGGA GAgGAGtJ C uuCCAGc GAGGaGU C UUCCAGC G~aGtJCU U CCAGCUG GaGUCtJU C CAGCUGG ACCaACU C AGCGCUG Ct2GAGgtJ C AATJCuGC GgUCAAU C uG;CCCaA CC.CaAgU A cut~aGAC AgUAcuU a GACUrJUG ut~aGACtJ U UGCgGAG UaGACUtJ U GCgGAGU GCgGAGU C cGGGCAG GGCAGGU C UACUUtUG CAGGUCY A CUUtJGGa CAGguU a CUugGA cagGuCY a CUUtUgGA GUCtJACU U UGGagUJC UCE3ACUrJ U GGagUCA UUGGagU C AUJGC GagUJCAU U GCuCtJGt AUCCaUU c ucJACCC AuucuCU a CCCaGCC CCcCaCU C UgaCCCC cCCCactJ c UgACCCC CCCCACu c uGACC G-AcCCcU U uacUCUG ACCCCuU u acUCUIGA CUUtJAcU c ugaCCC GACCcCU u Uat~ugLJC gAcCCCU U tYAtJtguC CCUUUJAU U gu.cuaCU 940 343 973 984 984 985 997 .0179 :107 U.08 1:33 16 4 1i80 =203 1210 128 121.8 122.9 1226 1226 1227 1227 2.228 1238 1262 1.283 1283 2.285 12287 1287 1288 1289 1293 1293 2.294 GuctYACU a A\CUCCV: C .U,aaU u ucUaaCJ u CUaACU-U A AGg GgAU U AGC-GqaU *J GGGGautJ a U;cAGAgU C.IucC c CAG.CAgcU ACGAgcuU u C-AgCUU ugGGCU z AAGgAcldC at~c'GG~cU U tUGCGcUU u Gc~gCUUU c cGcAAutU C ClGAAugu gag(UGgU c ucugtucu c aaGAi.,CU c cAGGCCU7 U AkGc.cCjTj C CCCCU a Ccu.ACcU u C CuaC CU U cCUpACCU u C~jacCU u CuaCCUU C C*.AcCUUW c CagACC'U U CAG-Acct U agA.CCUjU u ACGAcctut U GACCuuu C gACtCuU c CAC-Cl-uY C CCCCCcU C ccclCC C cCCCUC A CctiCUAU L, =,=tYu cUt.7l-nt.tJ u ucZ:A~urju A Lt~aU U uLUaUaU u UtJcaruU U ctUCAGaG3 AGaGcCC,- AqAAAG:1 AGuaAgG GAAAggG auG.3cuc C-:A~vcu wcAaCAA cAaCAAC uc-3ugr-A AAAugG uccAANU ccGAAt~u cGaaJUC ACCGGaG

CAUUCCU

AgGUJUGc agaAUGA

AGC-UU

CC-UacCU C'JacCUu cM'uCAG CaGACCu

CAGACCU~

cAgACCtJ CAG-AccrJ AGACcuu agACctU uCCagAC tUCCAGAC CCAgACu CcAGakCU CACGAct.c cC'XGAGG C'JC~caG uaUUAU

TJAUTJAU

Ut~jAUaU tjauAuUU UAUaULU AUa~tjt.IG UaUUUGC U:C-CAkC'Ju tUGc~lct~u GC.ACUUa SUBSTITUTE SHEET (RULE 26) wo 9SI23225 PCIr/11195100156 252 1300 LUGCACU U alluAUUu 1462 aCCuCU u ~CiC C 1303 CAcu3aJU AuUuAUU 1470 GCcCUO UMtGcu 1304 acuuAuU A UUUAUTA 1472 cuCcUCU U UtJGcUUA 1306 UuAt3UAU U UAUUEJU 1473 uCcTurJ U UJJMJ 1307 UAUUAUU U AUCYAUU 1474 cc==u U GcUUAUX; 1307 UaUrat3TJ U AUUAUUU 1478 LUUUUGcU U AUMU~ta 1308 AUAUUTJ A UUAUtJUA 1479 t~TGcUU a UGuuu1a 1310 UauUUAU U AUUUAUU 1479 UVUGcVU A t;GUCUaa 1310 uAutJTj U AUUUAU;U 1484 UTJAU7,Tj aaAcAA 1310 DAuuuAu U AUUUAUTU 1498 AAAuauU U AC~aAc 1311 Au3uAu3 A UUt3AULTJ' 1511 Acc-cAaUJ GUCUUAA 1311 AUUAUU A UVUJAtJUU 1514 cAaUUGU C UuAAuAA 1311 AuuUAUU-T A UuUautJU 1515 atGuCJ u AAuAAcG 1313 tUTAUUAU U U-AUUUAU 1529 CgcugATJ u UGGuGAC 1313 UUAtUtAU U UAUU.UAU 1529 cGCUGAU U UGGtGCAC 1313 uAUUAU u UautUtAu 1530 gCUGAUtJ u gGUgacC 1314 tUAMT3U U AUtUMJU 1530 GCUGALTU U GGUGACC 1314 tuAuLTA3 U AUUUATJU 1563 tUgaAcCU c TUGcUCCC 1315 AMUUALI A UUtAThJA 1563 ugaaCC'J C UGCUCCC^ 1317 tr.uuuAu U UAUUAUJU 1568 CIUC"t'GC C CC=%cGG 1318 AUUUAUU U AUUAUUJU 1589 UGaCUGtJ A ;UuGcCC 1319 UMUUU A t3UAUEJtIA 1592 CUGUAAU u GcCCtJAC 1326 Aut3Autu A UUUAUtJU 1617 GAGAAAU A .AAGaUcG 1328 tJAUUUTAU U tJAUUUgC 1623 UAAAGaU c GCU.UAaa 1329 AUUUJ U AUUUgCU 1633 tJUAaaaU a aaAAaCC 1330 UUUAUUU A U-UUgCuu 25 AgGgaC'U a gCCagGA 1332 UAUUUAU U UgCuUAU 1333 AUUUAUU U gCuUAUG !337 auUUGCtJ U AUGAAUG 1338 uUUGCt3U A UGAAUGu 1346 uGAAuGu A tJUUAUtU 1348 AAUGUAU U UAUUJUGG 1349 AUjGUAUU U AUUUGGa 1350 UGTJAUUU A UUUGGaA 1352 uAUuUAU u UGGaAGG 1352 UAUUUAU U UGGaAGg 1353 A UUaAUU U GGaAGgC 1369 CGGgU C CUJGGaGG 1398 gcUguCU U cAGACAg 1398 GcuGuCt3 U cagaCAG 1412 GACAUGU U TJUCUGUG 1413 ACAUGUU U UCuGUGA 1414 CAUGUuu U CuGUGAA 1415 AuGuuuu C uGUGAAA 1415 AUGUUtJU c UgugAaA 1438 gaGCUGU c CCCACCtJ 1451 ctJ'GGCCU C UcE~aCCU 1453 ggcCtICJ C UaCCuUG SUBSTITUTE SHEET (RULE 26) WO 95/23225 1'(7/1105/00156 253 Table 26: 'Mouse T.NF-a Hammerhead Rib ozyme Sequences mt Mouse Mi Piboz'mc Soquonce Position UCCGC CALGAC-G3CC-AAAC-GCCGAA AG%"CCCV 66 UGGAGC CCGAUG-AC-GCCGAAAGGCCGAA ALTJtJ'%c 2.2.GGGACAG CLGAtLGAGCCGAAAGGCCGAA ACCjC-, :0.G=GCAG CtGAUGAGGCCGAvAAGGCCGAA ACCUGCC 2.C 2 AGG~2C'~ Ct P3AGCCGAAAGGCCGAA AACTCC 2.02 AGGGACA Ct7GAUGAGGCCGAAAGGCCCAA ?.ACCUGC i.06 tIGAAAGG Cc:GAU'GAC7GCCGAAAGGCCGAA ACAGAAC 1.2.0 UGAGUGA CtGJGAGv%-GCCGAAAGGCCGAA ?.G=GCk i -I -I UGAGCU3 CEtGAUGAGGCCGAAAGGCCGAA AAGGGAC 2.2.2 GLUGAUG Ct.UGAG3CCGAAAGGCCGAA AAGGGAC 112 AGUGAGU CUC-AGAC-GCCGAAAC-GCCGAA AAAGGGA I S GCCAGU CU.GAC-AGGCCGAuAAGGCCGAA AGUGAAA .37/ GGAGGGA CUGAtUGAGGCC"GAAAGGCCGAA AL'GtjGGC 1.39 C=7GGGG CtJGAUGAGGCCGAAAGGCCGtAA AGAU~GtG !.77 CG;UCGCG CUGAt;GAGGCCGAAALGGCCGAA AUCAUGC 207 UTOtJGGGG Ct;GAUGAGCCGAAAGGCCGAA AGIGCCtJ 228 A~TwUCItG C!,GAGAGGCCA.AGGCCGAA AAGCCCC 228 AGtWtCUG CUGAJGAGGC-CGAAAGGCCGAA AAGC-CC 236 CCGCC',. C,;GAUGAGCCCGAAAGGCCGA AGw-UCUG 236 C=-CCUG CUCGAVGAGGCCGAAAGrGCCGAA AGL'UCUG 249 UAGACA CtUGAtGAGG-CCAAAGGCC-GAA AGGCACC 249 UGAGACA Ct;GAUGAGCCCGAAACGGCCGAA AGGCACC 262. AUGAAA CUGALGAGGCCGAAAGGCCGAA AGCVGA 262. AUC-AGAA CUJGALGAGGCCG-AAAGGCCGAA AGCUG-VA 263 GZAAUGAG CUGAtJGAGGCCGAAGGCCGAA AGIAGGCU 263 GAAUGAG CCGAUGAG7GCCG-AAAGG3C CG AA A GAGGC 264 GGAAUGA CUGAUGAGGCCGAAAGGCCGA AAGAGGC 264 GGAAUGA CC G A UGA G G CCCAAA G GC CG AA AAG-AGGC 266 CAGGAAU CUGAUGAGGCCGAAGGCCGA AGAAGAG 269 AAGCAGG CUGAUGAGGCCG-AAAGGCCGAA AUGAGAA 270 CAAGCAG CUC-AtGAGGCCGAA.AGGCCGiAA AAUGAGA 276 CUGCCAC CUIGAUGAGGCCGAAAGGCCGAA AGCAGGA 297 GACAGAA CUGAUGAGGCCGAAAG7GCCGAA AGCGtJGG 299 tJAGACAG CUGAUJGAGGCCGAAAGGCCGAA AGAGCGu 300 GtJAGACA Ct;GAUGAGGCCG-AAAGGCCGAA AAGAGCG 304 tJUCAGt7A M*AUGAGc-CCGAAAGGCCGAA ACAGAAG 306 AGUUCAG CtL'GAUGAGC-CCGAAAGGCCGAA AGACAGA 321. CACCCCG CCGA7GAGCCCAAAGGCCGAA AGucAG tCCCCC, CUG-AUGCC-GCCG-AALAC-GSCCGAA AG~TCA SUBSTITUTE SHEET (RULE 26)~ WO 95/23225 254 UCACCCC CUGAUGAGGCCGAAACGCC=A AA=~tCA 324 GGGGACC CUGAUGAGGCCGAAAG=CGAA AUCACCC 324 GGGGACC CUGATJGAGGCCGAAAGGCCGAA AtCACCC 347 AUUt7GGG CUGAUGAGGCCGwALAG3CCG-AA ACUC 3 S4 CE;GAUGA CtUGAUGAGGCCIGAAA =CAA AGGGAG 366 ?ACUGAU CUGAt AGGCC-GAAGC-GA AGAGGGA 366 AACt3GAUJ CUGAUGAGGCCGAAAGGCCGAA AGAGGGA 369 U7kr.GA=C CtGAUGA C.GA.AGGCGAA AtUGAGAG 376 UGGGCCA MtGAUGAGGCCGQAAAGGCC-AA AGAACiG 390 UGAGtUGU CtlGAUGAGGCCGAAGGCC-GAA AGG~T%:U 396 AUGAUCCJ CUGAUGAGCCGAAAGCC AGAG 401 AGAAGAU CL;GALGAGGCGAAGCCAA AUCUG 404 UUGAGAA CUGAUjGAGGCCGAAAGCC,-AA AtG-AUCU 406 =7UtG CUGALGAGGCCGAAGGCC=AA AGAUGAU 406 UUUUGAG CLGAUGAGGCCGAAAGCCGMA AGAL'GAL 407 AUUUJUGA CUGCtM3GCCGAAAGGCCGAA AAGATJGA 409 GAAUtUU CUGAUGAGGCCGAAAGGCCGAAM AGAAGAU 409 GAAUtUU CUGALGAGGCCGAAAGGCCGQAA AGAAG~AU 409 GAAtUUU CUGAUGAGGCCGAAGGCCGAA AGAAGAU 432 CGUGGGC CEJGAUGAGGCCCGAAAGGCCGAA AQAkGcC 444 GG M~rUG C CUGAUCIAGGCCGAAAGGCCGAA ACGAC~TJ 501 UGGCCAG CUGAGGCCGAAA.GGCCGAA AGGG=G 560 GACAAC73 CUGAUGAGGCCG =GCGAA ACAACCC 560 GACAAGG CUGAUGAGGCCGAAAVGCGAA ACAACCC 564 AGUAGAC CUGAUGAGGCCGAAAGGCCAA AGGUACA 567 GGGAGUA C1;GAUGAGGCCG-AAGGCCGAA ACAAGG1J 569 CUOJGGAG CUGAUGAGGCCGAAAGGCCGAA AGACAAG 572 AACCUGG CUGAUGAGGCCGAAAGGCCGAA AGUAGAC 572 ACCUGG CUGAUJGAGGCCGAAAGGCCGAA AGUAGIAC 572 AACCUGG CUGAUGAGGCCGAAAGGCCGAA AGUAGAC 579 UTGAAGAG CUGAUGAGGCCGAAAGGCCGAA ACCUGGG 58 ao tGAAGA CUGAUGAGGCCvAAGGCCGAA AACCUGG 580 UUGAAGA CUGAUGAGGCCGAAAGGCCGAA AACCUGG 582 CCUUGA CUGAUGAGG;CCGAAAC-GCCGAA AGAACCU 582 CCUUGA CUGAUGAGGCCGAAGGCCGA AGAACCU 584 UCCCUG CUGCAUGAGGCCGAAGGCCGuXA A~uGAAC 585 GUCC=U CUGAUGAGGCCGAAGGCCGAA AGAGAA 608 GAGCACG CUGAUGAGGCCGAAAGzGCCGA AGUCGGG 62.5 GGGUGAG CUGAUGCAGGCCGAAAGGCCGAA AGCACG;U GGGUGAG CGAUGAGGCCGukAGGCCGAA AGCACGIJ 618 UGUGGUt CUGAUGAGGCCGAAAGGCCGAA AGGAGCA 630 AUCGGCU CUGAUGAGGCCGuAAGGCCG-AA ACGGUGU 630 AUCGGCU CUGAUGAGGCCGAAAGGCCGAA ACGGUGTJ 638 GAUAGCA CUGAUGAGGCCGAAGGCCCG; AUCGGCU 643 UAUGAGA CUGAUGAGGCCG-AAAGCCGA AGCAAAU 645 C-G-UAUG-A CUGAUJGAGGCCGAAAGGCCGAA AUAGCA 647 CUGGUJAU CUGAUGAGGCCG--AAGGCCAA AGALMGC PC'T/I B')5O()I SC SUBSTITUTE SHEET (RULE 26) WO 95123225 255 663 GGAGGUU CtJGAUGAG7GCCGuAAG7GC CGA ACLtL'CCJ 669 CAGNAG CLGAUGAGGCCGAAAGCC,-AA AG-t=tCA 669 CAGAGAG CtJGAUGAGGCCGAAAGGCCGAA AGYGtUCG% 672, CGGCAGA CUGAUGAGGCCGAAAGGCCGAA AGGAGGU 674 GACGGCA CUGAUJGAGGCCGAAAGGCCGAA AGAGGAGk 681 G G C tJ.CtL CUGAJGAGGCCGAAAGGCCGAA ACGGCAG 681 GC-UCUU CUGAUGAGGCCGAAAGCCGAA ACGGCAG 681 GG--CtJUJ CUJGAUGAXGGCCGAAAGGCCGAA ACGGCAG 734 GGGC-UCA CUGAUGAGGCCGAAAGGCCG,-%A ACCAGGG 734 GG=CCA CUGAUGAGGCCGAAAGTGCCGAA ACCaGCG 744 CCAGGUA CUGAUGAGGCCGAAAGGCCGAA ACGGGC'J 746 UCCCAGG CUGAUGAGGCCGAAAGGCCCGAA AL'ArC-GG 759 GCtGGAA CUGAUGAGGCC A,%GCCGAA A=CCC 759 GCLGGAA CGAUGAGGCCL-AA. CCGAA AC=-C' 761 CAGC-UGG CtJGAUGAGGCCGAAAGGCCGAA AGAC-XC 762 CCAGC-UG CUGAtJGAGGCCGAAAC-GCCGAA AAGAZC 786 CAGCGCU CUGAUGAGGCCGAAAGGCCGAA, A=GTJjGU 798 GCAGAUU CUGAUGAGGCCGAAGGCCCAA ACCUCAfl 802 UUG3GGCA CUGAUGAGGCCGAAAGGCCGAA AUMGACC- 81 GUCUAAG CUGAUGAGGCCGAAAGGCCGAA ACLUGG 816 CAAAGUC CtIGAUGAGGCCGAAAGGCCCAA6 AAGUAC~J 821 CUCCGCA CtJGADGACGCCGAAAGGCCGA G 822 ACt3CCGC CtJGAUGAGGCCGAAAWGGCCGAA AACUCt:A 830 CUGCCCG CUGAUGAGGCCGAAAGGCCGAA ACUC=G 840 CAAAGUA CtUGAGGCCGAAAGGCCGAA ACCMGCC 842 UCCAAAG CUGAUGAGGCCGAAAGGCCGAA AGACMt; 842 UJCCAAAG CUGAUGAGGCCGAAAG7GCCGAA AGACCrX- 842 tCCAAAG CUGAUGAGGCCGAAALGGCCGAA AGAC,-,G 845 GACUCCA CUJGAUGAtGGCCGAAAGGCCGAA AGUtAGAC 846 UGACI3CC CUGAUIGAGGCCGAAAGGCCGAA AAGt7AGA 852 GAGCA6AU CUGAUGAGGCCGAAAGGCCGAA ACTJCCAA 855 ACAGAGC CtJGAUGAGGCCGAAAGGCCGAA AUGACC 887 GGGUAGA CtJGAUGAGGCCGAAAGGCCGAA AAUGGAU 891 GGC-UGGG CUGAUGaAGGCCGAAAGGCCGAA AGAGA,-tJ 905 GGGGUCA CUGAUGAGGCCGAAAGGCCG-AA AGUGGGG 905 GGGGtJCA CtJGAUGAGGCCGAAAGGCCGaA AGUGGGG 905 GGGGUCA CUJGAUGAGGCCGAAAGGCCGAA AGtJGGG 914 CAGAGtJA CUGAUGAGGCCGAAAGGCCGAA AGGGtC 915 IC.AGAGU% Ct7GAUGAGGCCGAAAGGCCGAA AAGGGGU 919 GGGGUCA CUGAUGAGGCCGAAAGGCCGAA AGIAAAG 928 a GACAAUA CUGAUGAGGCCGAAAGCCGAA AGGGGtCC 928 GACAAUA CUGAUGAGGCCGAAAGGCCGAA AGGG~t~c 932 AGtIAGAC CtJGAUGAGGCCGAAAGGCCGAA AtJAAAGG 940 CtJCUGAG CTUGAUGAGGCCGAAAGGCCGA.A AGUAGAC 943 GGGCUUtJ CUGAtJGAGGCCGAAAGGCCGAA AGGAGtJA 972 CCUUIJCU CUGAUGAGGCCGAAAGGCCGAA AGUULAGSA 972 CCUUUCU CUGAUGAGGCCGAAAGGCCGAA AGUUAZA 973 CCCUUUC CUGAUGAGrCCGAAAGGCCGAA AAGUL7JAG 984 GAGCCAUJ CUGAUGAGGCCGAAAGGCCCAA AUCCCCU PCT'1IB95O()156 SUBSTITUTE SHEET (ROLE 26) WO 95123225 256 984 GAGCCAU CTJGAUGAGGCCGAAAGGCCGAA AUCCCCtJ 985 L'GAGCCA CUGAtMAGGCCGAAAGGCCGAA AALCCCC 997 AGAGUt7G CUGAUGAGGCCGAAAGGCCGAA ACVCUGA 1.010 AAGCucu CUGAtJGAGGCCGAAAGGCCGAA AGCACAG 1017 UUrtGt7GA CUGAUGAGGCCGAAAGGCCGAA AC-UCtIG 102.8 =jtUGGG CUGAUGA=GCGAAAGGCCGAA AAGCJCrJ 1019 AGVU2TJU CUIGAUGAGGCCGAAAGGCCGAA AAAGC-UC 1073 tIGCAUGA CUGAUGAGGCCGAAAGGCCGAA AG=CCCA 1096 CCCAtUtJ CUGAtuGGCCGAAAGGCC-GAA AGUCCTjTJ 1106 ALUCCGGA CUGAt;GAGGCCGAAAGGCCGAA ACCCCANU 1107 AUtUCGG CtJGAUGAGGCCGAAAGGCCGAA

AAGCCCA

U208 C-AAUrJCG CUGALMGGGCCGAAAGGCCAA

AAAGC:CC

CtCCAGU CtJGAUGAGGCGAAAG!CCU AAU l-- 1133 A.GGAATJG CUGAUGAGGCCGA AGGCCGAA ACAUUCG 1.164 GCAACCt CUGAuGAGGCCGAAA=GCCuAA ACCACJC 11280 UJCAUTUCU CUGAUGAGGCCGAAAGGCCGAA AGAC-%Gh 1203 A=GCUt CUGAUGAGGCCGAAAGGCCGAA AGAUJCUU 1.210 AGGtJAGG CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 121i AAGGUAG CEJGAUGAGGCCGAAAGGC-.GAA AAGGCCU 1214 CUGAAGG CUGAL1GAGGCCGAAAGGCCGAA AGGAAGG 1.218 AGGUCUG CUGAUGAGGCCGAAAGGCCGAA AGGtJAGG 1218 AGGUCtJG CUCAUGAGGCCGAAAGGCCGAA AGGUAGG 2.218 AGGUCUG CUGAUGAGGCCGAAAGGCCGAA AG%-tJAGG 1218 AGGUCCG CTJGAUGAGGCCGAAAGGCCGAA AGGtJAGG 2.219 AAGGUCU CUGAUGAGGCCGAAAGGCCGAA AAflGAG 2.219 AAZGGUCtJ CUJGAUGAGGCCGAAAGGCCGAA AAGGUAGM 1226 GUCUGGA CUGAUG-AGGCCGAAAGGCCGAA AGGUCUG 2.226 GUCUGGCA CUGAUGAGGCCGAAAGGCCGAA AGGU;CUG 1227 AGUCUGG Ct7GAUGAGGCCGAAAGG;CCGAA AAG~CJ 2.227 AGUCUGG CUGAUJGAGGCCGAAAGGCCGAA AACAGUCtJ 2.228 GAGUCUG CUGAUGAGGCCGAAAGGCCGAA AAAGGUC 2.238 CCUCAGG CUGAUGAGGCCGAAAGGCCGAA AAGAGUC 1262 =t~GAG CUAUGAGCUAGGAA AAGGCUjG 1283 AUAAAUA CJGAtrAGGCCGAAAGGCCGAA AGGGGGG 1283 AUAAAUrA CUGAU;GAGGCCGAAAGGCCGAA AGGGGG 1285 ATJAUAAA CUGAUGAGGCCGAAAGGCCGAA AGAGGGG 1287 AAAUAUA CUGAUGAGGCCGAAAGGCCGAA ArJGAGG 2.287 AAAUAUJA CUGAUGAGGCCGP 1 AAGGCCGAA AUAGAGG 2.288 CAAAUAU CUGAUGAGGCCGAAAGGCCGAA AAUAGAG 1.289 GCAAAUA CUGAUGAGGCCGAAAGGCCGAA AAAUAGA 2.293 AAG;UGCA CUGAUGAGCGCCGAAAGGCCGAA AUAUAAA 1293 AAGUGCA CUGAUGAGGCCGAAAGGCCGAA AUAUAAA 1294 UAAGUGC CUGAUGAGGCCGAAAGGCCGAA AAtJAUAA 2.300 AAAUAAU CUGAtJGAGGCCGAAGGCCGAA AGUGCAA 2.303 AAUAAAU CUGAUGAGGCCGAAAGGCCGAA AUAAGUG 2.304 UAAUAAA CUGAUGAGGCCGAAAGGCCGAA AAUAACU 1306 AAUTAAUA CUGAUGAGGCCGAAAGGCCGAA AUAAUAA 1307 AAX3AAU CUJGAUGAGGCCGAAAGGCCGAA PAUAAtJA 1307 AAAUAAU CUTG=UGAGGCCGuAAGGCCGAA -A AUAAUA 11C11111 IMAM 156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 257 i308 T,"AAAUAA CLUGALGAG:C CAAAGGC- AA AAtJAAU CUGAUGA%=CCGCuAA.GGC C A A AUAAAUA 1:310 AAAA CUGAUGAGG CrGAAACGGC CGAA AUAAAUA 1310 AAUAAA CUGAUGACGCCGAA=CCCAA AUAAAU 1311. AAAUAAA CUGAtUGAGGCCGAAAGCG. AA17A 2.311AAAUAAA CUGAUGAtGGCCGAAAGGC~uAA AAUAAAU 1311 AAUA CUG AUGAGGC GAA AGGfCCGA A AUAAAU .313 kAAMUA C~jGALGAGGCGAAAGGCCGA AUAAIJAA 12313 .AUAAMUA CUGAUGAGGCCGAAAGGCCGAA AUAA 1314 AAUAAAUJ CtTGAUGAGGCCGAAAGGCCGAA k.AUAAUA 1314 AAUAAAU CUGAT3GA-GCCG AC-C;CCM AA X.L'A 1315 UAtAhAA CL'GAM3AGGCCGAAGGCCAA AAAUAAU i13:7 AAUA.AUA CUGAUGAGGCC-GCGCC-GAA ALM'WUA 131.8 AAtJAAU CUGAUGAGGCCGAAGGCCGk AAUAU 1319 t3AAAA CtUGAtUGAGGCCCAAAGGClCGA.A AAAUA 1325 AAUAAA CUGAUGAGGCCGAAAGGCCGAA AAAUAAU 1328 GCAAAUA CUGAUGAGCCCGAAAGG;CCGAA AUAAAUA 1329 ACAU CUGAL'GAGGCGkAAGGCCM AAAAAU 1330 AAGCAAA CUGAUGAGGCCGAGGCCZGA. AAAtUAA 1332 AUJAAGCA CUGAUGAGGCCC-AAAAGGCCGAA AUAUA 1333 CAtUhAGC CUGAUGAGGCCGAAGGCGAA AAUAAAU 1337 CALUCAU CUGAUGAGGCC C-A -GCGAA ALGCAAAU 1338 ACAUUCA CUGACAGGCCGAAGCCCA. AAGCAAA 1346 AAAtUAA CUGAUCAGCCGA.GGCCGA ACAkUUCA.

1348 CCAA.UA CUGAUGAGGCCGAAGGCCGA AUACATU 1349 UCCU CUJGALGAGiGCCGAA GCC-GA AAUACAU 1350 UEJCCAAA CUGAUGAGGCCGAAAGGCCGA.A AA.AUACA 1352 CCUUCCA CUGAUGAGGCCGuAAGGCAA AUWAAAT 1352 CCtJUCCA CUGAUGAGGCGAAGGCCGA AtJAAUA 1.353 GCCUUCC CUGAUGAGGCCGAAAGCCG-AA PAUAAAU 1369 CCJCCAG CUGAUIGAGGCcGAAAWGGccG-AA AcAcccc 1398 CUGUCUG CUGAUGAGGCCGAAAGGCCGAA AGACAGC 1398 CUCt7CUG Ct3GAUGAGGCCGAAAGGCCGAA AGACAGO 1412 CACAGA CTUGAUGAGGCCGAAAGGCC-GA ACAUGUC 1413 UCACAGA CUGAt7GAGGCCGAAAGGCCGAA AACAUGU 1414 UUJCACAG CUGAUJGAGGCCGAAAGGCCGAA AAACAUG 141.5 LTUUCACA CTJGAUGAGGCCGuAAGGCCGA AAAACAU 1415 tUUUCACA CUGAUGAGGCCGAGGCCGAA AAACAU 1438 AGGtJGGG CUGAUGAGGCCGAAAGGCCGAA ACAG%-UC 1451 AGGUAGA CUGAUGAGGCCG AC-GCCGAA AGGCCAG l- CAAGGUA CUAGGCGAcGcA AGAGGcc 1455 AACAAGG CLGAUGAGGCCGAAAGGCCGAA AGAGAGG 1462 AGGAGGC CUGAUGAGGCCGAAAGGCCGAA ACAflGGU 1470 AGCAAAA CUrGAUGAGGCCGAAAGGCCGAA AGGAGGC 1472 UAAGC7AA GUGAUGAGGCCGALtGGCCGAA AG-AGGAG.

1473 AUP.AGCA CL'GAUGAGGCCGWAAGGCCCGAA AAGAGGA 14A74 C-AUAAGC CUGAUGAcGCCGLAGGCcG.. AAAiGAG 'A73 8 AAACAU CUGAUGAGGCCGAAA-GCCGAA AGCAAAA PC('7I 9510()156 SUBSTITUTE SHEET (RULE 26) WVO 95/23225 258 1479 tUAAACA CtUGAGGCCGAAAGGCCGAA AAGCAAA i479 UtAAACA C'JG AtrAGGC CGA AAGGC CGAA AAGCAAA 1484 ttUJU U CUGAUGAGGCCGAAAGG3CCGAA AACALUAA 1498 GUUAGAU CUGAUGAGGCCGAAAGGCCGAiA AAUAUUEJ 1511 UIAGAC C3GA7LriAGCCGAAGGCCGAA AUJUGGGU "1314 L-UU~ CUGALGAGGCCGAAAGGCCGAA ACAAUUG 1516 CGUUAUU CUGAT;GAGGCCGAAAGGCCGAA AGACAAU 1529 GUCACC.A C'GAUGAGGCCGuAAGGCCGAA AUCAGCG 1529 GLCACCA CGATJGAGCCGQAAAGGCCGAA AUCAkGCG 1530 GGUCACC CUGAUGAGGCCGAAAGGCC-'AA AAUCAGC 1530 GGUCACC CUGAUGAGGCCGAAAGGCCGAtN AAL;CAGC 1563 GGGAGCA CUGAUGAGGCCGAAAGGCCGA AGGUUCA 1563 CTSGAC C'GAUGAGGCCGAAAGGCCGAA AGTUUCA 1568 CCGE2GGG CUGAL'GAGGCCCGAAAGCrC-lGAA AGCA-GAG i589 GGGC2AAU CUGAUGAGGCCGAAAGGCCGAA ACAGUCA 1592 GUAGGGC CUGAUGAGGCCGAGGCCGAA AUUACAG 1617 CGAkUCUU CUGAUGAGGCCGAAAGGCCGAA AUUtJCUC 1623 U=AGC CUGAUGAGGCCGAAAGGCCGAA AUCEJUA 1633 GGMt~7tJ CUGAUGAGGCCGAAAGGCCGAA ALUUUAA 11('1711195/00156 SUBSTITUTE SHEET (RULE 26) TIable 27: IltiniianT'INF-ct Hairpi n Ribozyinc Sequences Cl, 0i cfc

-Z

rrl rri

I-

46 54 185 201 230 234 254 296 317 387 404 453 518 554 565 576 687 704 726 730 824 1042 1168 1178 1202 1220 1284 1340 1390 Hlairpin Ribozyme Sequence AGCCGUGG AGAA GUAUGU ACCAGAGAAzACACACGUUGIGGUACAUUACCUM3UA GAGCGUGG AGAA GUGGGU ACCAGAGAAAC-ACACGUUGUCGUACAUUACCUCGGUA GGACAAGA AGAA GAGGAA ACCAGAGAAACACACGUUGrJGGUACAUUACCU)GGUA CUJGCCACG AGAA GGAAGG ACCAGAGAAACACACGUUGrJGGUACAUUACCU3GGUA GUGCAGCA AGAA GAAGAG ACCAGAGAAACACACGUUIGUGGUACAUUACCUJGGUA CAAAGUGC AGAA GGCAGA ACCAGAGAAACACACGUUIGUGGUACAUUACCUGGUA CCUCUCG AGAA GAUCAC ACCAGAGAAACACACGUUCULGGUACAUUACCUIGGUA GGCCAGAG AGAA GAUUAG ACCAGAGAAACACACGUU3GUGGUACAUUACCUGGUA AGAAGAUG AGAA GACUGC ACCACAGAAACACACGUUGUGGUACAUUACCUCGGUA GCCACUGG AGAA GCCCCU ACCACGAAACACACGLJGUGGLJACAUUACCUGGUA AUUGGCCC AGAA GUUCAG ACCAGAGAAACACACGUU)GUGGUACAUUACCUCGGUA GCACCACC AGAA GGUJUAU ACCAGAGAAAC-ACACGIJUGUGGUACAUUACCGGJA GUGAGG AGAA GCCUUG ACCAGAGAAACACACGUUJGUGGUACAUUACCUGGUA GCGAUGC AGAA GAUGGtJ ACCAGAGAAACACACGUUGUGLIACAUUACCUJGGUA UGGUAGGZA ACAA GCGAUG ACCADGAGAAACACACGUUGUGGUACAUUA CCUCGGUA UGACCUUG ACAA C-GUAGG ACCAGAGAAACACACGUUGUGCUACAUUACCUGGUA CCUUCUCC AGAA CGAAGA ACCAGAGAAACACACGUUGUGGUACAUUACCCGJA ACCUGA AG-AA GUCACC ACCAGAGAAACAkCACGUUCGUGGUACAUUACCU)GGUA GAUJACUCG AGAA GADUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGEJA UCGAGAUA AGAA GGCCGA ACCAGAkGAAACACACGUUGUGGUACAUUACCU3 GUA GGGAUUGG AGAA GGGGAG ACCAGAGAAACACACGUUJGUGGUACAUUACC)GGEJA GGGAUCAA AGAA GUAGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGGAAAC AGAA GGACAG ACCAGAGAAACACACGUUGUGGUACAUUACCU)GGJAL UCAAGGAA ACMA GGAAAC ACCACGAAACACACGUUGUGGUACAUUACCUIGGtJA AUGOGGAG AGAA GCUC ACCAGAGAAACACACGUUGUGCEJACAUUACCUCGUA AUAGAGGC AGAA GGCUCC ACCAGAGAAACACACCGUUGUCGGJACAUUACCUGGUA AUACAUUC AGAA GUAAAU ACCAGAGAAACACACGUVGUGC[JACAUUACCUCCGUA UGACCCAA AGAA GCUCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUIGGEJA UACAUGGG AGAA GCCUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUJGGUA Substrate ACAUACU CAC CCACCGCU ACCCACG GCU CCACCCUC UUCCUCA CCC UCUUCUCC CCUUJCCU GAU CGLJGGCAG C(JCLUCU CCC UGCUGCAC UCUGCCU GCU GCACUUUG GUGAUCG CCC CCCAGAGG CUAAUCA CCC CUCUGGCC GCAGUCA GAU CAUCUUCU AGGGGCA CCU CCAGUGGC CUGAACC GCC GGGCCAAU AUAACCA CCU GGCUGGUGC C.AAGGCU GCC CCLJCCACC ACCAUJCA GCC GCAUCGCC CAUCGCC GUC UCCUACCA CCUACCA GAC CAAGGUCA UCUUCCA GCU GGAGAAGG GGUGACC GAC UCAGCGCU UCAAUCG CCC CGACUAUC UCGGCCC GAC UAUCUCGA CUCCCCU GCC CCAAUCCC GCCUACA GCU UUGAUCCC CUCUCCA GAU GUUUCCAG GUUUCCA CAC UUCCUU)GA GACCCCA CCC CUCCCCAU GCAGCCA GCU CCCUCLJUAU AUEJUJACA GAUl GAAUCUAU AGGAGCU GCC UUGCCUCA AUAGGCU GUIJ CCCAIUGUA

LA

%DJ

1452 ACAACUUA AGAA G-AUAAU ACCAGAGAAACACACGUUGUGGJA~UUACCUGGUA AIJUAUCLJ GAU UAAGUUGU 1475 GUCACCAA, AGAA CCAUUJG ACAAAAAAGUUGAAUACCUGGIJA CAAUGCU GAU UUGGUGAC0 1513 CCCUGGGG AGAA GAGGCC ACAAAAAAGUUGAAUACCUGGUA GGCCUCU GCU CCCCAGGG 1541 GAAUAGUA AGAA GAUUAC ACCGrGAAACACACGUUGUGGUACAUUACCLJGGUA GUAAUCG GCC UACUAUUC r-tN m r -4 m 0 WO 95/23225 WO 95/23225 r/ii1195/0(1156 261 II ii4 4 'l ii4 ii1 o4uI10

'IN

U

Ii ~ii

DIII

liii' ~1 (N N m r- '-i N C-4 U.I 0 r 0o Ui o0N r- H1NFN rnm im mm 4 ul N wo G G G %o 80 C'o M 'C SUBSTITUTE SHEET (RULE 26) WO 95/23225 262 0ill Uill ii n nC I r K11r1/11195/00156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 263 Table 29: Human bcr/czbl HR Target Sequence PC'T/11395/OO 156 ID No.

vlc:-Q~ HH axget Sequence M-CAL PIM AM-AAGAAC AAG-AA=C M-~C AG&('-CTh LIM=- G UC AAAAICWOU tMAAk=OC CVJE CA.C-C.=ZJ CAAAA=CC LM. AGG=A-J SUBSTITUTE SHEET (RULE 26) WO 95/23225 264 Table 30: Human bcr-abl EH Ribozyme Sequences IC'T/1 1395/00156 S equencea 11) No.

HH Ribozyme Sequence GGCUiUCCIt CJGAUGAGGCCGAAAGGCCGAA AUUGAUGGUCA ACUGGCCGCUG CQGAUGAC-GCCGAAAGGCC::AA AGGGC=UC~ UACUGGCCGCrU CUGAUGACCGAAA GC&? MGGGCUUj GAAGGGCUUUU7 CtGAUGAGGCCGAAAGGCCGAA AACUCUGCUUA ACUGGCCGCTJG CtUGAUGAGGCCG"AAAGGCCGAA AGGGCEIUUUGA UACUGGCCGCU CUC-AUGAGGCCGAAkAGGCCCGAA AAGGCUUUUG SUBSTITUTE SHEET (RULE 26) PC'T/11195/OO 156 WVO 95123225 265 Table 31: RSV (iB) ERH Target Sequence nt.

Position H Tax-get Sequence GGCAAAUJ A AAUCAAU AAtUhAAU C AAUJCAG AAUCAAU U CAGCCAA AUCAADU C AGCCAAC C-%AUGAU A AUACACC UGAUAAU A CACCACAL UGAUGAU C ACAGACA AGACCGU U GUCACUU CCGUUGU C ACUUGAG UGUCACU U GAGACCA AGACCAkU A AUAACAU CCAUAAU A ACAUCAC AUAACATJ C ACUAACC CAUJCACU A ACCAGAG GAGACAU C AUAACAC ACAUCAU A ACACACA CACAAAU U t7AUAUAC ACAAAUJU U AOAACU CAAAUUUJ A UAtUhCtUt AAUUUU A tJACUUGA UUUMtMU A CtJUGAUA AUAUACU U GAUAAAU ACUUGAU A AAUCAUG GAU.AAU C AUGAAUG AAUGCAU A GUGAGAA GAAAACU U GAUGAAA GCCACAU U UACAUUC CCACAUU U ACAUUCC CACAUUU A CAUUCCU UUtJACAU U CCUGGUC UUACAUU C CUGGUCA UCCUGCtJ C AACItAUG GUCAACTJ A UGAAAUG UGAA.AZU A UUACACA AAACUAtJ U ACACAAA AACUJAUU A CACAAAG ACAAAGTJ A GGAAGCA nt.

Position 276 283 295 303 304 305 309 317 319 320 323 327 337 338 340 341 350 356 357 363 372 375 380 383 385 391 396 398 402 406 410 411 412 421 423 424 432 434 446 448 454 HE Taxgot Sequence AAAAUAU A CUG-AAUA ACUGAAU A CAACACA ACAAAAU A UGrGCACU UGCACU U UCCU=AU GGC\CjJ U CCMUG GCA-CUUU C C=t;AUGC UUMCCCU A UGCCkAU LUGC-CAU A UC-AUCA CCAAUAU U CAkUCAAU CAAtUt3U C AUCAAUC UAUUCAU C AAUCAUG CAUCAtI C AUGAUGG GkUGGG~U U CUUAGA AUGGGMJ C UUAGAAU G-GUtC U AGAAUC GCJU A GAAUC-CA AAUGCAkU U GGCAULA UUGGCAU U AAGCZ7o.

UGCCUU A AGCCUAC L7AAGCCU A CAAAC-CA% AAAGCAU A CUCCCAU GCALACU C CCAUA.AU C'UCCrAU A AUAUACA CCAUAAU A UACAAGU AUAAtJAU A CAAGUAU UACAAGU A UGAUCUC GTO-AUGAU C UCAAUCC- AUGAL'CU C AAUCCAU UCUCAAU C CAUAAAU AAUCCAU A AAUUCrA C-kLYAAAU U UCA.ACA~c AUAAAUU U CAACACA UAA-A=U C AACACAA ACAkCAAU A tJUCACAkC ACAAUAU U CACACAA CA.AUAU-U C ACACAAU ACACAAU C tJAAAACA ACAAEJCU A AAACAC AACAACU C UAtJGCAU CAACtJCU A UGCAUAA,.

tIAUC-CAU A ACUAUAC

AAGCACU

ACUAAAU

UAAAUAU

AAAAAAU

A AAUAUAA A UAAAAAA A AAAAAUA A UACUGAA SUBSTITUTE SHEET (RULE 26) WO 95/23W WO 5/2225PcTl/I1395/00 266 458 460 463 467 470 489 490 492 495 CAtUhACU A UAACtU.U A CUAUACU C ACUCCAU A CCAUAGU C UGAAAAtJ U GAAAAUU A AAAut3AU A UUAUL.G A

UACUCCA

CUCCAUA

CAUAGUC

GUCCAGA

CAGAUGG

AA.GtAA

U'AGUAAU

GUAAUUU

ALVEAA

SUBSITUTE SHEET (RUILE 26) PCT/I1105100156 WO 95/23225 267 Table 32: RSV (1B) HH Ribozynie Sequence nt KH Ribozyme Sequence Position AUUGAtJU CtTGAUGAGGCCGAAAGGCCGAA AUU*UGCC 14 CLTGAAUU CUGAUGAGGCCGAAAGGCCGAA At3UUAUU i8 UUGG=tG CUGAUGAGGCCGAAAGGCCCAA AtUGAUU 19 GUUCG%-CU CtJGAUGAGGCCGJAAGGCCG?.A AAUUGAU 54 GGtIGtJAU CUGAtJ'GAGGCCGAAAGGCCGAA kLCAUUG 37 t.GUGGUG CUGAMGGCCGAAAGGCCruAA AUUAUCA 77 tL=CJGU CUGALtGAGGCCGAAAGGCCGAA AUCAtJCA 94 AAG!JGAC CUGAtJGAGGCCGAAAGGCCGAA ACGGUCU 97 CUCAAGU CUJGAUG=ACGCCGAAAGGCCGAA AcAACGG 101 UGMUC CUG UAGGCCGAAAGGCCGAA AGLGACA 110 AUGUUJAU CUJGAUGAGGCCGAAAGGC~rAA AUGGUCU 113 GUGA6TGU CUGAUGAGGCCGAAAGGCG AUUAUGG 1U8 GGUUAGU CUrAUGGGCCGAAGGCCGA6 AUGUt3AU 122 CUCUGGU CtJGAUGAGGCCGAAGGCGAA AGUGAUG 134 GUGJAU CUGAUGAGGCCGA.'AGGCCG2 AUGUCLC 137 UGUJGUGU CUGAUGAGGCCGAAAGGCCGA AUGAUGU 148 GUJAUAUA CUGAUGAGGCCGAAAGGCCGAA AtJUUGUG 149 AGUAUAU CUGAUGAGGCCGAAAGGCCGAA AAUUUTGU 150 AAGUAIJA CU7GAUGAGGCCGAGGCCGAA ?AAAUULG 152 UCAAGUA CUGAUGAGGCCGAGGCCCGAA AUAAAUrJ 2154 tUUCAAG CUGAUGAGCCGAGGCCGAA AW.UAA 157 AUUUAUC CUGAUGAGGCCGAAAGGCCGAA AGUATUAU 161 CAUGAUU CUGAUGAG-GCCGAAAGG-CCGAA AUCAAGU 165 C =JUCAU CUGAUGAGGCCGAAAGGCCGAA AUUUAUC 176 UUCUCAC CUGAUGAGGCCGAAAGGCGA AUGCAUU 188 UtJUCAUC CUGAUJGAGGCCGAAAGGCCGAA AGUUJtc 208 GAATUGUA CtJGAUGAGGCCGAAGGCGAA AUGUGGC 209 GGAAtJGU CUTGAUTGAGGCCGAAAGGCCGAA AAUGUGG 210 AGGAAUG CUGAUGAGGCCGAAAGGCCGAA AAAUGtJG 21.4 GACCAGG CUJGAUGAGGCCGAAAGGCCGAA AUGtJAAA 215 UGACCAG CtJGAUGAGGCCGAAAGGCCGAA AAUGUAA 221 CAUJAGUU CUGAUGAGGCCGAAAGGCCGAA ACCAGGA 226 CAUUUCA CUGAUGAGGCCGAAAGGCCGAA AGUJUGAC 239 UGUGUAA CUGAUGAGGCCGAAAGGCCGAA- AGUUCA 241 UUUGUGU CUGAUGAGGCCGAAAGGCCGAA AUA=UUU 242 CUUUGUG CUGAUGLAGGCCGAAAGGCCGAA AAUAGUU 251 UGCUJUCC CUGAUGAGGCCGAAAGGCCGAA ACUUUGU 261 UUAUAUU CUGAUGAGGCCGAAAGGCCGAA AGUGCUU 265 UUUUUUA CUGAUJGAGGCCGAAAGGCCGAA AUUUAGU 267 UAUUUUU CUGAUGAGGCCGAAAGGCCG-AA AUAUUUA 274 UUJCAGUA CUGAUGAGGCCGAAAGGCCGAA AUUUUUUJ 276 UAUUJCAG CUGAUGAGGCCGAAAGGCCGAA AUAUrUUTJ SUBSTITUTE SHEET (RULE 26) WO 95/23225 268 283 tTGUGUE7G CtJGALtXAGGCCGAAAGGCCGAA AUUCAGU 295 A~T%.GCCA CUC=GAflCCGAAAGC-CCGAA AUUrJUGU 303 AUTAGGGA CrJGAtLAGGCCGAAAGGCCGAA AGEUG=C 304 J7kUAGG CUGAUGAGGCCGAAAGGCCGAA AAGUJGCC 305 GCAtUhGG CUJGAtGAGGCCGAAAGGCCGAA AAAGJIGC 309 AUUGGCA CLGAUGGCCGAAAGCG3AA AGGGAAA 317 UiGAUGAA Ct3GAUIGAGGCCGAAAGG;CCGAA AUE2GGCA 319 AuUiGAUG CUGAUGAGGCCGAAAWGCCCAA AU=U 320 GAtJUGAt7 CUGAtJGAGGCCGAAAGGCCGAA AAtULWG 323 CAUGAtJU CrJGAt3GAGGCCGAAAGGCCGAA ALGAAUA 327 CC-t7CAtJ CtGAMA=GCGAAAGGCCGAA, A0TUGAUG 337 iucUAAG CUGATGAGGCCGAAAGCCGAA ACCCAC 338 AtUMAA CUCAT~AGGCCGA AGCCGAA AACCCAU 340 GCAUUCU CUG-AtJGGCCGAAAGGCCG-AA AI3AACCC 34. 1UGCAtj-UC CUGAtvW3GCCGAAAGGCCGAA AAGAACC 350 UAAUJGCC CUGAUGAGGCCGAAAGGCCGAA AUGCAUU 356 UA.GCrt CUG UGAGCGAAAGGCCCGAA AUGCCAA 357 GtL.GCU CUGAUGAGGCGAAGGCCCAA AAtflCCA 363 LTGCUG CTUGAUGiAGGCCGAAAGGCCGAA AGGCUUA 372 AL'GGGAG CUGANUGAGGCCGAAAGGCCGAA, ADGCl=J 375 AUUAUGG CUGAL*GAGCGAAAGGCCGAA AGOA.UGC 380 LGrJAUAU CUGATJGAGCCGAAAGGCCGALA AUGGGAG 383 ACUUGUA CU'GAt~aGGCCGAAAGGCCGAA AULDXUG 385 ATJACUUJG CUGAUGAGGCCGAAAGGCCGAA A~IJEMU 391 GAGAUCA CUGAtrG-AGGCGAAAGGCCGAA ACUUGUA 396 GG-AUUGA CUGAtrzAGGCCGAAGGCGA AUCAJUAC 398 ALGGAUJU CLGAt;GAGGCCGAAAGGCCGA AGAU 402 AUUUAUG CUGAUG.GGCCGAAAGGCCGAA AMt-GA 406 !JGAAAUtJ CUGAUGAGiGCCGAAAGGCCGAA ATJGGAtU 410 GuGUtJGA CUGAUGAGGCCGAAAGGCCGAA AUUUAL'G 411 UGUWG CUGAUGAGGCCCGAAAGGCCGAA AAflUUAU 412 tJUGUGUU CUJGAUGAGGCCGAAAGGC-CGAA AAA=UU 421 GUGUGAA CUGAUGAGGCCGAAAGGCCGAA AUU~tGGU 423 UtMtGtG CUGAUGAGGC-CGAAAGGCCGAA ADJAEUGU 424 AtJUGUGU CDGAUGAGGCCGAAAGGCCGAA AAUUG 432 UGtJE3UA CUGAUGAGGCCGAGGCCGAA AULIGUGU 434 GEJUGUUU CtJGAtruIGGCCGAAAGGCCGAA AGAUtTGU 446 AUCMUA CUGAUGAGGCCGAAAGGCCGAA AGtJUGUtJ 448 UUAUGCA Ct7GAUGAGGCCGAAAGGCCGAA AGAGtJG 454 GtMhUAGU CUGAU)GAGGCCGAAAGGCCGAA AtJGCAUA 458 UGGAGUA CUGAtJGAGGCCCAAAGGCCGAA AGUUAUG 460 UAUJGGAG CtJGAUGAGGCCGAAAGGCCGAA AUAGUUA 463 GACUAUG CUGAUGAGGCCGAAAGGCCGAA AGUAUAG 467 TJCUGGAC CUGAUGAGGCCGAAAGGCCGAA AUGGA t 470 CCAUCUG CUGAUGaAGGC-CGAAAGGCCGAA ACUAUGG 489 UTJACUAUJ CUGATTGAGGCCGAAAGGCCGAA AUUUUCA 490 AUUACUA CUGATUGAGGCCGAAAGGCCGAA AA~Tj-UUC 492 AAT.C CUGAUGAGGCCGAGGCCGAA AUAAUUU 495 UTJUAAAtJ CUG-AUGAGGCCG-AAAGGCCG-AA ACUAtJA PC'T/I 95/01)I56 SUBSTITUTE SHEET (RULE 26) WO 95123225 269 Table 33 RSV (IC) EM target Sequence 1IT/B95/0()156 nt.

Ponition i6 17 21 31.

32 36 37 38.

42 46 s0 51 67 68 71 76 81 87 88 92 93 100 101 104 105 120 125 128 129 135 143 145 151 155 156 i59 163 164 Target sequence GGCAAAT A AGAAUj7t3 UAAGAAU U UGAUAAG AAGAALTU U GAUAAGJ AUUUGAU A AGUrACCA% GAUAAGt7 A CCACUTUA UACCACU U AAAUUUA ACCACUU A AAUUUAA CtUUAAAU U UAACUCC UTUAAAUU U AACUCCC UAAAUU A ACUCCCU UUUAACUJ C CCUUGGUj ACUCCCtJ U GG-UUJAGA CCJGGU U AGAGAUG CGGUU A GAGAUGG CAGCAAU U CAUUGAG AGCAAUJU C AMUGAGU AAUUCAU U GAGUAUG AUjUG.AGU A UGAUIAAA GJAUGAU A AAAGUUA UAAAAGU U AGAUUAC AAAAGUU A GAUUACA GUUAGAU U ACAAAAt7 UUJAGAUUJ A CAAAAUtJ ACAAAAU U UGUUUGA CAAAAtJU U GUUTUGAC AAUUUGU U UGACAAUJ AUUUGUU U GACAAUG AUGAAGU A GCAUUGU GUAGCAU U GUUAAAA GCAUUGUJ U AAAAAUJA CAUJUGUJU A AAAAUAA UAAAAAU A ACAUGCU ACAUGCU A UACUGAU AUGCUAU A CUGAUAA UACUGAU A AAUUAAU GAUAAAU U AAUACAU AUTAAAUU A AUACAUU AAUUAAU A CAUUUAA AAUACAU U UAACUAA AUACAUU U AACUAAC nt.

poqsition 176 2.89 2 2i76 2081 12 216 221 222 231 232 234 235 241 247 249 250 256 259 262 265 267 270 273 278 283 284 285 300 303 316 317 319 321 338 339 346 Target Sequence UACAtLUU A ACJAACG7 MIIAACJ A ACGCZUU UAACGCUJ U UGGCU;.

AACG-CUU U GIGCU'A.G UUUGcCJ A, AC-CCAGU CA.GUGAU A C.ALUAC.A GAUACAU A CAAUC:A AUACAAU C AAAUUGA AUCAAAU U GAAUGGC AUGGCAU U GUGUOUG AUUGUGU U UGUGCAU UUGUG"UU U Gt3C-CAL'G tUC-CALGZU U AL'UACkA GCAUGUU A LEJCAAG AUGUUAU U ACAAGUA UGUUAU'U A C-AAGUAG UACAAGUJ A GUGAUAU UAGUG-AU A UUCGCCC- GUGAUAU U UGCCCLUA UGAUALUU U C-CCCUAA UUGCC-CU A AMAA-A CCCUAAU A AUAAUAU UAAUAAU A AUAU3UGU UjAAUAAU A 0UUGUAGU AUAAUAU U GtJAGMA AUALU'U A GUAAAAUv UUIGUAGU A AAAUJCCAk GUAAAAU C CAAUuCC AUCCAAU U UC.ACAAC UCCAAUU U CACAACA2 CCAAUUU C ACAACA.A UC-CCAG*% A CUACAAA CAGUACU A CAAzAAUG UGGAGGU U AUAUAUG GGAGGUU A UAUAUGG AGGUUA=U A UAUGGGA GUUTAUAU A UGGGAAA AUGGAAU U AACAcAU UGG.AAULT A ACACAUR: AACACAU U C-CUCUCAk SUBSTITUTE SHEET (RULE 26) WO 95123225 270 350 CAOUGCU C TJCAACCt3 352 LUGrCUCU C AACCU.-A 358 UCAACCU A AUGGT-JCUJ 364 tJAAUGGU C tIACEUGA 366 AUGGUC A CEJAGAUG 369 GUCtTAat A GAtJGACAX 379 UGAV0,11 U GUGAAhU 3 87 GUGAAAU U AAATJUCU 388 UGAAIJU A AAUJTCrJC 392 AUtMhAU U CUCCAAA 393 UjUAAAIJU C UCCAAAA 395 AAAtJUCU C CAAAAAA 405 AAAAACU A AGUGAUU 412 AAGUGAU U CAACAAU 413 AGUGAtUU C AACAAU;G 427 -ACCA.AU UJ AUAUGAA 428 ACCAAUU A UJAUGAAUJ 430 CAAUtDMU A UGAAUCA 436 UATJGAAU C AAUEMhUC 440 AAUCAAU U AUCtGAA 441 AUCAAUU A UCUGAAU 443 CAAUUAU C UGAAUUA 449 UCUGAAU U ACUUGGA 450 CUGAAUJU A CUUGGAU 453 AAUUACU U GGAUUUG 458 CUUGGAU U UGAUCUU 459 Utj"GGAUU U GAUCUUA 463 AUEJUGAU C UUAAUCC 465 tJUGAUCU U AAUCCAU 466 UGAUCUUJ A AUCCAUJA 469 UCUUAAU C CAUAAAIJ 473 AAUCCAU A AAUUAUA 477 CAUAAAU U AUAAUUtA 478 AUAAAUU A UAAUUAA 480 AAAU!TJ A AUUAAUA 483 UUAUAAU U AAtJAUCA 484 UAUAAflU A AUAUCAA 487 AAUUL.AU A UCAACUA 489 UUrAAUAU C AACUAGC 494 AUCAACU A GCAAAUC 501 AGCAAAU C AAlUGUCA 507 UCAAUGU C ACUAACA 511 UGUCACU A ACACCAU 519 ACACCAU U AGUtJAAU 520 CACCAIJU A GtU7rAAtJA 523 CAUUAGU U AAUAUTAA 524 ALMA=U A AUAUAAA PICT/I B195/00156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 271 Table 34: RSV (10) HH Ribozyme Sequence nt. HE Ribozymne seque.nca AAAUUCU CUGA0GAGGCCGAAAGG.-CCAA AUtJUGCC i.6 CUUAUCAk CEUGA:GACGCCCAAAGGCCG2.A AIJUCUL-2 i.7 ACUUAtJC C7UGAtraAGGCCGAAAGGCCGA A AAM=CU 2i. UGCytACtJ CUGAtnACYGCCGAAAGGCC-,AA A.tJC.AUt tJAAGUGG CUGAUG-A(2GCCGAAACGGCCGA A Ac~TmALc 32. UAAAUtJU CUGAVGAGGCGaMCGCC-AA AGr,,-3UA 32 tU-JAAflt3 CUGAUGGC=CGAAAGGC-CGAA AGUCGU 36 GCAGTJUA CLCGAGGCCGAAAC-GCCGAA AUUtJAAG 37 GC-GAGUU CUG-AUG-AGG-CCGAAAGGCCGAA ;kAUUUAA 38 AGGGAGtJ CtJGAUzACGCCGAAAGGCCGAA AAAUUUA 42 ACCAGG CUCAtMAGGCcGAAAGrCccAA AGuUAA 46 U~CUAACC CtJGAUGAGGC-CGAAAGGCCWA kGGALGu CAtTCtCU CLGAtrAGGCCGAAAGGCCGAA ACCAAG 51. CCACUC CUGAUCGr-CCCAAAGGCCGXA AACCAAG 67 CUCAAL'G CLIGAt7GAGGCCGAAAGGCCGAA AL7;CGt,; 68 ACt7C.AU CtGAUGAC-GC-CGAAGGCCGAA AAUU-rGCJ 72. CNUACtC CtGAJGAGC-CCGAAAGGCCGAA AtGAAXU 76 UUUAUCAk Ct3GALGAGC-CCGAAAGGCcGAA A ct.rCAU 81. UAACUtJU CL'GAUGAGGCCGAAAGGCCGA A AUCAt2AC 87 GUJAAUCU C'JCALTCJGCGCCGAAAGGCCGAA ACTUMtA 88 UGUAAUC CUGAUGAGGCCGAAAGGCCaAA AActumi-U 92 AUTj"UUGU CtJ'GAUG-AGGCC-GAAAGG ACUtAAC 93 AAtUM-'G CtJGAUGAGGCCCGAAAC,-CCGAA AArJCUAA NO0 UC-AAACA CUG-AUGAC-GCCGAAAGGCCGAA AUTjtJGU 2.02 GUCAAAC CUGAtGAGGCCGAAAGGCCGAA AAUUUUG 2.04 ATjt7GULCA CUGAUGAGCrCGAAAGGCCGAA AcAAtuu CAUJUGUC CUGAUG.AGGCCGAAAGGCCGAA AA~CAAAu 2.20 ACAAUGC CUGAUGXGGCCGAAAGGCvAA ACL-UCAU 2.25 TUUUAAC CUGAUGAGGCCGAAAGGCCGAA AUG-tJAC 2.28 UAtMtUU CUGAUG=AGGCCGAAAOGCCGAA ACAAUGC 2.29 UTjUUULT CUGAUGAGG3CCGAAAGGCCGAA AACAAUG 2.35 AC-CAUGU CUGAUGACGCCGAAAGGCCGAA AUUUU[JA 2.43 AUCAGtJA CUGAUG-ACGGCCGAAAGGrCCGAA AGCAUGTJ 2.45 ULMTjCAG CUiGAUGAGGCCGAAAGGCCGAA AUAGCAU 2.52. AUMAU CUGAruGCGCCCGAAAGGCCGAA AUCAGt3A 2.55 AtJGUUU CUGAUGAGGCCGAAAGGCCGAA AUUUUC 2.56 AAUGtJAU COJGAUGAAGGCcGAAAGc-ccGAA AAuuTJAu i359 T.jThAAUG CUGAUGAGGCCG-AAAGGCCGAA AUUAATU 3 UEAGUUA CTJGAtJ'GAC-GCCGAAAGGCCG;AA AuGuAU 2.64 GLUAGTtJ CUJGAUG-AGGCCGAAAGGCCGALkA AUtGTJAU 2.65 C~TJU4-GU CUGAUGC-AGCCGAAAGGC C GAA AtVLJUA;, PCIA/1195/00156 SUBSTITUTE SHEET (RULE 26)

I

WVO 95/23225 272 169 AAAGCGU CUGAL'GAGGCCGAAAGGCCGAA AGUUAAA 175 UUAGCCA CUGAUGAGGCCGAAAGGCCGAA A~C-GUEJA 176 CTJAGCC CUJGAIJGAGGCCGAUAGGCCGAA AAGCGU 181 ACUGCCtJ CUGAUGAGGCCGAAIAGGCCGAA AG=MAA 192 UTJGU'AUG CUGAUAGCGCCG?.AAGGCCGAA AUJC.C0G i96 UUGAUUG CUGAU7GAGGCCGAAAGGCCGAA AL'GUAUC 201 UCAAUUU CUGAUGAGjGCCGAAAGCCGAA AUUGtUt7 206 GCCAt3UC CUGAUGAGGCCGAAAGGCCGAA At3UUGAU 216 CAAACAC CLUGAUGAGGCCGAAAGGCCGAA AUGCCAU 221 AUGCACA CUGAUGAGGCGAGGCCGAA ACArC;AU 222 CAUGC-AC CtUGAUGA=GCGAAAGGCCGAA A~ACA 231 UUGUAU CGAUGAC7GCCGAAGGCCGAA ACAUJGCA 232 CtJUGUA CUGAUGAGGCCGAGCrCaAA AACAUC-C 234 UACUUGU Ct7GAUGAGGCCGAAAGGCCGA)A AUAACAmU 235 CA=hJG C3GAUGAGGCCGAGGCCGA. AAUAACA 241 AUAUCAC CUGAUGAGCCGAAGGCCGAA A=UG"JA 247 GGGCA.AA CUGAUGAGGCCGMAAGGCCGAA AUACA 249 UAGGGCA CUJGAUGAGGCCGAAAGGCCGAA AtJAUCAC 250 UUA.GGGC CUAUAGC~%A CA AUAUCA 256 UtJAtUAU CUGAUGAGGCCrAAAGGCCGAA AGGGCAA 259 AUAUt CUGAUGAGGCCGAAAGGCCGAA AUTJAGGG 262 ACAAUAU CUGAUGAGGCCGAAAGGCCGAA AUUAtWtA 265 ACUJACAA CUGAUGAGGC-CGAAAGGCCGAA AT,7AUrjA 267 ULJACEJAC CUGAUGAGGCCGAAAGGCCGAA At7AUtU 270 ALTIUUAC CUGAtJGAGGCCGAAAGGCCGAA ACAAULAU 273 UGGAUtJU CUGAUGAGGCCGAAAGGCCGAA ACtU.CAA 278 GAAPAUUG CtJGAUGAGGCCGAAAGGCCGAA AUUt3UAC 283 GUUGtJGA CUGAUGAGGCCGA?.AGGCCG-AA AUUGGAU 284 UGUUGUG CUGAUGAGGCCGAAAGGCCGAA AAUUGGA 285 UUGUt7GU CUGAUGAGGCCGAAAGGCCGAA AAAUtGG 300 tUtrJGUAG CUJGAUGAGGCCGAAAGGCCGAA ACtJGGCA 303 CAUUTG CtJGAUGAGGCGAAGGCCGAA AGUACUG 316 CAUAUAt7 CtJGAUGAGGCCGAAAGGCCGAA ACCTJCcA 317 CCADAUA CtJGAUGAGGCCGAAAGGCCGAA AACCUCC 319 UCCCAUA CUJGAUGAGGCCGAAAGGCCGAA AUAACCU 321 UUJUCCCA CUGAUGAGGCCGAAGGCCGAA AUAUAAC 3 -S AUGtJGtU CtJGAUGAGGCCGAAAGGC:CGAA AUUCCAU 32 AAtJGUGU CUJGAUGAGGCCGAAAGGCCGAA AAUUCCA 346 UGAGAGC CUGAUGAGGCCGAAAGGCCGA AJGUGtU 350 AGGUUGA CUGAUGAGGCCGAAAGGCCG-AA AGCAAUG 352 U(AGGUU CUGAUGAGGCCGAAAGGCCGAA AGAGCAA 358 AGACCAU CUJGAUJGAGGCCGAAAGGCCCAA AGGtJUA 364 UCUAGUtA CUGAUGAGGCCGAA6AGGCCGAA ACCAUUtA 366 CAUCaMG CUGAUGAGGCCGAAAGGCCGAA AGACCAU 369 UGUCAUC CUGAUGAGGCCGAAAGGCCGAA AGUAGAC 379 AUUUCAC CUGAUGAGGCCGAAAGGCCGAA AUUGUCA 387 AGAAUUU CUGAUGAGGCCGAAAGGCCGAA AUUUCAC 388 GAGAAUU CtUGAUGAGGCCCGAAAGGCCCGAA AAUUtJCA 392 UUUJGGAG CUGAUGAGGCCGAAAGGCCGAA AUtJUAAU SUSIUISE 6 IPC"T'/ 1195/010 SUBSTITUTE SNEET 26) 11CIA 1195/0 0 156 WO 95/23225 273 393 UUjt7GGA CtJGAUGAGGCCGAAAGGCCGAA AAUUt3A 395 tUtUUMG CUCGAGGCCGAAAGGCCGAA .GAAiUU 405 AAUCACU Ct7GAtMAGGCCGAAAGGCCGAA AGUUUUU 412 AUUGUG CUGAUGAGGCCGAAAGGCCGA'A AUCACU 413 CAUUGUU CUGAUGA.GGCCGAAAGGCCGAA AAUCACtJ 427 tJUCAUAU CUGALGAGGCCGAAAGGCCGAA AUt3GG%-UC 428 AUtJCAtJA CUGA~r3AGCCGA.AAGGCCGAA AAtMtGG 430 tUGAUt7CA CUGAUGAGGCCGAAAGGCCGAA AtJ1AVTJG 436 GAIJAAUU CUGAUIGAGGCCGAAAGGCCGAA AtUtCAUA 440 UITCAGAU CUGALGAGGCCGAAAGGCCGAA AUUGAUU 441 AUUC.AGA CUGAUGAGGCCGAAAGGCCGAA AAUUJGAU 443 UAATJUCA CUGA1JGGGCCGAAAGGCCGAA AUAAUUG 449 UCCA.AGU CUGAUCAGGCCGAAAGGCCGAA AUUCAC-A 450 AUCCAAG CiGALGAGGCCGAAAGGCCGAA AA.UUCAG 453 CAAAUCC CU~~MC~wJ CA A~TJAA~Tj 458 AAGAUCA CU~GAt7G?.GGCCGAAAGGCCGAA AUCCAAG 459 tJAAGAUC CUGAUGAGGCCGAAAGGCCGAA AAUCA 463 GGAWtAA CUGAUGAGGCCGAAAGGCCGAPA AUCAAAU 465 AM-GAUU CUGAUGAGGCCGAAAG;GCCGAA AGAXJCAA 466 UAUGGAU CUGAU~uGGCGAAAGGCCGAA AAGAUJCA 469 AUMAUG CUG-AW3AGGC~rGAAAGGCCGAA AUUEAGA 473 tAUATJU CL'GAUGAGGCCGAAGGCCGAA AUGGAUU 477 UA.tJUAU CUGAUGAGGCCG-AAGGCCGAA AUUUJAUG 478 UUAAtYUA CUGAUGAGGCCGAAAGGCCGAA AAUtJUAU 480 UAtJUAAtJ CU=AGAGGCCGAAAGGCCGAA AtJAAUUUt 483 UGAJUU CUGAUGAGGCCGAAAGGCCGAA AUUAUAA 484 UUjGAUAU CUGAUGAGGCCGAAAGGCCGAA, AAUt7AUA 487 UAGUUGA Ct3GAUGAGGCCGAAAGGCCGAA AUTJAAUtJ 489 GCUAGt3U CUGMUGA~fGCCGALAAGGCCGAA AUAUUAA.

494 GAtUUGC CUGAUGAGGCC-GAAAGGCCGAA AGUTUGAU 501 UGACAUtJ CUGAUGAGGCCGAAAGGCCGAA ALUU 507 UGLTUAGU CtJGAUGAGCGCCGAAAGGCCGAA ACAUUGA 51i AUG=UGU CUGAUGAGGCCGAAAGGCCGAA AGUGACA 519 AUUAACU CtEGAUGAGGCCGAAAGGCCGAA AtJGGUG 520 tJAUUAAC CUGAtJGAO3GCCGAAAGGCCGAA AAUGGUG 523 UtUhI.tUU CUGAUGAGGCCG AGCCGA ACtU.AUG 524 UUUAI3AU CUGAUGAGGCCGAAAGGCCGAiA AACtJAAU SUBSTITUTE SHEET (RULE 26) WO 95/23225 274 Table 36: RSV liE Target Sequence 11C11i/11195/00, 156 at. HU Target: Sequence GCAAAU A CAAAGAU GALUGGCU C UUAGCAA UGG-CC'7 U AG-CAAAG GGCtJCUUt A GCAAAGU GAAUC AAGUUG-A GUCAAGU U GAAUGAU GAAUGAU A CACUCAX AUACACU C AACAAAG CAAAGAU C AACLTUCU AUCAACU U CUGUCATJ UCAACTUU C UGUJCAUC CUUCUG C .AUCCAGC CUGUCAUT C CAGCAAA AGCAAAU A CACCAUC ACACCAU C CAACGGA .'GfGAGAU A GUfAUUGA AGAUAGU A UUGAI7AC AUAGUAU U GAMAC UAUUGAU A CUCCUA UGAUACU C CtUhAUUA UACUCCU A AUUJAUGA UCCtrAhAJ U AUGAUGU CCUAAUU A UGAUGUG AACACAU C AAUTAACU CAUCAAU A AGJAUG AAUAAGU U AUGUGGC AUAAGUU A UGUGGCA CGGCA'GU U AUUAAUC GCAUGUU A UUAAUCA AUGAJU U AAUCACA UGUUAUUJ A AUCACAG UAUTAAU C ACAX3AAG AGAUGCU A AUCAUAA UCUAAU C AUAAAUU UAAUCAU A AAUUJCAC CAUA.AAU U CACUGGG AUAAAUU C ACT3GGGU ACUGG U AAUAGG;U CUIGGGUU A AUAGGUA GrGUUAAU A GGUAUGU AAU;AGGU A UGUUAUA at.

Positi~on 217 218 220 229 231 235 236 254 260 263 277 279 284 299 305 315 318 326 327 346 347 355 356 361 370 371 383 384 389 395 401.

406 408 415 418 431 449 453 460 472 474 HH TDarget Sequence GGUJAUGU U AUAL*GCG GUAL'U A UA'u"CA AUGUUAU A UCCAUG GC=AUGU C UAC-G"%t'A CAUGUCJ A GC- 1G G UCUw-G5-U U AGGAAGA CUJAGG'%U A C-C-AAGAG ACACCAU A AAAALAC U7LAAAAU A CUC3AGAG AAAtACU C AGAIALIG GCGCGAU A UCAUGUA GGGAUAU C AtUGUAAA AUCAUGU A AAACCAA AUGGAGU A GAOSTA UAGAUGU A ACA?.CAC AACACAU C GUCAAG-A ACAUCCU C AAGACAU AAC-AC.AU Ui AALUGGAA AGACALU A A UCGAA A AUGAAAU U UGAAGG UGAAAUUJ U C-AAG%:GU G-AAGL'GU U AACAUUG AAGUGUUr A ACAUUGG UUAACAU U GGCAG G(XAGC1J U AACAACU C-AAGCUUr A ACAACtUG CUGAAAU U CAAAUCA U;GAAAUU C AAAUCAA UT2CAAAU C AACAUCG CCAACAU U GAGAUAG UCGAGAU A GAAUCUA AUAGAAU C UAGAAAA AGAAUC'J A GAAjAAUC AGAAAAU C CUACAAA AAAUCCU A CAAAAAA AAAUGCU A AAAGAA CA GAGt= A GCUCCAG C-GUAGCU C C-AGAAUA CCA =AAU A CAGSCAU CAL'GA=L C UCCUGAU tMAGC~j C CUGALVG SUBSTITUTE SHEET (RULe 26) WO 95/23225 r'n19/05 PUMB95/00150 275 480 491 494 496 497 501 503 511 512 515 518 522 526 527 544 549 551 552 563 564 573 576 581 584 603 604 613 614 617 629 640 641 643 652 653 663 670 671 672 674 680 681 682 683 686 687 690 691 692 UCCUGAU U GUGGGAU GGAUGAUJ A AUAUtJAU UGAUAAU A UUAUTJtA AUAAUJAU U AUGUAUA tUAMAM) A LIGUAIAG AUUU A UJAGCAGC tAUG3AU A GCAG-CAU GC?.GCAU U AGUThAUA CAGCAUU A GUAAUAA CAUUAGU A AUAACUA U~wGUAAU A ACUAAAU AA!ThACU A AAUCAGC ACUAAAU U AGCAGCA CLVAAUIU A GCAGCAG GACAGAU C UGGUCUU AUCUGGU C TJUACAGC CUGGUJCU U ACAGCCG MGM=CU A CAGCCGU CCGt3GAU U AGfAG CGUGAUJU A GGAGAGC GAGAGCU A AUA~AUGU AGCUAAU A AU-GUCCU AUAAVIGt C CUAAAAA AUGUCCU A AAAAAUG GAAACGU U ACAAAGG AAACGUU A CA.AAGC AAAGGCU U ACUACCC AAGGCU A CUACCCA GCUUACU A CCCAAGG AGGACAU A GCCAACA AACAGCU U CUAUGA ?ICAGCUJU C UAUGAAG AGCUUCU A UGAAGUG GAAGUGU U UGAAAAA AAGUGUU U GAAAAAC AAAACAU C CCC=~J CCCCACU U UAUA.GAU CCCACUU U AUAGAUG CCACUUU A UAGAUGU ACUUUAU A GAU7GUUU UAGAUGU U UUUGUUC AGAUGtJU U UUGUUCA GAUGUUU U UGUUCAU AUGUUtJU U GUUCAtUU UUMMUU U CAUUUtUG UU=JGUU C AUUUUGG UGtUCAU U tJUGGUAU GTUCAUUT U UGGUAUA UUCAUOU U GGUAUAG 696 698 706 708 709 721 726 731 740 741 742 743 751 754 755 756 766 787 788 800 802 803 811 815 816 822 824 825 829 830 840 866 869 875 876 877 883 895 913 914 916 921 923 925 943 946 947 949 950 UUULUGGU A UAGCAC-A UUGGUJAU A GCACAAU GCACAAU C UUCJACC ACAAUJCU U CUACCAG C-AAUCUU C UACCAGA AUCUt7CU A CCAGAGG UGGCCAGU A GAGUUGA GUAGAGU U GAAGGGA AAGGGAU U UUUGCAG AGGGAUU U UUGCAGG GGGAUUU U UCCAkGGA GGAUUUU U GCAGGAU =CAGGAU U TLAUG GGAUUGU U UAUGAAU GAUUGUU U AUGAAL'G AUUG=U A UGAAUGC .AAUGCCU A UGGUGCA GTJGAUGU U ACGGtJGG UGAUGU A CGGUGGG GGGGAGU C UUAGCAA GGAGUCU U AGCAAAA GAGUCU A GCAAAAU GCAAAAU C AGUtUhAA AAUCAGU U AAAAAUA AUCAGrUU A AAAAUAU UAAAAAU A UUAJGtU AAAAUAU U AUGUUJAG AAAUAUU A UJGUUAGG AUUAUGU U AGGACAU UUAU=U A GaACAUG ACAUGCU A GUGUGCk AACAAGU U GUUGAGG AAGUUGU U GAGG%-UUU UUGAGGU U UJAUGA.AU UGAGGUU U AUGAAUA GAGGUUU A UGAAUAU UAUGAAU A UGCCCAA CAAAAAU U GGGUGGU GCAGGAU U CUACCAU CAGGAUU C UACCAUA GGAUUCU A CCAUAUA CUACCAU A UAUUGAA ACCAUAU A UlUGAACA CAUAUAU U GAACAAC AAAGCAU C AUUAUUA GCAUCAU U AUUJAUCU CAUCAUU A UU7AUCrU tiCAUUAU U AUCUUIUG CAUUATUU A UCIUUC-A SUBSTITUTE SHEET (RULE 26) WO 95/23225 276 952 UtAUUAU C UUGACU 954 AtJUAUCU U UGACUCA 955 UUAUCUU U GACtJCAA 960 UUTUGACU C AAUUUJCC 964 ACUCAAU U UCCUCAC 965 CUCAUU U CCUCkCU 966 UCAALJUU C CUJCACUU 969 A==CC C ACUUCUC 973 CCUCACU U CCCCAGU 974 CTUCAMCUUJ C UCCAGUG 976 CACUUCU C CAGUGUA 983 CCAG=G A GUAUUAG 986 GUGUAGU A UUAGGCA 988 Gtl.GUAU U AGGCAAU 989 UAGUAUU A GGCAAt3G 1007 CUGG-CU A GGC-AUAA 1013 UAGGCAU A AUGGGAG 1024 GGAGAGU A CAGAGGU 1032 CAGAGGU A CACCGAG 1044 GAGGAAU C AAGAUCU 1050 UCAAGAU C ZMUAUGA 1052 ?AGAUCU A =DGAUG 1054 GNU=t. A UGAUGCAk 1072 AGGCAU A UGCCUGA 1085 AACAACU C AAAGAAA 1103 G7ZUGUGAU U AACtACA 1104 UGUGAUJU A ACUACAG :108 AUUA.CU A CAGUGUA 1,115 AQAGUGU A CDAGACU 118 GUGUAC"J A GACUUGA 11.23 CtJAGA=t U GACAGCA U139 AAGAACU A GAGGCUt, 1146 AGAGGC-U A UCAAACA 1148 AGGCOAU C AAACAUC 1155 CAACAU C AGCtJUAA 2160 AUCAGCU U AAUCOAA 1161 UCAGCUU A AUCCAA 1164 cCUUAAU C CAAG 1173 AAAGAU A AUGAUGU 1181 AUGAUG A GAG=UU 1187 UAGAGC-U U UGAGUUA 1188 AGAGCUU U GAIGUJA 1193 UUUGAGU U AAAA 1194 UUGAGUU A AUAAA PCT/B95/OO 156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 277 Table 36: RSV EB Ribozyme Sequence nt. HH Ribozyme sequence Position 9 AUCUUUG CtJGAUJGAGGCCGAAAGGCCGAA AUutJGCC 21 UtICUAA CUGAUGAGGCCGAAAC-GCCaAA AGCCALC 23 CUUUGCU CUGAUGAGGCCGAAAGGCCGA,;A ACGCCA 24 ACtUUGC CUGAUGAGGCCGAAAGGCCG-AA AAGAI3cC 32 UCAAcUU CUGAUGAGGCCGAAGGcCCAA ACruOuGc 37 AUCAUUC CUGAUGAGGCCGAAAGGCCGAA ACUUGAC TUUGAGUG CUGAUGAGGCCGAAAGGCCGAA AUCAUUC CUUtJGUU CUGAUJGAGGCCGAAAGGCCGA-A AGUG~tI AGAAGUU CUJGAUJGAGGCCGAAAGGCCGAA AUCtJUUG AUJGACAG CUGAM7GCCGAAAGGCCGAA AGUUTGAU 66 GAUGACA CUGAUGAGGCCGAAAGGCCGAX A-AGEJGA GCtJGGATJ CUGAUGAGGCCGAAAGGCCG-AA ACAGAAG 73 tJUUGCUG CUGAtUGAGGCCGAAAGGCCGAA AuGACAkG 82 GAUGGt7G CUGAUGAGGCCG-AAAGG-CCGAA A~jUUGCU 89 tJCCGUUG CUGAUGAGGCCGAAGGCCGAA AuGGuGUr 108 UCAAUAC CUGAUJGAGGCCGAAAGGCCGAA AuUccuCJ 111 GUAUCAA CUTGAUrGAGGCCGAAAGGCCGAA ACUAUJ 113 GA.GUAUC CUGAUGAGGCCGAAAGGccG-AA AuACUAU 117 UUAGGAG CUGAUGAGGCCGA6AAGGCCGAA AUCA6AUA 120 UAAUUAG CUGAUGAGGCCGAAAGGccGcAA AGJAucA6 123 UCAUAAU CTUGAUGAGGCCGAAAGGCCGAA AGCAGtJA 126 ACAUCAU CUGAUGAGGCCGAAAGGCCGAA AUUAGGA 127 CACAUJCA CUGAUGAGGCCGAAAGGCcGAA AAuUAGG 146 ACUUAUU CUJGAUGAGGCCGAAAGGCCGAA AuGuGut 150 CAtUhACU CUGAUGAGGCCGAAAGGCCGAA ADuGAUG 154 GCCACAU CUGALUGAGGCCGAAAGGCcGAA ACUUAU 155 UGCCACA CUGAUGAGGCCGAGGccGAA AACU 166 GAUUAAU CUGAUJGAGGCCGAAAGGCCGAA ACAUGCC 167 UC-AUUAA CtJGAUGAGGCCGAAAGGCCGA AA-AU'GC 169 UGUGAUU CUG-AUGAGGCCGAAAGGCCGAA AUrAACAU 170 CUGUGAU CUGAUGAGGCCGAAAGGC-cGAA pAAuAACA 173 CUUCUGU CUGAUGAGGCCGAAAGGCcGAA AUUAAUA 186 UUAUGAU CUGAUGAGGCCGAAAGGCCGAA AGcAuctJ i89 AAUUUAU CUGAUGAGGCCGAAAGGCCGAA AuUGC:A 192 GUGAAUU CUGAUGAGGCCGAGGCCGAA AUGAUUA 196 CCCAGUG C GAVGAGGCCGAAAGGCCGAA AUUUAUG 197 ACCCAGU CtJGAUJGAGGCCGAAAGGCCGA;A AATUUULAU 205 ACCUAUU CUGAtJGAGGCCGAAAGGCCCGAA ACCCAGU 206 tJACCUAU CUGAUGAGGCCGAAAGGCCGAA AACCCAG 209 ACAUACC CtJGAUGAGGCCGAAAGGCcGAA AjuUAACC- 213 UAUTAACA CUGAUGAGGCCG-AAAGGCCGAA ACCUAUU I'CT/11395/0()150 SUBSTITUTE SHEET (RULE 26) WO 95/23225 278 217 CGCAUAU CUGAUGAGGCCGAAAGGCCGAA AcAuACC 218 UCGCAUA CUGAfLGAGGCCGAAAGGCCGAA AACAUAC 220 CAUXCGCA CUGAUGAGGCCGAAAGGCCGAA AUAACAU 229 u=ACCA CUGAUGAGGCCGAAAGGCCGAA Ac~J3cGc 231 CCUAACC CUGAUGAGGCCGAAAGGCCGAA AGACAUC 235 UtJUCCU CUGAUGAGGCCGAAAGGCCGAA ACCUAGA 236 CU=UCC CUGAtGAGCGCCGAAAGGCCGAA AACCOAG 254 GUtJU CUGAUGAGGCCGAAAGGCCGAA AUC-,-UGU 260 CUCUGAG CUGAT.aAGGCCGAAAGCGCCGAA AUUUUUA 263 CAUC=C CUGAtrwtGGCCGAAAGGCCGAA AGUAUUtJ 277 uA.QAuGA cUiGATUGAGGccGAAAGGccr.AA AUtCCCGc 279 UutI.CAU CUGAUG-AGGCCGAAAGGCCGAA AuAuccC.

284 Ur)GC-T7U CUTGAUGAGGCCGAAAGGCCGAA ACAIJGAU 299 UUACACC CCUGAUGAGGCCGAAAGCCGAA ACUCCAU 305 GLIGUUGU CrUGAUGAGGCCGAAAGGCCGALA ACAucuA 315 UCUUGAC CUGAUGAGGCCGAAAGG;CCGAA AUGUGUU 318 AUGUCUU CUGAUGAGGCCGAAAGGCCGAA ACGAUGU 326 UUJCCAUJU CrJGALtAGGCCGAAAGGCCGAA AtJGUCUU 327 MJUCCAU COGMAGGCCGAAAGGCcGAA AAUGUCU 346 CACCUCA CUGAUGAGGCCGAAAQGCCGAA AuuucAu 347 ACACEJUC CUGAAUAGGCCGAAAGGCCGAA AAutuCrA 355 CAAUJGUU CUTGAUGAGGCCGAAAGGCCGAA AcAQuuc 356 CCAAUGU CUGAUGAGGCCGAAAGGCCGAA AAcACUU 361 GCUUGCC CUGAtJGAGGCCGAAAGGCCGAA AUGUUAA 370 AGUUGUU CUJGAUGAGGCCGAAAGG;CCGAA AGCUUGC 371 CAGUUGU CUTGAUGAGGCCGAAAGrGCCGAA AAGCUUG 383 UGAtJ=UG CUGAUGAGGCCGAAAGGCCGAA AUuUCAG 384 ULM=JU CUGAUGAGGCCGAAAGGCCGAA AAuuuCA 389 CAAUGUU CUGAUGAGGCCGAAAGGCCGAA AuuuGAA 395 CEMUCUC CUGAUGAGGCCGAAAGGCCGAA AUGUUGA 402. UAGAtJUC CLUGAUAGGCCGAAGCcGAA AucuCAA 406 U =JUCUA CUGAUGAGGCCGAAAGGCCGAA AUUCUAU 408 GAUUUUC CUGAtJGAGGCCGAAAGGccGAA AGAuucTJ 415 UTMJGUAG CUGAUG.AGGCCGAAAGGCCGAA AuUUCU 418 UUUUUG CUGAUGAGGCCGAAAGGCCGAA AGGAUu 431 UUUCUUU CUGAUGAGGCCGAAAGGCCGAA AGCAUu 449 CtrGAGC CUGAUGAGGCCGAAAGGCCGAA ACCuUCC 453 UAUUCUG CUGAUGGGCCGAAAGGCCGAA ACCUACC 460 AUJGCCtJG CUGAUGAGGCCGAAAGGCCGAA AUCUGG 472 AUCAGGA CUGAUGAGGCCGAAAGGccGAA AGUiCAUG 474 CAAUCAG CUGAUGAGGCCGAAAGGCCGAA AGAGUCA 480 AUCCCAC CUGAU3GAGGCCGAAAGGCCGAA AuCAGGA 491 AUAAUAU CUGAUGAGGCCGAAAGGCCGA.A AUCAUCC 494 UACAIIAA CUGAUGAGGCcGAAAGGccGAA AuUjAUCA 496 UJAUACAU CUGAUGAGGrCCGAAAGGccGAI. AuAumUA 497 CUATJACA CUGAUGAGGCCGAAAGGCCGAA AAUAUUA 501 GCUGCUA CUGATJGAGGCCGAAAGGC~CGAA AcAuAAU' 503 AUGC-UGC CUGAUGAGGCCGAAAGGccGAA AuAcAUA 511 UAUUACU cUGAUGAGGCCGAAAGGCCGAA AUGCUGC P*CT/1B95/OO 156 SUBSTITUTE SHEET (RULe 26) WO 95/23225 279 512 UUAUtJAC CUGAUGAAGGCCGuAAGGCCGAA AAUG%-tUG 515 UAtJT3AU CUGAtJGA=GCGAAAGGCCGAA ACtJAAUG 518 AUUUAGtI CUG?.GGCCGAAAGCGAA AtJUACUA 522 GCUAAUJU CtJGAUGAGGCCGAAAGGCCGAA AGUUJAUU 526 UGCTJGCU CUGAL;GAGGCCGAAAGGCCGAA AUUUAGU 527 CUGCUGC CUGAT2GAGGCCGAAAGGCCGAA AAUUUAG 544 AAGACC CtJGAUGAGGCCGAAAGGCCGAA AUCUGUC 549 GCUGtJAA CUGAUGAGGCCGAAAGGCCGAA ACCAGAL7 551 CGGCt(J CTJGAIGAGGCCGAAAGGCCGAA AGACCAG 552 ACGGCUG CLTGAt~AGGCGAAGGCCGAA AAGACCA 563 CUJCUCCU CGALAGGCCAAAGGCCGAA AUCACGG 564 GC-UCUCC CUGA.UGAGGCGAAAGGCCG-AA AAUC-ACG 573 ACAUUAU CtJGAUGAGGCCGAAAGGCCGAA AGCUCUCT 576 AGGACAU CUCAUGAGGCCGAAGGCCCGAA AtJUAGCU 581 UUUtJUAG CUGAUGNGGCCCAAAGGCCGAA ACAU 584 CAUUUUU CUGAUGAGGCCGAAAGGCCGAA AGGACAU 603 CL'JWJGU CUGAUGAGGCCG?.AAGGCCGAA ACGUUUC 604 GCCUUUG CUGAUGAGGCCGAAAGGCCGAA AACGUU 613 GGGUV.GU CUGAUGAGGCCG?%AA=GCGAA AGCCUUU 614 UGGGUAG CUGAUGAGGCCCGAAAGGCCGAA AAGCCU 617 CCUUGGG CT3GAUGAGGCCGAAAGGCCGAA AGUAGC 629 UGUUGGC CUGAD'GAGGCCGAAAGGCCGAA AUGUCCU 640 UTJCAUAG CUGAUGAGGCCGAAAGGCCGAA AGCUGUU-r 641 CtJCAUA CUGAUGAflGCCGAAAGGCCGAA AAGCUGU 643 CACUEJCA CUGAUGAGGCCGAAAGGCCGAA AGAAGCtJ 652 UUUUUCA CUGAUGAGGCCGAAAGGCCGAA ACAC= C 653 GUU=tJC CUGAUGAGCCGAGGCCGAA AACACUUJ 663 AAGUGGG CUGAUGAGGCCGAAAGGCCGAA6 AUGUUUU 670 AUCUAUA CUGAU-GAGGCCGAAAGGCCGAA AGUGGG 671 CAUCtJAU CUGAUGAGGCCG-AAAGGCCGAA AAGUGGG 672 ACAUCtJA CUGAUGAGGCCGAAAGGCCGAA AAAGtJGG 674 AAACAUC CUGAUGAGGCCGAAAGGCCGAA AUAAAGU 680 GAACAAA CUGAUGAGGCCGAAAGGCCGAA ACAUCUA 681 UGAACAA CUGAUGAG7GCCGAAAGGCCGAA AACAUCU 682 AUGAACA CUGAUGAGGCCGAAAGGCCGAA AAACAUJC 683 AAUGAAC CUGAUGAGGCCGAAAG7GCCGAA AAAACAU 686 CAAAAUG CUGAUGAGGCCGAAAGGCCGAA AC.AAAAA 687 CCAAAAU CUGAUGAGGCCGAAAGGCCGAA AACAAA 690 AUJACCAA CUGAUGAGGCCGAAAGGCCGAA AUGAACA 691 UAUACCA CUGAtuG.GCGAAAGGCCGAA AAUGAAC 692 CUAUACC CUGAUTGAGGCCGAAAGGCCGAA AAAEJGAA 696 UGUGCUA CUGAUGAGGCCGAAAGGCCGAA ACCAAAA 698 AUUGUGC CUGAUGAGGCCGAAAGGCCGAA AUACCAA 706 GGUAGAA CUGAUGAGGCCGAAAGGCCGAA AUUGUGC 708 CUGGUAG CUGAUJGAGGCCGAAAGGCCGAA AGAUUGU 709 TUCUGGTYA CUGAUGAGGCCGAAAGGCCGAA AAGAUUG 711 CCUCUGG CTJGAUGAGGCCGAAAGGCCGAA AGAAGAU 726 UCAACUC CUGAUGAGGCCGAAAGGCCGAA ACUGCCA 731 UCCCUUC CUGAUGAGGCCGA.AAGGCCGAA kCUCUAC PCTr/1B95/00 156 SUBSTITUTE SHEET (RULE 261 WO 95/23225 280 740 cUGcAAA CUGAUiGAGGcCCAAAGGCCG-AA AUCCCUU 741 CCUGCAA cuGAuGAG~cCGAAAGGCCGAA AAUCCMt3 742 UCCUGCA ctJGAtGAGGCCGAAAGGCCGAA AAAUCCZ- 743 AUCCUG CTJGAUGAGGCCGAAGCGCCGAA AAAAUCC 751 cAtJAAAc cUGAUGAGGCCCGAAQGCCGAA AUCCGC 754 AUUCAUA CUGAUG-AGGCGAAAGGCCGAA ACAAtJCC 755 CAUtJC.U CUGAUGAGGCCGAAAGCGAA AACAAUC 756 GCAUUCA CUGAUGAGGCCGANAGGCCGAA AAACAAU 766 UGCACCA% CtGAuGAGGC-CGAAAGGCCGAA AGAUU 787 CCACCGU CUJGAUGAGGCCGAAAGGCCC-M AC-NUCAC 788 CCCACCG C3GUCAAGCCGAAAGGCCGAA AACAtJCA B00 UUGCUAA CUTGAUGAGGC-CGAAAGGCCGAA ACUCCCC 802 LUU=~C CEJTGAUCAGGCCGAAAGGCCGALA AGACUCC 803 AUUUU-rGC CUGAUGAGGCCGAAGGCCGAA AAGACUC 81i UUU~ACU CTGAGAXGGCCGAAAGGCCGAA AUUUUrjGC 815 uAUtU= UG GAGC AA=C ACLGATJU 816 A~UUUU cUGArAV.GCCGA? GGCCGAA AACtrJGAU 822 AACAUA7L CtUGAAGCCGAAAGGCCGAkA AUCUUUA 824 CtACAU CUGAUGAGGCCGAAAGGCCGAA At3AUCUUt 825 cthAC CUGAUGAGGCCGAAAGGCCGAA AAUAUUU 829 AU~tCCt CUGCAUGAGGCCGAAAGGCCGAA ACAUAAU 830 cAuGt3CC cUGAUGAGGCCCAAAGGCCGAA AACADAA 840 jGCACAC CUGAUGAGGCCGAAAGGCCGAA AGCAUGJ 866 CCUCAAC CUG-AUGGCCGAAAGCCG-AA ACUUGUU 869 AAACCtJC CUGAUGAGGCCGAAAGrVXCCGAA ACAACUE 875 AUE.7cAUA CUGAUGAGGCCGAAAGGCCGAA ACCUCA 876 UJAUUCAU CUGAUGAGGCCGAAAAGCCG,,A AACCUCA 877 AIUAUUCA. CUCATUGGCCGAAAGGCCGAA AAACCUC 883 UUXGrG CUGAUGAGGCCGAAAGGCCGAA AUTUCAUA 895 ACCACCC CUAGGCGAAGCM AUUUUUG 913 AUGGUAG CUGALtGAGGCCGAAGGCCGAA AUCCUGC 914 UAUGGtJA CUGAUGAGGCCGAAAGGCCGAA AAUCCUG 916 UmhIAhGG CUUGArGG=ccAAAGGCCGAA AGAAIJCC 921 uucAAUA CtIGALUGAGGCCGAAAGGCCGAA AUGGUAG 923 OJGUUC.AA CUGAUGAGGCCGAAAGGCCGAA AUAUGGU 925 GOUGUUJC CGAUGAGGCCGAAAGGCCGAA ADAtIAUG 943 UAAMUt CUGAUGAGGCCGAAAGGCCGAA AUCUTJU 946 AGAUAAU CUGAUGAGGCCGAAAGGCCGAA AUGAUGC 947 AAGALTAA CUGAUGAGGCCGAAAGGCCGAA AAUGAUG 949 CAAAGAU CUGAUGAGGCCGAAAGGCCGAA AUAAUGA 950 UCAAAGA CUGAUGAGGCCGAAAGGCCGAA AAUAAUG 952 AGUCAAA CUJGAUGAGGCCGAAAGGCCGAA AUAAUAA 954 UGAGUCA CUGAUGAGGCCGAAAGGCCGAA AGAUAAU 955 UUGAGUC CUGAUGAGGCCGAAAGGCCGAA AAGAUAA 960 GG-AAAUJU CUGAUGAGGCCGAAAGGCCGAA AGUCAAA 964 GUGAGGA CUGAUGAGGCCGAAAGGCCGAA AUUGAGU 969 AGAAGU CUG-AUGAGGCCGAAAGGCCGAA PAGAA 965 AGUGAG CUJGAUGAGGCCGAAAGGCCGAA AAUUGA 969 AAAG CUGAU-GAGGCCGAGGCCGAA AAUGA PICTIB95/OO 156 SUBSTITUTE SHEET (RULE 26) WO 95/23225 281 973 ACUGGAG CUGAUGAGGCCGAAAGGCCGAA ?.GUGAGG 974 CACUGGA CUGAt3GAGGCCGAAAGGCCGAA AAGUGAG 976 UJACACUG CUGAUGAGGCCGAAAGGCCGAA AGAAGUG 983 (7JAAUAC CtYGAUGAGGCCGAAAGGCCGAA ACANCUGG 986 UGCCtJAA CUGAUGAGGCCGAAAGGCCG-AA ACUACAC 988 AUUGCCU CUGAUGAGGCCGAAkAGGCC-GA-A AUAC 989 CAUUJGCC CUGAUGAGGCCGAAAGGCCGAA AAI3ACUA 1007 UtJAUGCC CUGAUGAGGCCGAAAGGCUGAXA AGGCC-A-G 1013 CUCCCAU CtYGAUGAGGCCGAAGC-CCGAA AUGCCUiA 1024 ACCUCUG CUGAUGAGGCCC-G%CCGAA ACUCC 1032 CUCG-GUG CUGAUGAGGCCGAAGGCCGA ACCUCtJG 1044 AGAUCU C GAUGAGGCCGAAAGCGCCAA AIX=~t :050 UCAUAUA CUGAUGAG-GCCGAAAGC-CCG.AA ACC!JUGA 1052 CAUCATJA CUGATUGAGGCCGAAAGGCC7AA AC-AUCrU 1054 L'GCAUCA CUGAUGAGGCCGAAAGGCCGAA ALt7C.GAUC 1072 UUCAGC-A CUGAUGAGGCCGAAAGGCCGAA kUC-CCTj-U 1085 UUEJCUUJU CUGAUGAGGCCGAAGGCCGAA AGUUCUEJ 1103 UGUGUU CUGAUGAGGCCGAAGCCG-A AUCAQC 1104 CUGMGU CUGAUJGAGGCCGAAAGGCCGAA AUCACA 1108 UACACUG CUGAUGAGGCCGAAAGGCCGAA AZtIUAAU 12.5 AGUCUAG CUGAUGAGGCCGAAAGGCCGAA ACACGU 1118 UJCAAGUC CUGAUGAGGCCGAAAGGCCGAA AG"JACAC '123 UGC-UGUC CUGAUGAGGCCGAAAGGCCG7AA AZUCUAG 1139 UAGCCUC CUGAUGAGGCCGAAACGCCGAA AMU=CU 1.146 UGUUUGA CUGAUJGAGGCCGAAGGCCGAA AGCCUCtJ 1148 GAUGUUU CUGA!JGAGGCCGAAAGGCCGAA AUAGCCtJ 1155 UUAAG--U CUGAUGAGGCCGAPA.GGCCGAA AUGUUtJG 1160 UUGGAUU CUGAUGAGGCCCGAAAGGCC-AA AGCUGAU 1161 UUUGGAU CUGAUGAGGCCGAAAGGCCGAA AAGCUG-A 1164 UCUUUUJG CUGAUGAGGCCGAAGGCCGAA ALTj7AGC 1173 ACAUCAU CUGAUGAGGCCGAA.GGCCGAA ALCUUO 1181 AAGCUC CUGAUGAGGCCGAAGGCCGA ACAUCAU 1187 UAACUCA CUGAUGAGCCGAAGGCG6 AGCU-rCUA 1188 UU~AGUC CUGAUGAGGCCGAGGCCGA AAGCJCJ 1193 UUUUAUU CUGAtJGAGGCCGAAAGGCCGAXA ACUCAAA 1194 UUUUUAU CUJGAUGAGGCCGAAAGGCCGAA AACUCA PCI/1B95/00 156 SUBSTITUTE SHEET (RULE 26) Table 37: RSV (1B) HIP Rib ozyrne/Stibstrate Sequence nt. IIP flibozyme Sequence Substrate Position CUGUGAUC AGAA GUCUUU ACCAGAGAAACACACGUIJGUGGUACAUUACCUGGUA AAAGACU GAU GAUCACAG 91 CAAGUGAC AGAA GUCUCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGAGACC GUI) GUCACUUG 472 CAGGCUCC AGAA GGACUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UAGUCCA GAU GGAGCCUG Cf) CN3

C/)

My-

ND

Table 38: RSV HIP RibozyrnelSubstrate Sequence

:E

nt. Hairpin Iibozymne sequence Substrate Position 476 AUCCCACA AGAA OGAGAG ACCAGAGAAACACACGUUGUGGUIACAUUACCUG-GUA CIOCUCCtJ GAUl IGUGGGA!J 540 AAGACCAG AGAA GUCCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGOGACA GAU CUGGUCUU 554 CUJAAUCAC AGAA GUAAGA ACCAGAGAAACACACGUUGUGGUACAUUACCUJGGUA UCUUACA GCC GUGI\UUAG 636 UUCAUAGA AGAA GUUGGC ACCAGAGAAACACACGUUGUGGIJACAUACCUGGUA GCCAACA GCU UCUJAUGAA 998 CCUAGGCC AGAA OCAUUG ACCAGAG3AAACACACGUUGUGGUACAUUACCUGGUA CAAUGCU GCU GGCCUAGG 1156 'UUGGAUUA AGAA GAUGUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AACAUCA GCU UAAUCCAJA

C:,

CO)

-Z

rrn rr-i

I-

N)1

P?,

WO 95/23225 PCTIIB39/OO156 284 Table 39: Large-Scale Synthesis Sequence

A

9

T

A9T

(GGU)

3

GGT

(GGU)

3

GGT

CgT

OST

U 9

T

U

9

T

Activator (Added/Final] (m i T (0.50/0.33] S (0.25/0.17] T [0.50/0.33] S [0.25/0.17] T [0.50/0.33] S (0.25/0.17] T [0.50/0.33) S [0.25/0.17] T [0.50/0.33] S (0.25/0.17] S (0.50/0.24] 5 (0.50/0.18] 5 (0.50/0.18a] Amidite [Added/Final] (min) (0.1/0.02] 1/0.021 (0.1/0.021 1/0.021 1/0.02] 1/0.021 1/0.02) [0.1/0.02] (0.1/0.02] 1/0.02] (0.1/0.03] 1/0.05] 1/0.05] 15 m 15 m 15 m 15 m 15 m 15 m 15 m 15 m 15/15Sm 15/15 m 15/15 m 15/15 m 10/5 m Time* Full Length Product (36-mer) (36-me r) (36-me r) (36-mer) (36-mer) *Where two coupling times are indicated the first refers to RNA coupling and the second to 2'-Q-methyl coupling. S 5-S-Ethyltetrazole, T tetrazole activator. A is 5' -ucu ccA UCU GAU GAG GOC GAA AGG COG AAA Auc ccu -3 where lowerecase represents 2'-O-methyinucleotides, SUBSTITUTE SHEET (RULE 26) IIC'/11B95/00)156 WO 95/23225 285 Table 40: Base Deprotection Sequence iBu(GGU) 4 Deprotection Reagent

NH

4 OHiEtOH

MA

AMA

MA

AMA

NH

4 OH/EtOH !PrP(GGU) 4

AMA

MA

AMA

Ti me (m 1 16 h 1Cmn 10M 10M 1Cm 4 h 1Cm 1Cm 1Cm 1Cm 4 h 1Cmn 10M 10M 1Cm 4 h 1Cm T 0 C Full Length Product 62.5 62.7 74.8 75.0 77.2 44.8 65.9 59.8 61.3 60.1 75.2 79.1 77.1 79.8 75.5 22.7 28.9 cqu

NH

4 O HIEIOH

MA

AMA

MA

AMA

NH

4 OHIEtOH

MA

A (36-mer) SUBSII01 S~w g E 26)

F

WO 95/23225 P('TIB95OO 156 286 Table 41: 2'-O-Alkylsilyl Deprotection Sequence AgT

(GGU)

4 C0

U

10 B (36-mer) A (36-mer) Deprotection Reagent

TEAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF

TBAF

1.4 M HF Time (min) 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 24 h 0.5 h 24 h 1.5 h 24 h T 0 C Full Length Product 20 84.5 65 81,0 20 60.9 65 67.8 20 86,2 65 86.1 20 84.8 65 84.5 20 25.2 65 30.6 20 29.7 1.5 h 65 30.4 B is UCU CCA UCU GAU GAG GCC GAA AGG COG AAA AUC CCU SUBSTITUTE SHEET (RULE 26) Table 42:. NMILDatz for UC Dimncrs containing Pltosplhorothioate Linkage Syntfhesis It 3524 3525 3530 3 5 2 3578 3529 Type ri bo ribo ribo ribo rib o ribo Delivery 2 x 3s 2 x3 s 2 x3 s 1x 5s Ix 5s Ix 5s Eq.

10.4 10-4 10.4 08.6 08.6 08.6 WVa it 2 x 100 s 2 x 75 s 2 x 75 s 1 x 300 s Ix 250 s 1 x 150 S

ASE.(%

95.9 92.6 92.1 100.0 100.0 73.7 Tabl e 43: NMR Data for fti-iner RNA containling Phosphorothioate Linkages Synthesis It 3581 3663 Type rib o ribo Delivery Ix 5s 2x4s Eq.

08-6 13-8 Wait I x250 s 2x300s AS E (01) 99.6 100.0 3582 3668 3682 2'-O-Me Ilx 6s 2'-O-Me 2 x4 s 2'-O-Me 1 x 5 s 08-6 13.8 08.6 1.x 250 s 2-,300s I x 300 s 99-7 99.8 99.8 IIC PCT/IB95/0(015> WO 95/23225 289 Table 44. Kinetics of Self-Processing In Vitro Self-Processing Constructs k (minI)* HH 1.16 0,08 HDV 0.56 0.15 HP(GC) 0.36 0.06 HP(GU) 0.054 0.003 k represents the unimolecular rate constant for ribozyme self-cleavage determined from a non-linear, least-squares fit (KaleidaGraph, Synergy Software, Reeding, PA) to the equation: (Fraction Uncleaved Transcript) (-ekt) The equation describes the extent of ribozyme processing in the presense of ongoing transcription (Long Uhlenbeck, 1994 Proc. Natl. Acad. Sci. USA 91, 6977) as a function of time and the unimolecular rate constant for cleavage Each value of k represents the average range) of values determined from two experiments.

SUBSTITUTE SHEET (RULE 26) PCTIB95/00 156 WO 95/23225 290 Table Entry Modification t1/ 2 (in) t 1 1 2 (in) tS/tA Activity stability x Nt) (ts) I U4& U7= U 1 0.1 1 2 U4 U7 2'-0-Me-U 4 260 650 3 U4 =2'=0H 2 -U 6.5 120 180 4 U7 =2'=CH 2 -U 8 280 350 U4 &U7 =2'=CH 2 -U 9.5 120 130 6 U4 2'=CF 2 -U 5 320 640 7 U7 2'=CF 2 -U 4 220 550 8 U4 &U7 =2'=CF 2 -U 20 320 160 9 U4 2'-F-U 4 320 800 U7 2'-F-U 8 400 500 11 U4 &U7 2-F-U 4 300 750 12 U4 2'-C-Allyl-U 3 >500 >1700 13 U7 2'-C-Allyl-U 3 220 730 14 U4 U7 2'-C-Allyl-U 3 120 400 1 5 U4 2 '-araF-U 5 >500 >1000 1 6 U7 2'-araF-U 4 350 875 17 U4 &U7 2'-araF-U 15 500 330 18 U4 2'-NH 2 -U 10 500 500 1 9 U7 2'-NH 2 -U 5 500 1000 U4 &U7 2-NH 2 -U 2 300 1500 21 U4 =dU 6 100 170 22 U4 &U7 =dU 4 '240 600 SUBSTITUTE SHEET (RULE 26)

Claims (4)

1. An enzymatic nucleic acid molecule comprising a nucleotide selected from the group consisting of 5'-C-alkylnucleotide, 2'-deoxy-2'-alkylnucleotide, methylnucleotide, 5'-deoxy-5'-difluoro-methylnucleotide, 3'-deoxy-3'-dihalo-methyl- nucleotide, and 5',3'-dideoxy-5',3'-bis(dihalo)-methylphosphonate.

2. Method for producing an enzymatic nucleic acid molecule having activity to cleave an RNA or single-stranded DNA molecule, comprising the step of forming said enzymatic molecule with at least one nucleotide having an alkyl group at its 5'-position or 2'-position,

3. An enzymatic nucleic acid molecule according to claim 1, substantially as hereinbefore described with reference to any one of the Examples.

4. A method according to claim 2, substantially as hereinbefore described with reference to any one of the Examples. Dated 20 April, 1999 Ribozyme Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON ee *i