CA2216284A1 - Thiono triester modified antisense oligodeoxynucleotide phosphorothioates - Google Patents

Abstract

Antisense oligonucleotides having improved cellular uptake, increased nuclease resistance, and thermodynamically more stable target-binding capacity are disclosed. These novel oligonucleotides are characterized by having from 1 to 10 thiono-triester phosphorothioate internucleoside linkages bearing lipophilic moieties.

Description

W 096~9~37 PCTrUS96/03843 TElIONO TRIESTER MODIlilED ANTISENSE OLIGODEOXYNUCLEOTIDE
PEOSPHOROTE[IOATES

BACKGROUND OF TEIE INVENTION
~ S Field ofthe Invention This invention relates to the field of modified oligodeoxynucleotides, methods for their ~yl~LLesis, and meths)tlc of their use to inhibit gene cA~.t;ssion.

y of the Related Art ~.. ~ e oli~omlcleotides and their modified analogs have been shown to regulate the s~ion of genes (7:~m.o-.nik in Prospects for Antisense Nucleic Acid T,22erapy for Cancer and AIDS, pp. 1-6 (Wick:,Llulll, E, Ed.,Wiley Liss, N.Y., 1991); Agrawal, Trends in Biotechnology 10, 152-158 (1992); Wickstom, Trends in Biofechnology 10, 281-286 (1992); Rapaport et al., Proc. Natl. Acad. Sci. USA 89, 8577-8580 (1992); and Agrawal, S. (1991) in Prospectsfor Antisense Nucleic Acid T,22erapy for Cancer and AIDS, pp. 145- 158, supra)) and to be effective il~h;l~iLol~ of HIV (Agrawal et al,. Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988); Agrawal et al., Proc. Natl. Acad. Sci. USA 86, 7790-7794 (1989); Agrawal and Sarin in Advanced Drug DiliveryReviews, pp. 251-270 (Juliano, R. J., Ed., Elsevier, Amsterdam 1991); Agrawal and Tang, ~nti~n~e Res. Dev. 4, 261-266 (1992); Liziewize et al., Proc. Natl. Acad. Sci. USA 89, 11209-11213 (1992); and Liziewize et al., Proc. Natl. Acad. Sci. USA 90, 3860-3864 (1992)), infl~--on7~ virus (Leiter et al., Proc. Natl. Acad Sci. USA 87, 3430-334 (1990)), human papilloma virus (Vickers, et al., NucleicAcidsRes. 19, 3359-3368 (1991)) and herpes simplex virus (Gao et al., Antimicrob. Agen~s Chem. 34, 808-812 (1990)) in tissue culture studies. To use antisense oligonucleotides as drugs it is necec.c~ry to develop compounds that are stable under biological and physiological conrliti~ n~, that are capable of effectively binding to a complim~nt~ry nucleic acid target, and that can be readily taken up by cells.
To meet these criteria, considerable efforts have been made recently in the stru&ture morlifir.~tion of oli~om~çleotides (IJhlmann and Peyman, Chem. Rev. 90, 543-584 (1990J and I t:r~l el.ces cited therein; Methods in Molecu7~7r Biology VoL 20: Protocols for Oligonucleotides and Analogs (Agrawal, S., Humana Press, New Jersey, 1993) and references cited therein;
A/,~ Research and Applicahons (Crooke, S.T. and Lebleu, B., Eds., CRC press, Ann Arbor 1993) and l~r~ ces cited therein), in~.l---lin~ modifications ofthe internucleoside link~es such as pl.~.5~ eLl,yl~ho:j,uhollales, pho:.l,hol~ tes~ phosphotriesters (Koziolkiewicz W 096/29337 P~TrUS96/03843 and Willc, in Protocols for Oligonucleofides and Analogs Vol. 20: Protocols for Oligu, -~eofides and Analogs, pp. 207-224, supra, and references cited therein), and many other non-phosphate intermlcleo~eitle link~gP,~s.
Miller et al., Biochem. 21, 5468 (1982), disclosed both isomeric forms of a ~leç,AmPr 5 having an internal ~;;LLyl~ lJl .. .lliester linlcage. It was found that after adding polyadenylate tails to the 3' end of the deç~ " the ethyl triester group inhibited polymerization by E. coli DNA
polymerase I.
Stec et al., Tetrahedron Left. 26, 2191 (1985), disclosed dimers linlced by an isopropyl phosphorothioate triester group. These dimers were synthP~ei7Pd via a 3'-O-isopropyl 10 phosphomorpholidite interm~ te Hau et al., Tefrahedron I,etf. 32, 2497 (1991), discloses thiolation of O-neopentylphosphite dimer using PlPmPnt,Al sulfur (S8) in pyridine to yield the corresponding neopentylphosphorothioate triester dimer. Four of these dimers were then linked via rhosrhodiester linlcages to yield an 3'-acri&e-capped octamer having alternating phosphodiester 15 and neopentyl phosphorothioate triester link~gPs T .~Pteingpr et al., Proc. Nafl. Acad. Sci. USA 86, 6553 (1989), disclosed oligonucleotides having one 3'-terminal r.hçilestf~.ryl phosphorothioate triester internucleoside linlcage. They observed inhibition of syncytia formation and ~ ession of viral proteins p 17, p24, and reverse transcriptase of H[V I in Molt-3 cells. The best results were found with an eicosomer, although 20 ~eA~mPrs and a hppt~n~ eotide where also active. These results, combined with the observation that the activity of the dec~mP-rs did not correlate with a specific sequence, suggested that a mPcl~ l,, other than ~ntiePnee inhibition may be operative.
T etsingP.r et al., NucleicAcidsRes. 14, 3487 (1986), synthpei7pd nucleotide dimers and a trimer with O-phosphotriester and phosphor~mid~te internucleotide link~ges bearing C2-C4 25 trichloroalkyl ~ ~ -A e They found that these dimers and trimers bound to target polynucleotides relatively stably.
El~yll~Lic degradation of cligon~ Qtitle phosphorothioates is primarily from the 3' end.
In an effort to circumvent this problem, various modifications at the oligonucleotide phosphorothioate 3' end have been made. Koziolkiewicz et al., supra; T~,l,.e~",~l-i et al., 30 Antisense Res. Dev. 3, 277 (1993); MS~ P1~Ar~ et al., Nucleic Acids Res. 20~ 3411 (1992); and Tang et al., NucleicAcidsRes. 21, 2729 (1993).

W 096129337 1~1/~ /03843 The rolegoil g demonsL-~Les the co..~ g interest in developing sintieen.ee oli~onllçleotides with increased efflcacy for hybridizing to target nucleic acids. Such oli~mlr.l~otirirc exhibit strong cellular uptake, high stability when bound to the target, and high les;~ r~e to nllr.lesiee attack.

SUMMARY OF T~IE INVENTION
The present invention cc,l.~;,-ises a novel class of modified sintie~nee oligonucleotides having increased cellular uptake, high stability when bound to the target nucleic acid, and high ,lr,e to mlrl~siee attack. These novel sintiernee oligonucleotides are characterized by having 10 one to ten thiono triester phosphorothioate internucleotide linkages. These internucleotide linksiges have the following structure:
S
--O-P-O--OR
15 where R is a large lipophilic moiety, such as a straight or branched chain alkyl group, a cholesteryl derivative, or an ~ isimsintyl derivative. Oligonucleotides having one or more of these types of linkziges exhibit increased recictsinr~ to degradation by T4 polymerase and DNA polymerase I. They also exhibit increased melting tt;l~lpel~L~Ires when hybridized to target nucleic acids as compared to oligonucleotide 20 phosphorothioates.
The oligonucleotides of the present invention are useful for both in vitro and in vivo Sl~)pl e~sion of nucleic acid expression. Among the in vitro uses for the present oligonucleotides is the dt;L~l...;..,.l;l n ofthe role particular proteins play in biological processes by modlllziting the expression of the genes encoding the protein under study. The rl~lcici~tinn of most biochemical pathways now known was accompliehPd by isolating and studying deletion mntsint.e in vitro.
Studying deletion mlltsmte is arduous, however. The ~;,lesellLly rlsiime i oligonucleotides provide an attractive altemative because it is much less laborious to modulate gene CA~le;S~iOn using sintie~onee oligon-lr.l~otides than it is using the deletion mutation approach. Thus, the oli~Qn-lçleotides ofthe present invention are useful lt;sea,~ tools.
The oligonil-,leotides of the present invention are suitable for in vivo use as well. The increased stability of the oligonucleotide-target hybridization compleY the increased reei~tsince to mlrlesiee attack and the i,lcleased ~.lec~pl;l.iliLy to cellular uptake all make the oligonucleotides _ W O 96/29337 PCTrUS9~'0 of the present invention ideal for L~ Ling infçctinn~ by a wide variety of di~e~çs caused by pathogens, inr.lllrlin~ viral and b~ct~ri~l agents. When dç~ignP~l for in vivo use, the presently disclosed ~ onllrleQtides will have at least one region whose sequence is sllffici~ntly comrl~.. l~.y to a region ofthe pathogen's nucleic acid (the target nucleic acid) to result in l-yi,- ;.1;,,.1 ;c n to the target nucleic acid and :jupplession of its CAIJ1 ession, both under intrac~ r c~ nrlition~
Rec~ e of their efflcacy at gene modulation, the ~. ese--Lly claimed oligonucleotides are also useful for treating r~ e~çs arising from genetic abnormalities that cause under- or over-expression of a gene. For diseases in which an al)nol,--al gene is over-expressed, for example, the presently claimed oli~omlrlçotides may be dPcigned to target the abnormal gene directly, or, in the ~h~rn~tive~ to target the gene encoding the protein that promotes ~A~ s~ion of the abnormal gene. Conversely, where an al~no----al gene is under-t:A,u-essed, one may design an oligonllr.leotide that :~u~p~es~es ~;A~,-t;ssion of a gene encoding a protein that suppresses ~A~ ssion of the abnormal gene.
To Sl~ , the p.est;.-Lly claimed oligonucleotides are useful both in vi~ro and in vivo in ç~sçnti~l1y any ~it~ tion in which one desires to modulate gene CA~ ssion.
The present invention also provides novel compositions comprising the inventive oligonucleotides as well as methods for employing the oligonucleotides to treat pathogenic es and other abnormal states arising from aberrant gene e;A~ S~iOn.
The foregoing merely su------~iGes certain aspects of the present invention and is not int~.n~ l not should it be construed, to limit the invention in any manner. All patents and other publir~ti- n~ referred to in this sperifir~tic)n are hereby incorporated by reference in their entirety.

~ - = ~

W O 96t29337 PCTrUS96/03843 _5_ BRIEF DESCRIPTION OF TElE DRAWINGS
Figure 1 displays the synthetic scheme for pro~llring ph~sl)hn~ liLe interme~ trc useful in the prodl~r,ti- n of oligf~mlr.leotides bearing S-triester internucleotide link~çs Figure 2 displays an autoradiogram of the dig~stion of modified S-triester-phosphorothioate oli~o~rleotides by T4 polymerase.
Figure 3 displays an autoradiogram of the ~li~stion of an O-ethyl triester phosl,horo~l~oate by DNA polymerase I.
Figure 4 displays an autoradiogram of the digestion of an Ol dill~ly phosphorothioate and an O-ethyl triester phosphorothioate oligonucleotide by T4 polymerase.

DETAILED DESCRIPTION OF T~E PREFERRED EMBODIMENTS
The present invention comprises, inter alia, a new class of ~nticence oligonucleotides having improved properties relat*e to prior art ~ntic~nce oligonucleotides. The present oligonucleotides exhibit incl~;ased s -cc~ y to cellular uptake when carrying a large lipophilic group, increased rt~cict~nre to ex~n~lcleolytic degradation, and increased stability of hybridization colll~ es formed between the oligQmlr.leotides and their targets. These properties make these novel oligonucleotides ideal for modl-l~ting gene expression, both in vi~ro and in viva Oli.~rnnrl~tides accol.lh~g to the invention may be anywhere from 2 to 100 nucleotides in length. In a plerelled embodiment, the oligonucleotides will be from 13 to 50 nucleotides in length. In a more p~erel- ed embodiment, the oligonucleotides will be from 20 to 35 nucleotides in length.
In one embodiment, the present invention cu-ll~lises ~nticrnce oligonucleotides having one to ten thiono triester co.~l~;";"g oligodeoxynucleotide phosphorothioate (S-triester-phosphorothioate) intemucleotide link~ges having the following structure:
S
--O-P-O--OR
where R is any large lipophilic group that doesn't ~ub~ ially change the ~eometry of the intemucleotide bond in a manner that 30 tlimiches the olig( nncleotide's efficacy at modlll~tinp gene e~leS~,iOn. In a plt:relled embodiment, R is a C2-C22 linear or branched chain alkyl group, a cholesteryl derivative having the structure:

W 096129337 PCTrUS96/03843 ~ (~
H >

--(cH2)nNi~o~

where n= 2-12, an ~d~ yl derivative having the structure:
~
~(CH2)n--where n = 2-12, a 1,2-di-O-alkyl-rac-3-glyceryl derivative having the structure:

~OR1 where R, is CnH2n,, and n = 2-22, or a DL a-tocopherol (Vitamin E) de~iv~Llive having the structure:

--(CH2)noJ~ l H3 CH3 CH3~ ,~(CH2)3CH(CH2)3CH(CH2)3CH(CH3)2 where n CH3 = 0-10.
In an oligonucleotide according to the invention having two or more S-triester internucleotide link~gPc, the R groups of di~, ~"L link~g~s may be the same or di~ llL.
In another plt;r~ d embodiment, R is chosen from the group coll~;~l;ll3~ of 1-~d~m~ntyl-2-ethyl, l-hPY~ecyl, and ~holpst~pryl-3-carbo~y~yamino-6-he~y~yl Oligonucleotides accolding to the invention are synth~.~i7ed via a precursor having the following structure:
-W O 96129337 PCTrUS96/03843 DMTO~ o B

O X
RO--P\
~N_<

where R is as described above, B is a suitably protected (if nece.e~,y) base, and X is H, F, benzyloxy, -OCH2CH=CH2, or a linear or b~ .hed alkyloxy moiety CnH2n+l, where n=1-5 Figure 10 1 displays the scheme for synthPei7inp these interm~ tes and representative protocols are provided in the F.Y~mrlçe, in~a In one aspect ofthis embodiment, the oligonucleotide will have from 1 to 10 S-triester pho~hol~Ll,ioate intemucleotide link~ges In a prt:re~ d embodiment, the oligonucleotides will have an S-triester ph~l,h--~oLl~ioate intemucleotide linkage at the 3' or 5' terminal link~ge, or at 15 both the 3' and 5' tf~rmin~l link~s Where an olig~nl-rleQtide ofthe invention has more than one S-triester link~g~7 all of the S-triester link~ges may have the same R substituent or they may all be d~ L
In d~Le~,ll.l ,.lg the number of S-triester link~ges and the identity of the R group substit~-ente to be incorporated into the oligonucleotides, the artisan will want to consider that 20 il.c.~:ased lipophilicity will result in de~ eased solubility in aqueous solution and steric interactions may change the conru..l.alion and affect hybridization By virtue ofthe new intemucleotide linl-~grs ~lierlosecl herein, the oligonucleotides of the present i--vellLioll exhibit i l, ~ased mlr~ ee rreiet~nre and, when having a bulky lipophilic group, exhibit subs~ lly increased cellular uptake relative to the corresponding phosphorothioate 25 internucleotide analogs while still ., ~i"l~ g the ability to specifically hybridize to a compl~ "~ ;. y target nucleic acid under normal stringency conditions Increases in melting temperature (T"~ of 5 ~C and more have been observed with a co~ ,onding increase in cellular uptake of 2 5 to 3 times that of the ph- sph~ rothioate analog As demonstrated below, it has been found that an increase in melting temperatures is observed even when large R moieties are 30 present, co..L.~y to the exrect~tinn that these large substih~rnts might interfere with hybri~li7~ti~n W 096129337 P~r/U~5G~ 843 The ~ ce oligc~nllcleotides of the present invention may be designP,d to incorporate a number of ~lc1ition~l feaLules that have been d~ dLed to hlcJlcase efficacy. For example, they may be dP~;gl~P~ to be "self-:il ;.l .;I;,P,A," i.e., having a first region s -ffic-iPntly cv~ ,lc-m~nt~3ry to a second region to allow for intr~mcnl~ll~r hy~ I;()n thereby rendering the olig~ mlc-leotide S less susceptible to nucleolytic attack. Such oligonucleotides are desclil,ed in PCT IllLel;.l;on~l ApplicationPublicationNo. WO 94/01550.
Alternatively, the plt;se"Lly disclosed oligonucleotides may be dPcignçcl to be "fold-back triplex rO",~ g," i.e., having a first region complpmpnt~ry to a target nucleic acid and a second region having a sP~lPnc-e that allows for triplex formation by Hoogsteen base pairing between it and the duplex formed by the first region and the target nucleic acid, as described in PCT
Intern~ticm~l ApplicationPublicationNo. WO 94/17091.
Oligonucleotides acco,ding to the invention are useful for both in vitro and in vivo applications. For in vitro applications, the present oligonucleotides are useful as,t;seal c h tools in d~ gene fimc~tic~n Because they can be prepared to be comple",e~Lal y to a particular sequence, the present oligc~m~clPotides can be used to selectively inhibit expression of a target gene. The present oligon~ çotides thus provide an attractive and easily used alternative to the laborious method of gene inhibition by mllt~tic n (e.g, deletion mutation). The signific~nce of this will be a~,~;dLed when one realizes that the elllc-~ tion of all biological pathways now known was deter nined by deletion mllt~tionc The oligonucleotides of the present invention are also useful as therapeutic agents for diseases or physiological conditions involving ~A~,es.,ion of specific genes. Oligonucleotides useful for L~eaLillg a disease or conc~iti(m will have a nucleotide sequence sufflciently co Fl Y to the target nucleic acid to bind under physiological conditions. As used herein, the terms "~c . ' ~ l y" and ''sllffic-iently compk~ y~ are used hlLe~ c ~ g~bly and, when used to describe the sequence of an ~ntict?nce oligonucleotide, mean that the oligonucleotide seqll~nc-.e is such that the oligonucleotide inhibits ~A~ll es~,ion of the target nucleic acid under the conc1itions of interest (e g, in vitro expe~i,,,c_,,Lal conditions and physiological conditions). In general, oligonucleotides acco,di,lg to the invention will have sequence complementary to a nucleic acid (e.g, a gene or mRNA) that is ess~nti~l to a biological process. As elaborated more fully below, such ~,ucesses include reproc~ncticnn and metabolic processes of pathogens and other disease-causing infectious agents. Or, the biological process can be a naturally occuring one W O 96~g337 PCTnUS96/03843 _9_ whose inhihition is desirable, e.g., ~cllllalogenesis in men and ovulation in women dçeiring contr~reption The oli~omlrleQtides of the invention can also be comrl~ y to a gene or other nucleic acid whose cA~Ies~;on causes or is involved in a tliee~ced or otherwise abnormal state ofthe or~,al~slll.
Rer~llee oftheir efflcacy at gene mo~ ticn the plt;s~llLly cl~imed oligonucleotides are also useful for treating tlic-?~erc arising from genetic abnormalities that cause under- or over-eA~JleS;~On of a gene. For diseases in which an al~ ., IAl gene is over~eA,ul ~ssed, for example, the presently el~imçcl oligonucleotides may be d~cign~d to target the abnormal gene directly, or, in the alternative, to target the gene encoding the protein that promotes CAI~1 t;s~ion of the abnormal 10 gene. Conversely, where an abnormal gene is under eA~lessed, one may design an oligonucleotide that suppresses expression of a gene encoding a protein that suppresses C~IJ1 es~ion of the abnormal gene.
In many cases the target nucleic acid sequence will be a virus nucleic acid sequence. The use of ~ ;e~l~ee oligonucleotides to inhibit various viruses is well known and has been reviewed 15 in Agrawal, Tibfech 10, 152 (1992). Viral nucleic acid sequences that hybridize to effective ~ntieenee oli~om-cleotides have been described for many viruses, inrll~fling human immllnod~ficienr.y virus type 1 (U.S. Patent No. 4,806,463), Herpes simplex virus (U.S. patent No. 4,689,320), Tnflllrn7~ virus (U.S. PatentNo. 5,194,428), and Human papilloma virus (Storey et al., Nucleic Acids Res. 19:4109-4114 (1991)). Sequences hybridizing to any ofthese nucleic 20 acid seq~lPnr~e can be used, as can nucleotide seq~l~nr~c comrlr~ llrl ~1~, y to nucleic acid sequences from any other virus. Additional viruses that have known nucleic acid sequences against which an ~ntieenee oligonucleotide accol.lillg to the invention can be prepared inr.llld~, but are not limited to, Foot and Mouth Disease Virus (See Robertson et al., J. Virology 54, 651 (1985);
Harris et al., J. Virology 36, 659 (1980)), Yellow Fever Virus (See Rice et al., Science 229, 726 25 (1985)), Varicella-Zoster Virus (See Davison and Scott, J. Gen. Virology 67, 2279 (1986), Cucumber Mosaic Virus (See Richards et al., Virology 89, 395 (1978)), Hepatitis B Virus (See Rane,v and ~ 'T .~rhl~n in Molecular Biology of Hepatitis B ~iirus (CRC Press, 1991)), Hepatitis C Vlrus (See Miller and Purcell, Proc. Natl. Acad. Sci. USA 87, 2057 (1990); Proc. Natl. Acad.
Sci. USA 89, 4942 (1992); J. General Virology 74, 661 (1993)), and Respitory Syncitial Virus 30 (See Collins, in Ihe Paramy~co Viruses, Chapter 4, pp. 103-162 (David W. Kingsbury, Ed., 1991)).

W 096/29337 PCTrUS96/03843 Alternatively, the oligonucleotides of the invention can have a nucleotide seq~lçnre t~t~ to a nucleic acid seq~lpnre of a p~thnE~nic organism. The nucleic acid sequences of many p~thn~enir. O~ ~ have been des-;l il,ed, inrlllrling the malaria Ol~ i ,lll, pl~rno~i~
fal.,-i~~, ~"., and many pathogenic b~ct~-ri~ F.Y~mrl~ of pathogenic eukaryotes having known S nucleic acid seq~nres against which olig~mlrleQtides of the present can be prepared inr.hld~, but are not limited to Trypanosoma brucei gambiense and Leishmania (See Campbell et al., Nature 311, 350 (1984)), and Fasciola hepa~ica (See Zurita et al., Proc. Natl. Acad. Sci. USA 84, 2340 (1987)). ~ntifimg~l oligonucleotides can be prepared having a nucleotide sequence that is comllt -~ y to a nudeic acid seq~ nre from, e.g, the chitin synth~-t~e gene, and ~ntihac.t~t ial 10 oligonucleotides accoldh~g to the invention can be prepared using, e.g, the alanine r~rem~ce gene.
In yet another embodiment, the oligonucleotides can have a nucleotide sequence c~ r~ ..ll;tly to a cellular gene or gene Ll~uls~ , the abnormal t;A~les~ion or product of which results in a disease state. The nucleic acid sequences of several such cellular genes have been 15 described, inr~ ling prion protein (Stahl and Prusiner, FASEB J. S, 2799 (1991)), the amyloid-like protein associated with ~l,l.~;.,,~,'S disease (U.S. Patent No. 5,015,570), and various well-known oncogenes and proto-oncogenes, such as c-myb, c-myc, c-abl, and n-ras.
In addition, oligonucleotides that inhibit the synthesis of structural proteins or enzymes involved largely or exclusively in ~ ,5 log~n~ , sperm motility, the binding of the sperm to the 20 egg or any other step ~ sperm viability may be used as contraceptives for men Similarly, contraceptives for women may be olignnnrleotides that inhibit production of proteins or enzymes involved in ovulation, fertilization, impl~nt~tion or in the biosynthesis of holmones involved in those processes Hypertension can be controlled by nligomlr.leotides that suppress the synthesis of angiotensin converting enzyme or related enzymes in the renin/angiotensin system; platelet 25 aggregation can be controlled by suppression of the synthesis of enzymes nece~ry for the synthesis of thromboxane A2 for use in myocardial and cerebral circulatory disorders, infarcts, arteriosclerosis, embolism and thrombosis; deposition of cholesterol in arterial wall can be inhibited by suppression ofthe synthesis offatty acyl co-enzyme A: çholestçrol acyl Ll~lsÇt;l~se in arteriosclerosis; inhibition of the ~yll~lle:iis of cholinephosph-)ll~lsrt;l~se may be useful in 30 hypolipiclr.mi~ There are numerous neural disorders in that oligonucleotides of the present invention can be used to reduce or ~.l;",;~ e adverse effects of the disorder. For example, W O 96/29337 PCTnUS96103843 ~u~les~ion of the synthesis of mc)n~ ~mine oxidase can be used in P~killson's ~1iee~ee;
s~p~,le~;on of r~terh~l O-methyl L~ sr~l~se can be used to treat depression; and su~ ion of indole N-methyl l~lsrt;l~se can be used in ll~;alillg s~,l~op~ellia. Suppression of selected el~yllles in the ar~ acid cascade (which leads to prost~gl~n~line and leukc~llienes) may be 5 useful in the control of platelet ag~G~alioll, allergy, infl~mm~tiQn, pain and asthma. Su~pression of the protein cA~lessed by the multi-drug rÇciet~nre (mdr) gene, which is le~oll~ le for development of re~iet~nr.e to a variety of anti-cancer drugs and is a major illlpe-l;...~,.l in chemotherapy may prove to be benefi~ in the tre~tment of cancer. Nucleotide sequences complementary to nucleic acid seq~lenre~s from any of these genes can be used for the 10 olignnllr.leoti~ e accold~lg to the invention, as can be oligonucleotide sequences complem~nt~ry to any other cellular gene or gene Llal~s~ L, the abnollllal C,~ ssion or product of which results in a disease state.
~ ntie-on.ee re~ ti~-n of gene c~ les:iion in plant cells has been described in U.S. Patent No. 5,107,065.
Since the nllrleotitle seq~lP.nre ofthe oli~mlr,leQtide can be adapted to form Watson-Crick base pairs with eesrnti~lly any gene, the thel~t;uLic spectrum of the oligonucleotides of the invention should be very broad. Still, certain rliee~ees are of particular interest. For eY~mple~ a variety of viral ~lieç~ees may be treated by oligonucleotides having one or more S-triester phosphorothioatçs internucleotide link~ge~e inr~ ing AIDS, ARC, oral or genital herpes, 20 papilloma warts, flu, foot and mouth disease, yellow fever, chicken pox, shingles, HTLV-le~lkemi~, and h~.p~titie Among fungal rliee~ee~e treatable by oligonucleotides according to the invention are ç~n~lirli~eie, histoplasmosis, cry-ptococcocis, blastomycosis, aspergillosis, sporotrichosis, ~,h~ulllolllycosis, delllaLophy-tosis and coccidioidomycosis. The method can also be used to treat rirL-e-ttci~ eç~es (e.g, typhus, Rocky Mountain spotted fever), as well as 25 sexually Ll;~ ---;lled diseases caused by Chlamydia ~rachoma~is orLymphogranuloma venereum.
A variety of parasitic ~liee~er~e can be treated by oligonucleotides of the present invention, inr,l~lrlin~ alll~;a~is, Chegas~ disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, and Pneumocysfis carini pneumonia; also worm (helminthic ~lieÇ~ees) such as ascariasis, filariasis, trichinosis, schistosomiasis and nematode or cestode 30 infections. Malaria can be treated by oligonllrleotides of the present invention, regardless of wllt:LIl~l it is caused byP. faL-i~u,u~, P. vivax, P. orale, or P. malariae. The infectious tlieÇ~ces i~lr.ntifiecl above can all be treated with oligon~lcleQtides having one or more S-triester pll~.Spllc,~ Qate link~e because the infectious agents for these tlieÇ~ees are known, and, thus, oligonucleotides accoldh~g to the invention can be plepaled having a nucleoticle seq ~nre that hybridizes to a nucleic acid seq~lpnre that is an eeeenti~l nucleic acid sequPnr,e for the prop~ tion S of the; - -r~l ;0~ ~5; agent, such as an ~ 1 gene. As used herein, an ç$ePnti~l gene or nucleic acid is one that is required for a biological process and without which the biological process does not occur.
The following eY~mrl~e are provided for illustrative purposes only and are not intPn~le~, nor should they be construed, as limiting the invention in any way.

EXAMPLES
The following is applicable to each of the examples presented below, unless indicated otherwise. Anhydrous ~cetonitrile, tetrahydrofuran, dichlorometh~nr, ethyl alcohol, 2-propanol, pentane, triethylamine, 1-hex~dec~nc-1, tetrazole, cholesteryl chlo~ aLe, 6-arnino-1-h~x~nol 15 bis(diisopropylamino)chlorophosphine and l-a~ toeth~n~l were purchased from Aldrich (Milwaukee, WI). ~.Y~ne, ethyl acetate, and meth~nol were purchased from J.T. Baker Inc.
(Phillip~11ulg, N.J.) 5'-DMT-deoxyadenosine (~BA) cyanoethyl phosphoramitlit~, 5'-DMT-deoxycytidine (~BA) cyanoethyl phosphor~mi(litç~ 5'-DMT-deoxyguanosine (~BA) cyanoethyl phosphor~mitlite, 5'-DMT-thymidine cyanoethyl phosphoramidite, Cap A, Cap B, 20 activator and C-18 SEP-PAK cartridge were purchased from Millipore (Bedford, MA).
Beaucage reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide) was purchased from R.I. Chemical (Orange, CA). Fluorescein-ON phos~,hc,l~lidite was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The reagents (Sequagel Sequ~onc~ing System: concentrate, diluent and buffer) used for PACE were purchased from National Diagnostics (Atlanta, GA). 3'P NMR
25 spectra (121.6~ MHz) were recorded on a Varian OEalo Alto, CA) UNITY 300 (the chemical shift was correlated to 85% H3PO4). Thermal melting data were collected from GBC 920 W-Vis spectrophotometer (Dandenong, Victoria 3175, Australia). Oligonucleotide synthesis was performed on a 8909 Expedite DNA synth~ei7:f!r (Mllipore). PAGE was carried out by using Model S2 seq~lPnring gel ele~ ,phc,le:~is ap~a~dL-Is (LIFE TECHNOLOGY, Gaithersburg, MD) 30 and EC600-90 power supply (E-C APPARATUS COPORATION, St. Petersburg, FL).
Fluoresc~nre was measured by Spectrofluorometer (PTI Technology, South Brunswick, N.J.).
The W al~sol~L~lce was measured W-160A W spectrometer (SHIMADZU, Columbia, MD).

-W 096/29337 PCTnUS96103843 Flow ~ J~ data was ac ~ d on Epics XL (Coulter, ~~ h FL). The plepdled-HPLC was performed on Waters (Milford, MA) 650E HPLC system with DYNAMX Model W-C
al~so~ ce deLe.,Lur. (Dyl~ lsLlulll~lll Co., Woburn, MA). Flash column chlc""aLography was carried out using silica gel 60 F254 (Merck, Gibbstown, N.J.) and t.l.c. on silica-gel 60 F2s4 - S (Merck). Products were vi~ li7ed on t.l.c. using either ultraviolet abso~lion at 264 nm or 5%
(NH4)6Mo,O24 and 0.2% Ce(SO4)2 in 5% H2SO4 (dark blue color for oxidizable compounds).

F.~
Synthesis of cholesteryl-3-carboxyamino-6 h.,~ ol Cholesteryl chloloro~ Le in CH2Cl2 (60 ml) was added dropwise at 0 ~C to a stirred solution of 6-amino-1-hexanol (2.40 g, 20.5 mmol) and triethylamine (3.6 ml, 2.6 g, 25.6 mmol) in CH2C12 (30 ml). The mixture was stirred overnight at room temperature. The reaction mixture was filtered to remove the resulting salt. Then 100 ml of CH2CI2 was added To the mixture. The solution was washed with saturated solutions of NaHCO3 and NaCI, dried over anhydrous Na2SO4, and filtered. The solvent was removed under reduced pressure to give a pale yellow solid crude product. This crude product was purified by flash silica gel chromatography (eluant:
CH2CI2/CH3OH 98:2) to obtain cholesteryl-3-carbo~y~lllillo-6-hexanol as a white solid (7.5 g, 80.1 % yield); TLC Rf 0.23 (CH2Cl2/CH3OH 95 :5).

FY~rl~ 2 Synthesis of N,N,N',N'-tetraisopropyl-O-et*yl-phosphorodiamidite Anhydrous ethanol (1.1 ml, 0.86 g, 18.7 mmol) in THF (4 ml) was added dropwise at 0 ~C to a stirred sollltion of bis(diiso~ ylamino)chlorophosphine (5.0 g, 18.7 mmol) and triethylamine (3.92 ml, 2.84 g, 28.1 mmol) in THF (19 ml). After stirring for 1 hour at room L~ ul~, the reaction mixture was filtered and the filtrate was conc~ L~d to an oil. The oil was dissolved in 15 ml of anhydrous pentane and filtered. The solvent was removed under the reduced plt:s:,ul ~;. The mixture was again dissolved in 15 ml of anhydrous pentane and filtered to remove the ~ g pre~ iL~Les. The solvent was evaporated under the reduced pressure to give O-ethyl-phosphortli~mitlite as a colorless oil (4.83 g, 93.3% yield); 31p NMR (CDCl3) ô 137.79.

W 096/29337 PCTrUS96/03843 ~ 3 SynfhesisofN,N,N N'-t~ v~l-O-isopropyl-~hc7~ )0rdiamidite In a manner similar to that presented in F.Y~mple 2, O-isopropyl-phosphor li~mirlite was ol~k illed as a colorless oil (4.00 g, 73.4% yield) by using bis(diisopropylamino)chlorophosphine S (5.0 g, 18.7 mmol), triethylamine (3.92 ml, 2.84 g, 28.1 mmol), anhydrous 2-propanol (1.43 ml, 1.13 g, 18.74mmol)andTHF(23 ml); 3IPNMR(CDC13) o 126.95.
F,-~mp'e 4 Synthesis of N,N,N',N'-tetraisopropyl-O-(l-adamantyl-2-ethyl) -phosphordiamiditeIn a manner similar to that presented in Example 2, O-(l-~ m~ntyl-2-ethyl)-10 phosphordiarnidite was obtained as a white solid (6.60 g, 86.3% yield) by usingbis(diisopropylamino)chlorophosphine (5.0 g, 18.7 mmol), triethylamine (3.92 ml, 2.84 g, 28.1 mmol), 1-~ eeth~n(ll (3.38 g, 18.74 mmol) and T~ (31 ml); 31p NMR (CDCl3) o136.68.

F~ S
Synthesis of N,N,N:N'-tetraisopropyl-O-(c*olesteryl-3-carboxyamino-6-hexyl)-phosphordiamidite In a manner similar to that ~, ~sellLed in Example 2, 20 0-(cholesteryl-3-carbo~y~llillo-6-hexyl)- phosphor ii~mic1ite was obtained as a colorless sticky oil (4.38 g, 98.4 % yield) by using bis(diisopropylamino)chlorophosphine (1.56 g, 5.84 mmol), triethylarnine (1.22 ml, 0.89 g, 8.76 mmol), cholesteryl-3-carboxyamino-6-hexanol (3.10 g, 5.84 mmol) and T~ (3.6 ml); 31p NMR (CDC13) o 136.17.

E_ample 6 Synthesis of N,N,N',N'-tetraiospropyl-O-(I-hexadecyl)-phosphordiamidite In a manner similar to that presented in Example 2, O-(1-hexadecyl)-phosphor~ mi~1ite was obtained as a colorless oil (7.82g, 88.1% yield) by using bis(diisoplo~yl~llillo)chlorophosphine (S.Og, 18.74 mmol), triethylamine (3.92 ml, 2.84 g, 28.1 30 mmol), 1-h~oY~1ec~n~1 (3.38 g, 18.74 mmol) and THF (29 ml); 31p NMR (CDC13) o 136.28.

W 096/29337 PCTnUS96/03843 -15_ F,Y~ P~7 Synthesis of S '-dimethoxytrityl-2' deoxythymidine-N,N-diisopropyl-O-ethyl-pho;.~h."~."~idite (5 '-DMT-dT-O-ethyl-phosphoramidite) O-ethyl-pho~hol-lih.,.;~lhe (1.02 g, 3.67 mmol) was added in one batch under nitrogen to a stirred suspension of 5'-DMT-T (1.0 g, 1. 84 mmol) in CH2CI2 (5.0 ml). A solution of tetrazole (0.129 g, 1.84 mmol) in act;LolliL-ile (4.7 ml) was added dropwise to the res-lltin~
mixture. A~er the mixture was stirred for 5 hours at room tel--pe-~ re, the solvent was removed at reduced p-es~u-e to give a foamy solid. The crude product was purified by flash column cl~l(,"~o{~raphy (eluant: CH2C12/EtOAc/Et3N 70:20:10) and pre~.ipit~ted from hexane at -78 ~C
to give 5'-DMT-dT-O- ethyl-pho~ lldite as a white foam (0.62 g, 47.0% yield): TLC Rf 0.66 (CH2CI2/EtOAc/NEt3 45:45:10); 31p NMR (CDC13) o 157.73, 158.52.

F,Y~PI~ 8 Synthesis of (5 '-dimet*oxytrityl-N4-(4tert-butylphenoxy)acetyl-2'-deoxycytidine-N,N-diisopropyl-O-ethyl-phosphoramidite ~5'-DMT-dC'BA-O-ethyl-phosphoramidite) O-ethyl-phosphortli~mitlite (1.71 g, 4.64 mmol) in CH2CI2 (9.7 ml) was added under nitrogen to a stirred pale yellow sl l~ ;on of 5'-DMT-dC~3A (2.25 g, 3 .08 mmol) in CH2CI2 ( 1 1.3 ml). A sol~ti~n oftt;Ll 1~ (0.216 g, 3.08 mmol) in acetonitrile (9.7 ml) was added dropwise to the resulting mixture. A~er the mixture was stirred for 5 hours at room ~elllpel ~ul e, the solvent was removed at reduced pressure to give a foamy solid. The crude product was purified by flash column ~,hlull~ographly (eluant: CH2CI2/EtOAc/Et3N 70:20:10) and precipitation from hexane at -78~C to give 5'-DMT-dCtBA-O-ethyl-phosphoramidite as a white foam (1.63 g, 58.5% yield):
TLCRfO.80 (CH2C12/EtOAc/NEt3 =45:45:10); 31p NMR (CDCl3) o 158 20, 159.08.

~,Y~ 9 Synthesis of 5'-dimethoxytrityl-2'-deoxy~hymidine-N,N-diisopropyl-O-isopropyl-pho~h~ ".idite (5'-DMT-dT-O-isopropyl-phosphoramidite) O-isopropyl-phosphor~i~mi~lite (0.80 g, 2.8 mmol) in CH2CI2 (2.0 ml) was added under nitrogen to a stirred suspension of 5'-DMT-T (1.0 g, 1.9 mmol) in CH2CI2 (5.0 ml). A solution ~ of tetrazole (0.129 g, 1.84 mlnol) in ~c~o.,;l~ile (5.7 ml) was added to the rf~ lting llli~Lult;.
After the mixture was stirred for 5 hours at room temperature, the solvent was removed at 35 reduced ples~ult; to give a foamy solid. The crude product was purified by flash column W 096/29337 PCTrUS96/03843 cll,.... ~ ~lo~raphy (eluant: CH2CI2/EtOAc/Et3N 70:20:10) and pl e~ (e~ frorn hexane at -78~C
to give 5'-DMT-dT-O- isoplu~yl-phos~ . ~nidite as a white foam (0.66 g,48.7% yield): TLC
RfO.70(CH2Cl2/EtOAC/NEt345:45:l0), 31p NMR (CDCl3) 8 156.63,157.14.

F~ 10 Synthesis of S '-dimethoxytriyl-N~-(4-tert-butylphenoxy)acetyl-2'-deoxycyfidine-N,N-diisopropyl-O-isopropyl-pho.,~hv,.~"~idite fS'-DMT-dC~A-O-isopropyl-pkosp*oramidite) O-iso~.upyl-phosphordi~mi~lite (0.60 g, 2.06 rnrnol) in CH2Cl2 (2.0 ml) was added to a stirred pale yellow :iu:,~,el,sion of 5'-DMT-dC'BA (1.0 g, 1.37 rnrnol) in CH2Cl2 (5.0 rnl) under nitrogen. To the resulting mixture was added dropwise a solutioin of tetrazole (0.096 g, 1.37 rnmol) in ~ ol ,;l . ;le (4.3 rnl). Af~er the rnixture was stirred for 5 hours at roorn t~ dLllre' the solvent was removed at reduced pressure to give a foamy solid. The crude product was purified by flash column cl~ol"dlography (eluant:CH2CI2/EtOAc/Et3N 70:20:10) and pre~ipit~tion from hexane at -78 ~C to give 5'-DMT-dC~3A-O-isopropyl-phosphoramidite as a white foam (0.84 g, 67.1% yield): TLC Rf O.75 (CH2C12/EtOAc/NEt3 45:45:10); 31p NMR (CDCl3) o 157.24, 157.56.

m~
Synthesis of S '-dimethoxytrityl-2'-deoxythymidine-N,N-diisopropyl-O-(I-adclmantyl-2-ethyl)-phosphoramidite ~5 '-DMT-dT-O-I-adamantyl-2-ethyl)-phosphoramidite) O-(l-~",~.,lyl-2-ethyl)-phosphordi~mi~ite (1.14 g, 2.76 mmol) in CH2CI2 (5.7 ml) was added under nitrogen to a stirred suspension of S'-DMT-dT (1.0 g, 1.8 mmol) in CH2CI2 (5.0 ml).
A solution oftetrazole (0.129 g, 1.84 mmol) in act;Lonil,ile (5.7 ml) was added dropwise to the resulting mixture. After the mixture was stirred for 5 hours at room temperature, the solvent was 25 removed at reduced pressure to give a foamy solid. The crude product was purified by flash column cl~,ulll~Lography (eluant: CH2C12/EtOAc/Et3N 70:20:10) and precipit~ted from hexane at -78~C to give 5'-DMT-dT-O-(l-~ m~ntyl-2-ethyl)-phosphoramidite as a white foam (0. 86 g, 54.7% yield): TLC Rf 0.65 (CH~C12/EtOAc/NEt., 45:45:10); 31p NMR ~CDC~ 157.93, 158.3 1.

W 096/29337 ~ 3843 Synthesis of 5 '-dimethoxytrityl-N4-(4-ter~-butylphenoxy)acetyl-2 '-deoxycytidine-N,N-diisopropyl-O-(I-adamantyl-2-ethylJ-~Ios~oramidite (5 '-DMT- dC~A-O-(l -adamantyl-2-ethyl)-pho~,c/, ~".idite) O-(~ yl-2-ethyl)-ph~sphc)rtli~mirlite (0.85 g, 2.06 mmol) in CH2Cl2 (4.3 ml) was added under nitrogen to a stirred pale yellow suspension of 5'-DMT-dC~'A (1.0 g, 1.4 mmol) in CH2Cl2 (5.0 ml). A solution oftetrazole (0.096 g, 1.37 mmol) in ~c~lo~ e (4.3 ml) was added dlu~lse to the resulting mixture. After the mixture was stirred for 5 hours at room temperature, 10 the solvent was removed at reduced pressure to give a foamy solid. The crude product was purified by flash column .,hl~ la~ography (eluant: CH2CI2/EtOActEt3N 70:20:10) and pre~ iL~Led from hexane at -78~C to give S~-DMT-dctBA-o-(l-~ lyl-2-ethyl)-phosphoramidite as a white foam (0.83 g, 58.6%, yield): TLC Rf 0.63 (CH2CI2/EtOAc/NEt3 45:45:10); 31p NMR (CDCI3) o 158.35, 158.67.
15F,-s~ p'~ 13 Synthesis of S 'dimethoxytrityl-2'-deoxythymidine-N,N-diisopropyl-O-~cholesteryl-carboxyamino-6-hexyl) -phosphoramidite (5'-DMT-dT-O-~cholesteryl-3-carboxyamino-6-hexyl)-phosphoramidite) O_(rhrlP~tpryl-3-carboxyamino-6-hexyl)-pho~llol~ te (2.1 g, 2.76 mmol) in CH2CI2 (11.9 ml) was added under nitrogen to a stirred suspension of 5'-DMT-T (1.0 g, 1.84 mmol) in CH2CI2 (5.0 ml). A solution oftetrazole (0.129 g, 1.84 mmol) in ~cetonitrile (7.5 ml) was added dropwise to the resulting mixture. After the mixture was stirred for 5 hours at room temperature, - the solvent was removed at reduced ~ ul ~ to give a white foamy solid. The crude product was 25 purified by flash column cl~c,l-l~Lography (eluant: CH2CI2/EtOAc/Etl~ 70:20:10) to give 5'-DMT-dT-O(cholesteryl-3-carbo~ycullil1o-6-hexyl)-phosphoramidite as a white foam (2.11 g, 95.2% yield): TLC Rf0.60 (CH2CI2/EtOActNEt3 45:45:10); 3'P NMR (CDCl3) o 158.60, 158.82.

W 096/2~337 PCTrUS96/03843 F, ~ 14 Synthesis of 5 '-dimethoxytrityl-N~-(4tert-butylphenoxy)acetyl-2 '-deoxycytidine-N,N-diisopropyl-O-(cholesteryl-3-car~oxyamino-6-hexyl)-pho~h.J,~".idite (5'-DMT-dC~A-O-(cholesteryl-3-carboxyamino-6-hexyl)-phosphoramidite) O-(cholesteryl-3-carbu~yal~ o-6-hexyl)-phosphor~ mi~lite (2.35 g, 3.09 mmol) in CH2CI2 (13.1 ml) was added under nitrogen to a stirred pale yellow suspension of S'-DMT-dCb'' (l.S g, 2.06 mmol) in CH2C12 (7.5 ml). A solution of tetrazole (0.144 g, 2.06 mmol) in ac~ ;le (8.4 ml) was added dropwise to the resulting mixture. After the rnixture was stirred 10 for 5 hours at room temperature, the solvent was removed at reduced pressure to give a foamy solid. The crude product was purified by flash column chromatography (eluant:
CH2 Cl2 /EtOAc/Et3 N 70:20: 1 O) to give S'-DMT-dC'~A-O-(~.holçsttoryl 3-carbuxydll~ino 6 hexyl)-phosphoramidite as a white foam ( 2.58 g, 90.4% yield):
TLC Rf 0.63 (CH2C12/EtOAc/NEt3 45:45:10); 31p NMR (CDC13) a 158.93, 159.25.
F.-~mll!e 15 Synthesis of 5'-dimethoxytrityl-2'-deoxythymidine-N,N-diisopropyl-O-(I-hexadecyl)-phosphoramidite (5'-DMT-dT-O-(I-hexadecyl)-phosphoramidite) O-(1-h~x~ecyl)-phosphortli~mi-lite (1.31 g, 2.76 mmol) in CH2CI2 (5.7 ml) was added 20 under nitrogen to a stirred suspension of S'-DMT-T (1.0 g, 1.84 mmol) in CH2CI2 (5.0 ml). A
solution of tetrazole (0.129 g, 1.84 mmol) in a~etonitrile (5.7 ml) was added dropwise to the resulting mixture. Af'cer the mixture was stirred for S hours at room temperature, the solvent was removed at reduced pressure to give a foamy solid. The crude product was purified by flash column cl..~,ll.d~ography (eluant: CH.CI2/EtOAc/Et3N 70:20: 10) to giveS'-DMT-dT-O-(1-h~x~(lecyl)-phos-phoramidite as a colorless sticky oil (1.65 g, 98.0% yield):
TLCRfO.63 (CH2C12/EtOAc/NEt345:45:10); 3IPNMR(CDC13) o 159.00, 158.32.

W 096/29337 PCTnUS96/03843 F ~~ .'~16 Synthesis of 5'-dimethoxytrityl-N'-(4 -tert-butylphenoxy)acetyl-2'-deoxycytidine-N,N-diisopropyl-O-(I -hexadecyl)-phG ,f ,~l v, ~".idite ~ (5'-DMT- dC~A-O-(l-hexadecyl)-pho~ ,,",.idite) O-(l-h~x~-lecyl)-phosphorli~m;~lite (0.97 g, 2.06 mmol) in CH2Clz (4.3 ml) undernitrogen was added under nitrogen to a stirred pale yellow suspension of 5'-DMT-dC~3A (1.0 g, 1.37 mmol) in CH2C12 (5.0 ml). A solution of tetrazole (0.096 g, 1.37 mmol) in acetonitrile (4.3 ml) was added dropwise to the resllltin~ mixture. After the ~ lul~ was stirred for S hours at 10 room te~ ,cl ~.L lre, the solvent was removed at reduced pressure to give a foamy solid. The crude product was purified by flash column chromatography (eluant: CH2Cl2/EtOAc/Et3N 70:20: 10) to give 5'-DMT-dCt~3A-O-(l-h~Y~-lecyl)-phosphoramidite as a pale yellow sticky oil (1.41 g, 93.5%
yield): TLC Rf 0.62 (CH2C12/EtOAc/NEt3 45:45:10); 31P NMR (CDC13) o 158.71, 159.47.

FY~ .'e17 Synthesis of O-alkyl-phosphoramidite The ~y~L}~~es of a number of O-allcyl-phrsp~ it~c have been reported, which include methyl, ethyl, trifluoroethyl, isopropyl and neopentyl derivat*es. Koziolkiewicz and Wilk in Protocols for Oligonucleotides and Analogs, supra, pp. 207-224, and references cited therein.
20 In a similar way, the O-alkyl-phosphorcli~mitlites were p~ epal ed by the reaction of bis(diisopropylamino)chlorophc sphin~ with the alcohols. In purification of the O-alkylphosphor~mitlitec, vacuum ~lictill~tion at rather high tt;.llpe.dLLIre was replaced by the l~eaLed filtration of the corresponding pentane solution to avoid the possible decomposition of the product. The phncrhitylation of the nucleosides by the res-llting O-alkyl-phosphordi~mitlit~c 25 gave the O-alkyl-phosphor~mi~itPs in good to excellent yield. The synthesis of the O-alkyl-phosphor~mi-lites is clepicted in Figure 1.

W 096/29337 PCTnUS96103843 -20-~ mp'~ 18 Synfhesis of S-friester-p*osphorothioate and O-friester-phosphofriester oligonucleotides The S-triester-phosphnrothioate and O-triester-phosphodiester oligonucleotides were synthesi7ed using the phosphor~mitlites of Example 17 protected by the base labile tert-S bulyl~ y~cetyl (tBA) group on the exocyclic amine (dA, dC, and dG). The increased liabilityof tBA group over the standard benzoyl and isobutyryl protection permitted use of rnilder condition for dep,ule-;Lion of the bases and release from the solid support with ~,-",onia hydroxide (room temperature, 2 hours), which was of crucial importance in the ~y"Ll~esis of the O-ethyl Co~ lg oligom~cleotides Oligonucleotides were purified by reverse-phase HPLC
10 and/or PAGE. The 31p NMR spectrum of the S-triester-phosphorothioate compound no. 1.1 showed that the corresponding peaks for S-triester-phosphorothioate and S-phosphorothioate eotide link~c appear at 62 and 53 ppm respectively. The ratio between S-triester and phosphorothioate was 19.8 :100.0 (The calculated value is 20:100). The content of O-phosphodiester link~gec was shown to be less than 2.5 percent.

~,,~mpl~ 19 Synfhesis andpurification of oligonucleotides All of unmodified or triester co,.l~ g phosphorothioate and phosphodiester olig n-~rleotides were syntheci7ed on a 1 ~Lmol scale following the standard protocol by using an 20 ~nlul..nled :jylllh~ (Millipore 8909 Expedite, Bedford, MA). The modified phosphor~mi~lites, S'-DMT-dT-O-ethyl-phosphoramidite, S'-DMT-dCtBA-O-ethyl-phosphoramidite, 5'-DMT-dT-O-iso-propyl-phosphor~mi~ite, and 5'-DMT-dC'BA-O-isopropyl-phosphoramidite were dissolved in dry ~retonitnile at a concel,L,~Lion of 50 mg/ml, and the others, S'-DMT-dT-O-(l-adamantyl-2-ethyl)- phosphoramidite, S'-DMT-dC'BA_ 25 adamantyl-2-ethyl)-phosphoramidite, S'-DMT-dT-O-(cholesteryl-3-carboxy~."i"o-6-hexyl)-phosphor~mti~1ite, 5'-DMT-dC'BA-O-(cholesteryl-3-carboxyamino-6-hexyl)-phosphor~mi~lite, Sl-DMT-dT-o-(l-he~ ecyl)-phosphoramiditç~ and S'-DMT-dC'BA-O-(l-h~Y~clecyl)-phosphoramidite were dissolved in dichlol~ h~l~e and diluted with acetonitrile to acetonitrile-dichlo~ h~ 1) at a final con~entration of 50 mg/ml. For 30 phnspht rothioate c-ligon~l~leotides, the iodine oYid~tion step was replaced by sulfurization with 3H-1,2-benzodithiol-3-one-1,1-dioxide (Be~l-c~e reagent). Iyer et al., J. Org Chem 55, 4693 (1990). Two-hour Il~Ll~ with s.. ~l~i.lm hydroxide at room temperature was carried out to PCTrUS96/03843 cleave the oli~ om the support and to deprotect nucleoside bases. Oligonucleotides were purified by reverse-phase HPLC and/or PAGE, and des~l~ecl by using C- 18 SEP-PAK cartridges.

Flu." ~s~ labeling of oligonucleotides PIUol't;~;eell~ was conj~ ted to the 5' end of the oli~onlleleotides by either an ~lltom~tecl DNA synth~ei7~r or by a manual procedure using a FLUORESCEIN-ONTM phosphor~mi~lite The effis~ ney of fluol~sceil~ l~helin~ was determined by using a spectrofluorometer (excitation 488 nm, emission 520 nm).
Esample 20 Exon~olen~e resistance of triester containing oligonucleotides Sensitivity to 3'-exomlcle~e degradation was measured by digestion with T4 DNA
polymerase and/or DNA polymerase I. The oligonucleotides tested are listed in Table 1.

Table 1 No. Sc~l~c-.ce' MO~Ijfi~.~tjOnb 1.1CTC TCG CAC CCA TCT CTC TCC TTC T --1 4CCTC TCG CAC CCA TCT CTC TCC TTC T Rl 1.6CTC TCG CAC CCA TCT CTC TCC TTC T R2 1.8CTC TCG CAC CCA TCT CTC TCC TTC T R3 1.10CTC TCG CAC CCA TCT CTC TCC _TC T R3 1.12CTC TCG CAC CCA TCT CTC TCC TTC T R3 1.15CTC TCG CAC CCA TCT CTC TCC TTC T R4 1.17CTC TCG CAC CCA TCT CTC TCC TTC T R5 25 ' All CU11I~JUU~ are oligùdc~)~lib.~ r..~ f c and are presented in the S' to 3' direction, lei~ to right; highligh n--rlF oti~lF e have S-triester linkages on the 3 ' side, except as otherwise in~lir~t~
~ b R~=ethyl; R2 ,~ lu~l, R3-l-adall~l1~1-2-ethyl; R4=Cholesteryl-3-carbuA~ 0-6-hexyl; R5=1-h~.A~-,yl, where one R group is listed, that R group is in each S-triester or O-triester linkage; where there are two or more R groups - listed, each R group ~1l~,;,~ll~ to one S-triester or O-triester linkage, with the first listed R group being in the S '-most S-triester or O-triester linkage and so on so that the last R group listed Cull~ JulldS to the 3'-most S-triester or O-triester linkage.
C ~i~hli~ L~ F ~ ha ~e O-triester linkages on the 3 ' side.

W O 96~9337 PCTrUS96/03843 To study the rçci~t~n-;e of T4 DNA polymerase, 5'-32P-labeled oligon-~rleQtides (45 pmole) were dissolved in 30 1ll of buffer (50 mM Tris, pH 8.0, MgCI2, 5 mM DTT, 0.05% BSA) and ~ b~ed with T4 DNA polymerase (7.5 units) at 37~C . Ali~uots (5 !11), inhibited by 6 ~ll 5 of the stop solution (95% ro~ ;de~ Io mM EDTA, 0.05% brolllGphenol blue, 0.05% Yylene cyanol), were removed at 0 and 60 mim1tç~ analyzed by PAGE (20% polyacrylarnide cn.,l;.i";"g 8.3 M urea), and followed by autoradiography. The results are shown in Fig. 2.
To study the l~ ln".-e of DNA polyllwlase I, 5~ 32p labeled oligonucleotides (30 pmole) were dissolved in 20 ~11 of buffer (50 mM Tris, pH 8.0, MgCI2, 5 mM DTT, 0.05% BSA) and 10 inc~b~ted with DNA polymerase I (5.0 units) at 37 ~C. Aliquots (5 1ll), inhibited by 6 ,ul of the stop solntic-n (95% r~.""~." ~ lo mMEDTA, 0.05% bromophenol blue, 0.05% xylene cyanol), were removed at 0, 60, 120, 180 and 240 mimltç~, analyzed by PAGE (20% polyacrylarnide cO,.n~ ;"g 8.3 M urea), and followed by autoradiography. Typical results are shown in Fig. 3.
The results clearly inriic~te that the modified S-triester-phosphorothioate is much more 15 stable than the unnodified S-phosphorothioate. Fig. 4 The ~yom~rle~e re~i~t~n~e of other modified S-triester-phosphorothioates and O-triester-phosphodiesters oligonucleotide was also studied by using T4 polymerase (Fig. 2). It is seen that not only the modified S-triester-phospl1oluLl~oate but also the O-triester-phosphodiester oligonucleotides have increased eYon--çlç~ce rP~ict~n-.e. This is important because the S-triester-phosphorothioate may convert 20 to O-triester-phosphodiester in cells (Wyrzykiewicz and Cole, Nucleic Acids Res. 22, 2667 (1994)), and the increased per~iet.on~.e of O-triester-phosphodiester would provide a longer duration of action in vivo.

W 096/29337 PCTnUS96/03843 Example 21 Melting T~".~,~.t~res (T"J
Melting te~ )e~ res were ~lt;lt;~ ...;.~d for the duplexes of unmodified and modified oligonucleotides with the complf!m~nt~ry DNA. ~ach oligonucleotide (0.2 A260 Units) and its 5 complçm~nt~ry DNA was ~nne~led in 1 ml buffer (10 mM Na2HPO4, pH 7.4, 0.1 M NaCl) by heating to 80 ~C and then cooling down to 40 ~C at a rate of 2 ~C/minute. The llli~lul ~ was then rt;he~Led to 80 ~C at a rate of 1~ C/minute and the A260 was continuously recorded. Melting profiles were obtained for oli~onllcleotides.
The results are presented in Table 2. Changes in affinity can be attributed directly to the 10 morlific~tiQn. Modification with triester linkage(s) at the 3' end and/or the 5' end of the S-phosphorothioate reduces the total negative charge of the oligonucleotide, which would be expected to ~nh~nce hybridization. As Table 2 shows, almost all of the modified S-triester-phosphorothio~tes have increased Tm col-lp~ed to the unmodified phosphorothioate (compound no. 1.0). The only exception is a chimeric oligomer (compound no. 1.2) modified at 15 both of the 3' and 5' ends with four ethyl triester link~ge,e The decreased Tm inrlic~te~e that the motlifit~.~ticn has caused c.l-~l-P~s in the geomP,try ofthe internucleotide bond. It is also shown that the melting le-..~ Lul ~s of O-triester-phosphodiesters are about 10 degrees higher than that ofthe corresponding S-triester-phosphorothioates, which is coneiet~nt with the results obtained from the unmodified oligon~ otides. Uhlmann and Peyman, supra; and Met*ods in Molecular 20 Biology 20: Protocols for Oligonucleotides and Analogs, supra.

Table 2 Melting T~".~, u~res of Various Modifications of SEQ ID NO I

No. Sc~ll ee~ Modificationb Tm(~C) 1.0 CTC TCG CAC CCA TCT CTC TCC TTC T -- 52.2 1.1 CTC TCG CAC CCA TCT CTC TCC TTC T Rl 56.8 W 096t29337 PCTtUS96tO3843 Tablc2(cont.) No. Sc~. _; Mo~ tionb Tm(~C) 1.2CTC TCG CAC CCA TCT CTC TCC TTC T R, 51.4 1.3CTC TCG CAC CCA TCT CTC TCC TTC T Rl 55.6 1.4CCTC TCG CAC CCA TCT CTC TCC TTC T Rl 65.8 .5cCTC TCG CAC CCA TCT CTC TCC TTC T Rl 65.4 1.6CTC TCG CAC CCA TCT CTC TCC TTC T R2 555 1.7CTC TCG CAC CCA TCT CTC TCC TTC T R2 54.2 1.8CTC TCG CAC CCA TCT CTC TCC TTC T R3 55.2 1.9CTC TCG CAC CCA TCT CTC TCC TTC T R3 54.1 1.10CTC TCG CAC CCA TCT CTC TCC _TC T R3 55.9 1.11CTC TCG CAC CCA TCT CTC TCC T_C T R3 57.0 1.12CTC TCG CAC CCA TCT CTC TCC TTC T R3 56.8 1.13CTC TCG CAC CCA TCT CTC TCC TTC T R3 55.8 l~i1.14CTC TCG CAC CCA TCT CTC TCC T_C T R3 SS.o 1.15CTC TCG CAC CCA TCT CTC TCC TTC T R4 57.5 1.16CTC TCG CAC CCA TCT CTC TCC TTC T R4 57.2 W 096129337 PCT/U~5~ 843 Table2(cont.) No. Sc~ ' Mo~lifi~7~ti~nb Tm(~C) 1.17CTC TCG CAC CCA TCT CTC TCC TTC T R5 56.1 1.18CTC TCG CAC CCA TCT CTC TCC T_C T R5 56.8 51.19CTC TCG CAC CCA TCT CTC TCC TTC T R5 54.4 1.20CTC TCG CAC CCA TCT CTC TCC T_C T R5 54.1 1.21C_C TCG CAC CCA TCT CTC TCC TTC T R" R4 53.6 1.22CTC TCG CAC CCA TCT CTC TCC TTC T Rl, R4 57.1 1.23CTC TCG CAC CCA TCT CTC _CC TTC T R" Rl, R4 55.2 101.24CTC TCG CAC CCA_CT CTC TCC TTC T R" Rl, R4 55.2 1.25CTC TCG CAC CCA TCT CTC TCC TTC T R" R" R4 56.4 1.26CTC TCG CAC CCA TCT CTC TCC _TC T R" R" R4 53.6 1.27'CTC TCG CAC CCA TCT CTC TCC TTC T R" R4 65.7 1.28'CTC TCG CAC CCA TCT CTC TCC TTC T R" R4 66.8 15l.29CCTC TCG CAC CCA TCT CTC TCC TTC T R5 66.0 1.30CTC TCG CAC CCA TCT CTC TCC TTC T R, 56.3 1.31CTC TCG CAC CCA TCT CTC TCC TTC T Rs 54.5 1.32CTC TCG CAC CCA TCT CTC TCC TTC T R5, Rl 52.5 W 096/29337 PC~rUS96103843 All f.'""l'U"'~'l' are f~ lf~y~ ;fl~ ~ and are ~ i7~ t~1 in the 5' to 3' direction, left to right; hi~hli~
""f 1. ~JI ;~lf ~ have an S-triester linkage at the 3 ' side, except as b R~=ethyl;R2-i~u~lu~l, R3=l-ad.~..l~Lyl-2-ethyl; R4=c~hrl yl-3-c~l,uA~ lo-6-hexyl; R5=l-h~A~f~yl; where 5 f ne R group is listed, th-at R group is in each S-triester or O-triester linkage; where there are two or more R groups listed, each R group CU11~;7IJU11~1S to one S-triester or O-triester linkage, with the first listed R group being in the 5'-most S-triester or O-t~iester linkage and so on so that the last R group listed .,ul 1 ~u~ds to the 3 ' -most S-triester or O-triester linkage.
C ~i~hli~htf-d mlrl~otifi-~ have an O-triestcr linkage on the 3' side.

F,~m~!~22 Cellular Uptake Cell culture: Human T cell and leukf-mi~ cell line H9 were used in the study. They were cultured in RPMI media sup~ ed with 10% fetal bovine serum (heat inactivated to 56 ~C for 15 30 minlltes to inactivate the nllrJP!~es), 2 mM glllt~min~, 100 ,ul streptomycin, 100 U/mL
penicillin and 6 x 10-5 M of 2-mercaptoethanol in an air inr~lb~tor (37D C, hl~mic1ifird by 5%
CO2-95% ~2) Cell uptake: The col~r~ Lions ofthe fluorescein labeled and unlabelled oligonucleotides in the samples were measured by a :,~e~;Llun~lo~ "ele~ and W spectroscopy, and adjusted to be 20 same by adding the corresponding unlabelled oligonucleotides. Oligonucleotide complexes (0.2 OD/100 111) were added to the cells (5 x 106 cells/ml, 0.5 ml) and set to culture. After 4 hours of culture, aliquots of cell culture mixtures were removed, washed, and resuspended in Hank's b~l~nr,ed salt solution (HBSS) supplrmlonted with 0.1 % BSA and 0.1 % sodium azide.
Propidium iodide (final concentration 10 ~l/ml) was used to rli~tin~ h viable cells from dead 25 cells. Flow ~,yl ~" "~l l ;c data on 5,000 viable cell was acquired in list mode on Epics XL (Coulter, Hialeah, FL), and data were analyzed by Epics XL (version 1.5 sonw~le) after gating on living cells by rc,l w~d scatter versus side scatter and propidium iodide st~ining The results are presented in Table 3. It is seen that the S-triester-phosphorothioates modified with a large lipophilic group (e.g., ~ m~ntyl-2-ethyl, 30 cholesteryl-3-carbo~y~ ino-6-hexyl and l-hexadecyl) have ~nh~nred cellular uptake.

PCTnUS96/03843 Table 3 Cellular Uptake of SEQ ID No. I

No. Sc~ Modificationb Uptake' 1.0 CTC TCG CAC CCA TCT CTC TCC TTC T -- 7 4 1.1 CTC TCG CAC CCA TCT CTC TCC TTC T R, 11.4 1.3 CTC TCG CAC CCA TCT CTC TCC TTC T Rl 5.7 1.6 CTC TCG CAC CCA TCT CTC TCC TTC T R2 7-9 1.8 CTC TCG CAC CCA TCT CTC TCC TTC T R3 14.1 1.12 CTC TCG CAC CCA TCT CTC TCC TTC T R3 1 1.6 1.15 CTC TCG CAC CCA TCT CTC TCC TTC T R4 19.9 1.17 CTC TCG CAC CCA TCT CTC TCC TTC T Rs 19.8 8 All c~ u---~ are .~ y~ and are presented in the 5' tû 3' direction, left to right; highlightt-~l m~ oti~ e have an S-triester linkage at the 3' side.
b Each of the S-triester linkages have the indicated R group, where R,=ethyl, R2--i~V~l U}Jyl~ R3= l -adcu.laLyl-2-ethyl, R4=(~hr' yl-3-c.ulJ~ lllo-6-hexyl, and R5=l-h.,.~e~,yl.
c Mean FITC nuul~,;~ l~.

Esample 23 Inhibition of CMV Gene Expression in Cell Culture A series of ml-~1ifircl CMV oli~on~lrleotides were synthrei7ed according to the invention.
These oli~ n~cleotides are ~lepicted in Table 4.

PCTnUS96/03843 Table4 Modification of SEQ ID NO 2 Used to Inhibit CMY Gene Expression No. Se~ ~' Mo~ifi~ -- b t 2.0TGG GGC TTA CCT TGC GAA CA R3 2.1TGG GGC TTA CCT TGC GAA CA Rl, R3 22cTGG GGC TTA CCT TGC GAA CA R3 23c_GG GGC TTA CCT TGC GAA CA Rl, R3 2.4TGG GGC TTA CCT TGC GAA CA Rl 2.5TGG GGC TTA CCT TGC GAA CA Rz 2.6TGG GGC TTA CCT TGC GAA CA Rl, R2 2.7TGG GGC TTA CCT TGC GAA CA R4 2.8TGG GGC TTA CCT TGC GAA CA Rl, R4 2.9TGG GGC TTA CCT TGC GAA CA R, 2l0cTGG GGC TTA CCT TGC GAA CA Rl,R4 2llcTGG GGC TTA CCT TGC GAA CA R4 2l2cTGG GGC TTA CCT TGC GAA CA R, 2l3CTGG GGC TTA CCT TGC GAA CA R, ' All c~ are ~ A~ y~ tiAP~ and are presented in the S' to 3' direction, lefl to right; hiEhliEhtl-d 20 .. ~ r'O~ '; have S-triester linkages on the 3 ' side, except as uLIIc;l w;~c in~linof~
b Rl=ethyl; Rz=l-~l~lLyl-2-ethyl, R3=~l--' yl-3-c~ u~ -o-6-hexyl; Rl=l-hexadecyl; where one R group is listed, that R group is in each S-triester or O-triester linkage; where there are two or more R groups listed, each R group ~~Ull~JUllJ:~ to one S-triester or O-triester linkage, with the first listed R group being in the S '-most S-triester or O-triester linkage and so on so that the last R group listed Cull~ ulld:~ to the 3'-most S-triester or O-triester 25 linkage.
C ~j~li"' ' ' .. 1. ~.1;~1~ ~ have O-triester linkages on the 3' side.
The s~~ e ~f, ' ~e d in Table 4, 5'-TGGGGCTTACCTTGCGAACA-3'(~L36 ANTI), is comr~ .y to the intron-exon boundary of UL36/37 and to the control sense 30 t~ omlcleoti~le 5'-TGTTCGCAAGGTAAGCCCCA-3', which is homologous to the UL36 mRNA These oligonucleotides co~ s~olld to HCMV genomic colldinales49,565 to 49,584.

W 096~9337 PCTnUS96/03843 Smith and Pari, J. Virology 69, 1925 (1995). In addition, two other compounds were used as controls in the cA~clilllt;llL~ described below: 5'-TCTGGGTAATTACACGCAAGC-3', anunrelated nonspecific control o1i~n-1rleotide, and 5'-ACAAGCGTTCCATTCGGGGT-3', al.o~ e control oli~nllrlrotide that is the same nucleotide sequence as UL36 ANTI, except in 5 reverse order.
Human folc;sLill fil,l~la~L~ (HFF) were plated at a density of 5,000 cells per well in a 96-well microtiter plate (Falcon, Franklin Lakes, N.J.) for 24 hr prior to tre~tm~nt Cells were prellt;aLed with ~nti~rn~e oli~onlleleotides at the various in~lir,~ted concentrations in CCM5 m~ illm (Hyclone, Logan, Utah) for up to 15 hr. The growth m~lillm was then removed, and the 10 cells were washed three times with Dulbecco's phosphate-buffered saline to remove any residual oligonucleotide. The cells were inr~lb~tecl with HCMV strain AD169 (ATCC VR-538) at a ml ~ ;ly of infection (MOI) of 0.1 for 1 hr at 37~C. Cells were washed again and refed in fresh growth medillm co"~ g serial dilutions of the ~nti~l?n~e oligonucleotide at the same concentrations used in the prrinr.~-b~tion. At 5 to 6 days po~,l;"recl;on (p.i.), cells were fixed 15 (100% ethanol) and reacted with a primary antibody specific for the HCMV UL44 gene product (Advanced Biotechnologies, Rivers Park, IL). The cells were then reacted with anti-mouse immllnoglobulin G conjugated to horseradish peroxidase-labeled (kerkgaard and Perry, Gaithersburg, MD) secondary antibody and developed with a TMB substrate, and the optical density at 450 nm was detell-lilled by using a plate reader (Ceres 900, Biotek, Winooski, VT).
These ELISA experiments showed that some of these modified oligonucleotides almost comrlrtrly inhibit HCMV DNA rrrlir~tic n when used at concentrations as low as 0.08 ~M. The results are presellled in Table 5.

Table 5 Thiono Triester Oligonucleotide Phosphorothioate Inhibition of HCMVDNA Replication No. Sc~ e Mo~ifie~ti~ Activity 0.08 ~lM 0.4 ~M
2.0TGG GGC TTA CCT TGC GAA CA R3 + +
~ 2.1TGG GGC TTA CCT TGC GAA CA Rl, R3 + +
2.2CTGG GGC TTA CCT TGC GAA CA R3 - n.t.

W 096129337 PCTrUS96/03843 Table5(cont.) No. Sequence MoAifi- ~...... Activity 0.0811M 0.4~1M
23cTGG CX~C TTA CCT TGC GAA CA R~, R3 - n.t.
2.4TGG GGC TTA CCT TGC GAA CA Rl - n.t.
2.5TGG GGC TTA CCT TGC GAA CA R2 2.6TGG GGC TTA CCT TGC GAA CA RI7R2 2.7TGG GGC TTA CCT TGC GAA CA R4 - +
2.8TGG GGC TTA CCT TGC GAA CA Rl, R4 - +
2.9TGG GGC TTA CCT TGC GAA C A R~ _ +

2l0cTGG GGC TTA CCT TGC GAA CA R~, R4 2llcTGG GGC TTA CCT TGC GAA C A R4 2l2cTGG GGC TTA CCT TGC GAA CA R~ _ n.t.
2l3CTGGGGC TTACCT TGC GAA CA R, n.t. n.t.
All c~ J~ are oli~ud~,~,Ayl ih. ~ r.lr~ ;riPc and are presented in the ~ ' to 3 ' direction, left to right; hi ghli ghtPd n- lr lf~otirir-c have S-triester linkages on the 3 ' side, except as Ulll~,. Wi~
b R~=ethyl; R2~ l~llyl-2-ethyl; R3=(~c ' yl-3-c~l~u~llillo-6-hexyl; R4=l-hexadecyl; where one R group is listed, that R group is in each S-triester or O-triester linkage; where there are two or more R groups listed, each R group CU11C;~U11~ to one S-triester or O-triester linkage, w ith the first listed R group 70eing in the ~'-most S-triester or O-triester linkage and so on so that the last R group listed Cull~ ul~d:i to the 3'-most S-triester or 0-triester linkage.
c ~ighli~htPCi "", .1. ~.1 ;ri~ have O-triester linkages on the 3 ' side.
n.t. = not tested CA 022l6284 l997-09-23 W 096/29337 31 PCTnUS96/03843 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Hybridon, Inc.
(ii) TITLE OF INVENTION: Thiono Triester Modified Antisense Oligodeoxynucleotide Phosphorothioates (iii) NUMBER OF SEQUENCES: 5 - (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Banner & Allegretti, Ltd.
(B) STREET: 10 S. Wacker Drive Suite 3000 (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: U.S.A.
(F) ZIP: 60606 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Greenfield, Michael S.
(B) REGISTRATION NUMBER: 37,142 (C) REFERENCE/DOCKET NUMBER: 94,1033 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (312)715-1000 (B) TELEFAX: (312)715-1234 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

(2) INFORMATION FOR SEQ ID No:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES

W 096129337 -32- PCTrUS96/03843 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Claims

We claim:
1. An oligonucleotide comprising 13 to 50 nucleotides and from 1 to about 10 thiono-triester phosphorothioate internucleotide linkages having the structure:

wherein the R group of each thiono-triester phosphorothioate internucleotide linkage is independently a C2-C22 linear or branched chain alkyl group. a cholesteryl derivative having the structure:

where n = 2-12, an adamantyl derivative having the strucutre:

where n = 2-12, a 1,2-di-O-alkyl-rac-3-glyceryl derivative having the structure:
where R1 is C~H2n+1 and n = 2-22, or a DL .alpha.-tocopherol derivative having the structure:

where n - 0-10.
2. An oligonucleotide according to claim 1 having from 20 to 35 nucleotides.
3. An oligonucleotide according to claim 1 wherein R is chosen from the group consisting of 1-adamantyl-2-ethyl, 1-hexadecyl, and cholesteryl-3-carboxyamino-6-hexyl.
4. An oligonucleotide according to claim 2 wherein R is chosen from the group consisting of 1-adamantyl-2-ethyl, 1-hexadecyl, and cholesteryl-3-carboxyamino-6-hexyl.
5. An oligonucleotide according to claim 2 having the formula 5'-TGGGGCTTACCTTGCGAACA-3' (SEQ ID NO 2).
6. A composition for inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising an oligonucleotide according to claim 1 and a pharmaceutically acceptable carrier wherein the oligonucleotide is complementary to a region of a nucleic acid essential to the replication of the infectious agent or expression of the gene.
7. A composition for inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising an oligonucleotide according to claim 2 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of a nucleic acid essential to the replication of the infectious agent or expression of the gene.
8. A composition for inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising an oligonucleotide according to claim 4 and a pharmaceutically acceptable carrier. wherein the oligonucleotide is complementary to a region of a nucleic acid essential to the replication of the infectious agent or expression of the gene.
9. A composition according to claim 6 wherein the infectious agent is CMV.
10. A composition for inhibiting the expression of a nucleic acid whose expression product causes or is involved in a diseased state comprising an oligonucleotide according to claim 1 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of the nucleic acid.
11. A composition for inhibiting the expression of a nucleic acid whose expression product causes a diseased state comprising and oligonucleotide according to claim 2 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of the nucleic acid.
12. A composition for inhibiting the expression of a nucleic acid whose expression product causes a diseased state comprising and oligonucleotide according to claim 4 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of the nucleic acid.
13. A method of inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising contacting the cell with an effective amount of an oligonucleotide according to claim 1, wherein the oligonucleotide is complementary to a region of a nucleic acid essential to the replication of the infectious agent or expression of the gene.

14. A method of inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising contacting the cell with an effective amount of an oligonucleotide according to claim 2, wherein the oligonucleotide is complementary to a region of a nucleic acid essential to the replication of the infectious agent or expression of the gene.
15. A method of inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising contacting the cell with an effective amount of an oligonucleotide according to any one of claims 4, wherein the oligonucleotide is complementary to a region of a nucleic acid essential to the replication of the infectious agent or expression of the gene, 18. A method of inhibiting the expression of a nucleic acid whose expression product results in a diseased state comprising contacting the nucleic acid with an effective amount of an oligonucleotide according to claim 1. wherein the oligonucleotide is complementary to a region of the nucleic acid.
19. A method of inhibiting the replication of an infectious agent or gene expression of an infectious agent in a cell comprising contacting the cell with an effective amount of an oligonucleotide according to claim 2, wherein the oligonucleotide is complementary to a region of the nucleic acid.

20. A method of inhibiting the replication of infectious agent or gene expression of an infectious agent in a cell comprising contacting the cell with an effective amount of an oligonucleotide according to claim 4, wherein the oligonucleotide is complementary to a region of the nucleic acid.