US20150240237A1

US20150240237A1 - Compositions and Methods for Inhibiting Expression of a Gene from the JC Virus

Info

Publication number: US20150240237A1
Application number: US14/645,922
Authority: US
Inventors: Pamela Tan; Dinah Sah; Birgit Bramlage
Original assignee: Alnylam Europe AG; Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Europe AG; Alnylam Pharmaceuticals Inc
Priority date: 2006-04-28
Filing date: 2015-03-12
Publication date: 2015-08-27
Also published as: JP5398769B2; US8410261B2; JP2011130772A; EP2013222A4; US20120022141A1; US8058257B2; WO2007127919A2; US20090062225A1; HK1127065A1; JP2014036665A; EP2013222A2; US20130158102A1; US20100227915A1; WO2007127919A3; JP4812874B2; US9012624B2; US7691824B2; JP2009535039A; EP2013222B1

Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a gene from the JC Virus (JC virus genome), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of a gene from the JC Virus. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by JC virus expression and the expression of a gene from the JC Virus using the pharmaceutical composition; and methods for inhibiting the expression of a gene from the JC Virus in a cell.

Description

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/779,116, filed on Feb. 27, 2013 (allowed), which is a continuation of U.S. application Ser. No. 13/252,414, filed on Oct. 4, 2011 (U.S. Pat. No. 8,410,261), which is a continuation of U.S. application Ser. No. 12/720,465, filed Mar. 9, 2010 (U.S. Pat. No. 8,058,257), which is a continuation of U.S. application Ser. No. 11/741,205, filed Apr. 27, 2007 (U.S. Pat. No. 7,691,824) and claims the benefit of U.S. Provisional Application No. 60/795,765, filed Apr. 28, 2006. All of the prior applications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of one of the genes of the JC virus and the use of the dsRNA to treat pathological processes mediated by JC virus infection, such as PML.

BACKGROUND OF THE INVENTION

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system which results from reactivation of the latent polyomavirus JC virus (JCV) and its productive replication in glial cells of the human brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18). Once a rare disease primarily seen in patients with impaired immune systems due to lymphoproliferative and myeloproliferative disorders, PML has become one of the major neurologic problems among patients with AIDS (Cinque, P., (2003). J. Neurovirol. 9(Suppl. 1):88-92).
It has been reported that between 4 and 8% of AIDS patients exhibit signs of PML, and JCV has been detected in the cerebrospinal fluid of affected patients, suggesting that there is active replication of the virus in the brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18, Clifford, D. B., (2001) J. Neurovirol. 4:279). In addition, PML has recently been seen in patients undergoing experimental treatment with Tsybari, an anti VLA4 antibody, in combination with interferon. The histological hallmarks of PML include multifocal demyelinated lesions with enlarged eosinophilic nuclei in oligodendrocytes and enlarged bizarre astrocytes with lobulated hyperchromatic nuclei within white matter tracts of the brain (Cinque, P., (2003). J. Neurovirol. 9(Suppl. 1):88-92), although in some instances atypical features that include a unifocal pattern of demyelination and involvement of the gray matter have been reported (Sweeney, B. J., (1994). J. Neurol. Neurosurg. Psychiatry 57:994-997). Earlier observations from in vitro cell culture studies and an in vivo evaluation of JCV in clinical samples led to early assumptions that oligodendrocytes and astrocytes are the only cells which support productive viral infections (Gordon, J. (1998) Int. J. Mol. Med. 1:647-655). Accordingly, molecular studies have provided evidence for cell-type-specific transcription of the viral early genome in cells derived from the central nervous system (Raj, G. V., (1995) Virology 10:283-291). However, subsequent studies have shown low, but detectable, levels of JCV gene expression in nonneural cells, including B cells, and noticeably high levels of production of the viral early protein in several neural and nonneural tumor cells in humans (Gordon, J. (1998) Int. J. Mol. Med. 1:647-655, Khalili, K., 2003. Oncogene 22:5181-5191).
Like the other polyomaviruses, JCV is a small DNA virus whose genome can be divided into three regions that encompass the transcription control region; the genes responsible for the expression of the viral early protein, T antigen; and the genes encoding the viral late proteins, VP1, VP2, and VP3. In addition, the late genome is also responsible for production of an auxiliary viral protein, agnoprotein. T-antigen expression is pivotal for initiation of the viral lytic cycle, as this protein stimulates transcription of the late genes and induces the process of viral DNA replication. Recent studies have ascribed an important role for agnoprotein in the transcription and replication of JCV, as inhibition of its production significantly reduced viral gene expression and replication (M. Safak et al., unpublished observations). Furthermore, the agnoprotein dysregulates the cell cycle by altering the expression of several cyclins and their associated kinases (Darbinyan, A., (2002) Oncogene 21:5574-5581).
Thus far, there are no effective therapies for the suppression of JCV replication and the treatment of PML. Cytosine arabinoside (AraC) has been tested for the treatment of PML patients, and the outcome in some instances revealed a remission of JCV-associated demyelination (Aksamit, A. (2001) J. Neurovirol. 7:386-390). Reports from the AIDS Clinical Trial Group Organized Trial 243, however, have suggested that there is no difference in the survival of human immunodeficiency virus type 1 (HIV-1)-infected patients with PML and that of the control population, although in other reports it has been suggested that the failure of AraC in the AIDS Clinical Trial Group trial may have been due to insufficient delivery of the AraC via the intravenous and intrathecal routes (Levy, R. M., (2001) J. Neurovirol. 7:382-385). Based on in vitro studies showing the ability of inhibitors of topoisomerase to suppress JCV DNA replication, the topoisomerase inhibitor topotecan was used for the treatment of AIDS-PML patients, and the results suggested that topotecan treatment may be associated with a decreased lesion size and prolonged survival (Royal, W., III, (2003) J. Neurovirol. 9:411-419).
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Recent reports have indicated that in vitro, RNAi may show some promise in reducing JC virus replication (Radhakrishnan, S. (2004) J. Vir. 78:7264-7269, Orba, Y. (2004) J. Vir. 78:7270-7273). However, the RNAi agents examined were not designed against all know JC Virus strains and were not selected for stability and other properties need for in vivo therapeutic RNAi agents. Accordingly, despite significant advances in the field of RNAi, there remains a need for an agent that can selectively and efficiently silence a gene in the JC virus using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit replication of the JC virus for use in treating pathological processes mediated by JC virus infection.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the JC virus in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by JC viral infection, such as PML. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a gene from the JC Virus.
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression one of the genes of the JC virus and viral replication. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoded by a gene from the JC Virus, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing infected with the JC virus, inhibits the expression of a gene from the JC Virus by at least 40%.
For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1a and b and the second sequence is selected from the group consisting of the antisense sequences of Tables 1a and b. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such modified sequence will be based on a first sequence of said dsRNA selected from the group consisting of the sense sequences of Tables 1a and b and a second sequence selected from the group consisting of the antisense sequences of Tables 1a and 1b.
In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is generally a mammalian cell, such as a human cell.
In another embodiment, the invention provides a pharmaceutical composition for inhibiting the replication of the JC virus in an organism, generally a human subject, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier or delivery vehicle.
In another embodiment, the invention provides a method for inhibiting the expression of a gene in the JC Virus in a cell, comprising the following steps:

- (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a region of complementarity which is substantially complementary to at least a part of a mRNA encoded by the JC virus, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein the dsRNA, upon contact with a cell infected with the JC virus, inhibits expression of a gene from the JC Virus by at least 40%; and
- (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a JC virus gene, thereby inhibiting expression of a gene from the JC Virus in the cell.

In another embodiment, the invention provides methods for treating, preventing or managing pathological processes mediated by JC virus infection, e.g. such as PML, comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.
In another embodiment, the invention provides vectors for inhibiting the expression of a gene of the JC virus in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of a gene of the JC virus in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of a gene from the JC Virus in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by JC virus infection using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a gene from the JC Virus. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and or maintenance of JC virus infection and the occurrence of PML in a subject infected with the JC virus. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a gene from the JC Virus. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by JC viral infection, e.g. cancer, by targeting a gene involved in JC virus replication and/or maintenance in a cell.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a gene from the JC virus, as well as compositions and methods for treating diseases and disorders caused by the infection with the JC virus, such as PML. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of a gene from the JC Virus, together with a pharmaceutically acceptable carrier.
Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a gene in a gene from the JC Virus, and methods of using the pharmaceutical compositions to treat diseases caused by infection with the JC virus.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “JC virus” refers to the latent polyomavirus JC Virus that has a reference sequence NC_—001699. In addition, further accession numbers of various JCVirus sequences are AB038249.1-AB038255.1, AB048545.1-AB048582.1, AB074575.1-AB074591.1, AB077855.1-AB077879.1, AB081005.1-AB081030.1, AB081600.1-AB081618.1, AB081654.1, AB092578.1-AB092587.1, AB103387.1, AB103402.1-AB103423.1, AB104487.1, AB113118.1-AB113145.1, AB118651.1-AB118659.1, AB126981.1-AB127027.1, AB127342.1, AB127344.1, AB127346.1-AB127349.1, AB127352.1-AB127353.1, AB198940.1-AB198954.1, AB220939.1-AB220943.1, AF004349.1-AF004350.1, AF015526.1-AF015537.1, AF015684.1, AF030085.1, AF281599.1-AF281626.1, AF295731.1-AF295739.1, AF300945.1-AF300967.1, AF363830.1-AF363834.1, AF396422.1-AF396435.1, AY121907.1-AY121915.1, NC_—001699.1, U61771.1, U73500.1-U73502.1.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene from the JC Virus, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding JC virus). For example, a polynucleotide is complementary to at least a part of a JC virus mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding JC virus.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence” and “inhibit the expression of”, in as far as they refer to a gene from the JC Virus, herein refer to the at least partial suppression of the expression of a gene from the JC Virus, as manifested by a reduction of the amount of mRNA transcribed from a gene from the JC Virus which may be isolated from a first cell or group of cells in which a gene from the JC Virus is transcribed and which has or have been treated such that the expression of a gene from the JC Virus is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of
$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$
Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to JC virus genome transcription, e.g. the amount of protein encoded by a gene from the JC Virus, or the number of cells displaying a certain phenotype, e.g. infection with the JC Virus. In principle, JC virus genome silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a gene from the JC Virus by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of a gene from the JC Virus is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, a gene from the JC Virus is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, a gene from the JC Virus is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention.
As used herein in the context of JC virus expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by JC virus infection. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by JC virus expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by JC virus infection or an overt symptom of pathological processes mediated by JC virus expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by JC virus infection, the patient's history and age, the stage of pathological processes mediated by JC virus infection, and the administration of other anti-pathological processes mediated by JC virus infection.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a gene from the JC Virus in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a gene from the JC Virus, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing the gene from the JC virus, inhibits the expression of the JC virus gene by at least 40%.
The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a gene from the JC Virus, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s).
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, a gene from the JC Virus is the human JC virus genome. In specific embodiments, the antisense strand of the dsRNA comprises the sense sequences of Tables 1a and b and the second sequence is selected from the group consisting of the antisense sequences of Tables 1a and b. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1a and b can readily be determined using the target sequence and the flanking JC virus sequence.
In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Tables 1a and b. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of a gene from the JC Virus. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1a and b and the second oligonucleotide is described as the antisense strand in Tables 1a and b
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1a and b, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1a and b minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1a and b, and differing in their ability to inhibit the expression of a gene from the JC Virus in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1a and b can readily be made using the JC virus sequence and the target sequence provided.
In addition, the RNAi agents provided in Tables 1a and b identify a site in the JC virus mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1a and b coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a gene from the JC Virus. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target JC virus genome produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1a and b.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a gene from the JC Virus, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a gene from the JC Virus. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a gene from the JC Virus is important, especially if the particular region of complementarity in a gene from the JC Virus is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_nO]_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C₁to C₁₀lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.
Other preferred modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. dsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
Vector Encoded RNAi Agents
The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.
dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a gene from the JC Virus and/or viral infection, such as PML. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.
The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of a gene from the JC Virus. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of a gene from the JC Virus.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by JC virus expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Liposomes
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_M1or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an dsRNA RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNA dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by JC virus expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Caused by Expression of a Gene from the JC Virus
The invention relates in particular to the use of a dsRNA or a pharmaceutical composition prepared therefrom for the treatment or prevention of pathological conditions associated with JC Virus infection, e.g., PML. Owing to the inhibitory effect on JC virus expression, an dsRNA according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life, particularly in a patient being treated with an anti-VLA4 antibody as part of treatment for MS.
The invention furthermore relates to the use of an dsRNA or a pharmaceutical composition thereof for treating PML in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.
Methods for Inhibiting Expression of a Gene from the JC Virus
In yet another aspect, the invention provides a method for inhibiting the expression of a gene from the JC Virus in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target JC virus genome is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target JC virus gene. Compositions and methods for inhibiting the expression of these JC virus genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of a gene from the JC Virus, to the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Design of JCV siRNAs

Full-length genome sequences to JC virus available on Apr. 10, 2006, were obtained, resulting in a target pool of 388 sequences (accession numbers: AB038249.1-AB038255.1; AB048545.1-AB048582.1; AB074575.1-AB074591.1; AB077855.1-AB077879.1; AB081005.1-AB081030.1; AB081600.1-AB081618.1; AB081654.1; AB092578.1-AB092587.1; AB103387.1; AB103402.1-AB103423.1; AB104487.1; AB113118.1-AB113145.1; AB118651.1-AB118659.1; AB126981.1-AB127027.1; AB127342.1; AB127344.1; AB127346.1-AB127349.1; AB127352.1-AB127353.1; AB198940.1-AB198954.1; AB220939.1-AB220943.1; AF004349.1-AF004350.1; AF015526.1-AF015537.1; AF015684.1; AF030085.1; AF281599.1-AF281626.1; AF295731.1-AF295739.1; AF300945.1-AF300967.1; AF363830.1-AF363834.1; AF396422.1-AF396435.1; AY121907.1-AY121915.1; NC_—001699.1; U61771.1; U73500.1-U73502.1). NC_—001699 was defined as reference sequence.
The siRNA selection process was run as follows: ClustalW multiple alignment was used to generate a global alignment of all sequences from the target pool. An IUPAC consensus sequence was then generated.
All conserved 19 mer target sequences from the IUPAC consensus represented by stretches containing only A, T, C or G bases, which are therefore present in all sequences of the target pool were selected. In order to only select siRNAs that target transcribed sequence parts of the JC virus, candidate target sequences were selected out of the pool of conserved 19 mer target sequences. For this, candidate target sequences covering regions between nucleotide 163-2594 and between 2527-5115 relative to reference sequence were extracted for late and early genes, respectively. Further, as sequences for early genes are in reverse complement orientation compared with genomic sequences, candidate target sequences of these genes were transferred to reverse complement sequences and replaced the former pool of candidate target sequences.
In order to rank candidate target sequences and their respective siRNAs and select appropriate ones, their predicted potential for interacting with irrelevant targets (off-target potential) was taken as a ranking parameter. siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.
For predicting siRNA-specific off-target potential, the following assumptions were made:

- 1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) may contribute more to off-target potential than rest of sequence (non-seed and cleavage site region)
- 2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage site region) may contribute more to off-target potential than non-seed region
- 3) an off-target score can be calculated for each hit, based on identity to siRNA sequence and position of mismatches
- 4) assuming potential abortion of sense strand activity by internal modifications introduced, only off-target potential of antisense strand will be relevant

To identify potential off-target genes, 19 mer input sequences were subjected to a homology search against publically available human mRNA sequences.
To this purpose, fastA (version 3.4) searches were performed with all 19 mer candidate target sequences against a human RefSeq database (downloaded available version from ftp://ftp.ncbi.nih.gov/refseq/on Nov. 7, 2006). FastA searches were executed with parameters-values-pairs -f 50 -g 50 in order to take into account the homology over the full length of the 19 mer without any gaps. In order to ensure the listing of all relevant off-target hits in the fastA output file the parameter -E 30000 was used in addition. A scoring matrix was applied for the run that assessed every nucleotide match with a score of 13 and every mismatch with a score of −7. The search resulted in a list of potential off-targets for each candidate siRNA.
To sort the resulting list of potential off-targets for each siRNA, fastA output files were analyzed to identify the best off-target and its off-target score. The following off-target properties for each 19 mer input sequence were extracted for each off-target to calculate the off-target score:
Number of mismatches in non-seed region
Number of mismatches in seed region
Number of mismatches in cleavage site region
The off-target score was calculated for considering assumption 1 to 3 as follows:
$Off - target score = number of seed mismatches * 10 + number of cleavage site mismatches * 1.2 + number of non - seed mismatches * 1$
The most relevant off-target gene for input each 19 mer input sequences was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as the relevant off-target score for the corresponding siRNA.
In order to generate a ranking for siRNAs, calculated relevant off-target scores were transferred into a result table. All siRNAs were sorted according to the off-target score (descending).
An off-target score of 2.2 was defined as cut-off for siRNA selection (specificity criterion). In addition, all sequences with only one mismatch in the seed region were eliminated from the screening set. The selection procedure resulted in a set of 93 JCV specific siRNAs (Table 1a).
An expanded screening was generated by re-calculating the predicted specificity based on the newly available human RefSeq database (Human mRNA sequences in RefSeq release version 21 (downloaded Jan. 12, 2007)) and selecting only 208 siRNAs that did not contain more than 3 G's in a row and had an off-target score of at least 2 for the antisense strand (Table 1b).
Synthesis of JCV siRNAs
All siRNAs were synthesized in 0.2 μmole synthesis scale on an ABI3900 DNA synthesizer according to standard procedures.
For the initial screening set (93 different siRNA sequences), 4 different strategies of chemical modification were used:
a) exo/endo light (EEL):

- sense strand: 2′-O-methyl @ all pyrimidines, PTO between nucleotides 20 and 21 (counting from 5′-end), dTdT at 3′-end (nucleotides 20 and 21)
- antisense strand: 2′-O-methyl at pyrimidines in 5′-UA-3′ and 5′-CA-3′ motives, PTO between nucleotides 20 and 21 (counting from 5′-end), dTdT at 3′-end (nucleotides 20 and 21)

b) EEL plus 2′-O-methyl in position 2 of antisense strand (only if no 5′-UA-3′ and 5′-CA-3′ at 5′-end, otherwise already covered by EEL)
c) EEL plus 2′-O-methyl in position 2 of sense strand (only if no pyrimidine in position 2, otherwise already covered by EEL)
d) EEL plus 2′-O-methyl in position 2 of sense and antisense strand (only if not already covered by a, b, and c) (Table 1a)
For the expanded screening set (208 different siRNA sequences), siRNAs were composed of unmodified RNA oligonucleotides with dT/dT overhangs (dTdT at 3′-end (nucleotides 20 and 21) of antisense and sense strands) (Table 1b).
Screening of JCV siRNAs
Construction of Reporter-Systems Encoding JCV Transcripts
The sequence of the early JCV transcript (E) was synthesized at GENEART (Regensburg, Germany) and cloned into GENEART standard vectors. The sequence of the late JCV transcript was subdivided in a first approach into two fragments: L1, including the transcript sequence of the VP1 protein, and LA23, including the sequences of VP2, VP3 and the Agnoprotein. Due to cloning problems with fragment LA23, this sequence was subdivided in a second approach into two fragments (LA23 1-700 and LA23 701-1438). All sequences were synthesized at GENEART and cloned into GENEART standard vectors. All fragments (E, L1, LA23 1-700 and LA23 701-1438) were subcloned into psiCheck-2 (Promega, Mannheim, Germany) via XhoI and NotI (both NEB, Frankfurt, Germany), resulting in constructs with the JCV sequences between the stop-codon and the polyA-signal of Renilla luciferase.
L1

	(SEQ ID NO: 931)
	CTCGAGACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTG

	AAGCATATGAAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAA

	GGACCCCGTGCAAGTTCCAAAACTTCTTATAAGAGGAGGAGTAG

	AAGTTCTAGAAGTTAAAACTGGGGTTGACTCAATTACAGAGGTA

	GAATGCTTTTTAACTCCAGAAATGGGTGACCCAGATGAGCATCT

	TAGGGGTTTTAGTAAGTCAATTTCTATATCAGATACATTTGAAA

	GTGACTCCCCAAATAAGGACATGCTTCCTTGTTACAGTGTGGCC

	AGAATTCCACTACCCAATCTAAATGAGGATCTAACCTGTGGAAA

	TATACTAATGTGGGAGGCTGTGACCTTAAAAACTGAGGTTCTAG

	GGGTGACAACTTTGATGAATGTGCACTCTAATGGTCAAGCAACT

	CATGACAATGGTGCAGGAAAGCCAGTGCAGGGCACCAGCTTTCA

	TTTTTTTTCTGTTGGCGGGGAGGCTTTAGAATTACAGGGGGTGG

	TTTTTAATTACAGAACAAAGTACCCAGATGGAACAATTTTTCCA

	AAGAATGCAACAGTGCAATCTCAAGTAATGAACACAGAGCACAA

	GGCGTACCTAGATAAGAACAAAGCATATCCTGTTGAATGTTGGG

	TTCCTGATCCCACCAGAAATGAAAACACAAGATATTTTGGGACA

	CTAACAGGAGGAGAAAATGTTCCTCCAGTTCTTCATATAACAAA

	CACTGCCACAACAGTGCTGCTTGATGAATTTGGTGTTGGGCCAC

	TTTGCAAAGGTGACAACTTGTATTTGTCAGCTGTTGATGTTTGT

	GGAATGTTTACTAACAGATCTGGTTCCCAGCAGTGGAGAGGACT

	GTCCAGATATTTTAAGGTTCAGCTCAGAAAAAGGAGGGTTAAAA

	ACCCCTACCCAATTTCTTTCCTTCTTACTGATTTGATTAACAGA

	AGGACCCCTAGAGTTGATGGGCAACCTATGTATGGTATGGATGC

	TCAGGTAGAGGAGGTTAGAGTTTTTGAGGGGACAGAGGAACTTC

	CAGGGGACCCAGACATGATGAGATATGTTGACAGATATGGACAG

	TTGCAAACAAAGATGCTGTAATCAAAATCCTTTATTGTAATATG

	CAGTACATTTTAATAAAGTATAACCAGCTTTACTTTACAGTTGC

	AGTCATGCGGCCGC

E

	(SEQ ID NO: 932)
	CTCGAGCCGCCTCCAAGCTTACTCAGAAGTAGTAAGGGC

	GTGGAGGCTTTTTAGGAGGCCAGGGAAATTCCCTTGTTTTTCCC

	TTTTTTGCAGTAATTTTTTGCTGCAAAAAGCTAAAATGGACAAA

	GTGCTGAATAGGGAGGAATCCATGGAGCTTATGGATTTATTAGG

	CCTTGATAGGTCTGCATGGGGGAACATTCCTGTCATGAGAAAAG

	CTTATCTGAAAAAATGCAAAGAACTCCACCCTGATAAAGGTGGG

	GACGAAGACAAGATGAAGAGAATGAATTTTTTATATAAAAAAAT

	GGAACAAGGTGTAAAAGTTGCTCATCAGCCTGATTTTGGTACAT

	GGAATAGTTCAGAGGTTGGTTGTGATTTTCCTCCTAATTCTGAT

	ACCCTTTATTGCAAGGAATGGCCTAACTGTGCCACTAATCCTTC

	AGTGCATTGCCCCTGTTTAATGTGCATGCTAAAATTAAGGCATA

	GAAACAGAAAATTTTTAAGAAGCAGCCCACTTGTGTGGATAGAT

	TGCTATTGCTTTGATTGCTTCAGACAATGGTTTGGGTGTGACTT

	AACCCAAGAAGCTCTTCATTGCTGGGAGAAAGTTCTTGGAGACA

	CCCCCTACAGGGATCTAAAGCTTTAAGTGCCAACCTATGGAACA

	GATGAATGGGAATCCTGGTGGAATACATTTAATGAGAAGTGGGA

	TGAAGACCTGTTTTGCCATGAAGAAATGTTTGCCAGTGATGATG

	AAAACACAGGATCCCAACACTCTACCCCACCTAAAAAGAAAAAA

	AAGGTAGAAGACCCTAAAGACTTTCCTGTAGATCTGCATGCATT

	CCTCAGTCAAGCTGTGTTTAGTAATAGAACTGTTGCTTCTTTTG

	CTGTGTATACCACTAAAGAAAAAGCTCAAATTTTATATAAGAAA

	CTTATGGAAAAATATTCTGTAACTTTTATAAGTAGACATGGTTT

	TGGGGGTCATAATATTTTGTTTTTCTTAACACCACATAGACATA

	GAGTGTCAGCAATTAATAACTACTGTCAAAAACTATGTACCTTT

	AGTTTTTTAATTTGTAAAGGTGTGAATAAGGAATACTTGTTTTA

	TAGTGCCCTGTGTAGACAGCCATATGCAGTAGTGGAAGAAAGTA

	TTCAGGGGGGCCTTAAGGAGCATGACTTTAACCCAGAAGAACCA

	GAAGAAACTAAGCAGGTTTCATGGAAATTAGTTACACAGTATGC

	CTTGGAAACCAAGTGTGAGGATGTTTTTTTGCTTATGGGCATGT

	ACTTAGACTTTCAGGAAAACCCACAGCAATGCAAAAAATGTGAA

	AAAAAGGATCAGCCAAATCACTTTAACCATCATGAAAAACACTA

	TTATAATGCCCAAATTTTTGCAGATAGCAAAAATCAAAAAAGCA

	TTTGCCAGCAGGCTGTTGATACTGTAGCAGCCAAACAAAGGGTT

	GACAGCATCCACATGACCAGAGAAGAAATGTTAGTTGAAAGGTT

	TAATTTCTTGCTTGATAAAATGGACTTAATTTTTGGGGCACATG

	GCAATGCTGTTTTAGAGCAATATATGGCTGGGGTGGCCTGGATT

	CATTGCTTGCTGCCTCAAATGGACACTGTTATTTATGACTTTCT

	AAAATGCATTGTATTAAACATTCCAAAAAAAAGGTACTGGCTAT

	TCAAGGGGCCAATAGACAGTGGCAAAACTACTTTAGCTGCAGCT

	TTACTTGATCTCTGTGGGGGAAAGTCATTAAATGTTAATATGCC

	ATTAGAAAGATTAAACTTTGAATTAGGAGTGGGTATAGATCAGT

	TTATGGTTGTATTTGAGGATGTAAAAGGCACTGGTGCAGAGTCA

	AGGGATTTACCTTCAGGGCATGGCATAAGCAACCTTGATTGCTT

	AAGAGATTACTTAGATGGAAGTGTAAAAGTTAATTTAGAGAGAA

	AACACCAAAACAAAAGAACACAGGTGTTTCCACCTGGAATTGTA

	ACCATGAATGAATATTCAGTGCCTAGAACTTTACAGGCCAGATT

	TGTAAGGCAGATAGATTTTAGACCAAAGGCCTACCTGAGAAAAT

	CACTAAGCTGCTCTGAGTATTTGCTAGAAAAAAGGATTTTGCAA

	AGTGGTATGACTTTGCTTTTGCTTTTAATCTGGTTTAGGCCAGT

	TGCTGACTTTGCAGCTGCCATTCATGAGAGGATTGTGCAGTGGA

	AAGAAAGGCTGGATTTAGAAATAAGCATGTATACATTTTCTACT

	ATGAAAGCTAATGTTGGTATGGGGAGACCCATTCTTGACTTTCC

	TAGAGAGGAAGATTCTGAAGCAGAAGACTCTGGACATGGATCAA

	GCACTGAATCACAATCACAATGCTTTTCCCAGGTCTCAGAAGCC

	TCTGGTGCAGACACACAGGAAAACTGCACTTTTCACATCTGTAA

	AGGCTTTCAATGTTTCAAAAAACCAAAGACCCCTCCCCCAAAAT

	AACTGCAACTGTGCGGCCGC

LA23 1-700

	(SEQ ID NO: 933)
	CTCGAGCAGCTAACAGCCAGTAAACAAAGCACAAGGGGAAGTGG

	AAAGCAGCCAAGGGAACATGTTTTGCGAGCCAGAGCTGTTTTGG

	CTTGTCACCAGCTGGCCATGGTTCTTCGCCAGCTGTCACGTAAG

	GCTTCTGTGAAAGTTAGTAAAACCTGGAGTGGAACTAAAAAAAG

	AGCTCAAAGGATTTTAATTTTTTTGTTAGAATTTTTGCTGGACT

	TTTGCACAGGTGAAGACAGTGTAGACGGGAAAAAAAGACAGAGA

	CACAGTGGTTTGACTGAGCAGACATACAGTGCTTTGCCTGAACC

	AAAAGCTACATAGGTAAGTAATGTTTTTTTTTGTGTTTTCAGGT

	TCATGGGTGCCGCACTTGCACTTTTGGGGGACCTAGTTGCTACT

	GTTTCTGAGGCTGCTGCTGCCACAGGATTTTCAGTAGCTGAAAT

	TGCTGCTGGAGAGGCTGCTGCTACTATAGAAGTTGAAATTGCAT

	CCCTTGCTACTGTAGAGGGGATTACAAGTACCTCTGAGGCTATA

	GCTGCTATAGGCCTTACTCCTGAAACATATGCTGTAATAACTGG

	AGCTCCGGGGGCTGTAGCTGGGTTTGCTGCATTGGTTCAAACTG

	TAACTGGTGGTAGTGCTATTGCTCAGTTGGGATATAGATTTTTT

	GCTGACTGGGATCATAAAGTTTCAACAGTTGGGCTTTTTCGCGG

	CCGC

LA23 701-1438

	(SEQ ID NO: 934)
	CTCGAGAGCAGCCAGCTATGGCTTTACAATTATTTAATCCAGAA

	GACTACTATGATATTTTATTTCCTGGAGTGAATGCCTTTGTTAA

	CAATATTCACTATTTAGATCCTAGACATTGGGGCCCGTCCTTGT

	TCTCCACAATCTCCCAGGCTTTTTGGAATCTTGTTAGAGATGAT

	TTGCCAGCCTTAACCTCTCAGGAAATTCAGAGAAGAACCCAAAA

	ACTATTTGTTGAAAGTTTAGCAAGGTTTTTGGAAGAAACTACTT

	GGGCAATAGTTAATTCACCAGCTAACTTATATAATTATATTTCA

	GACTATTATTCTAGATTGTCTCCAGTTAGGCCCTCTATGGTAAG

	GCAAGTTGCCCAAAGGGAGGGAACCTATATTTCTTTTGGCCACT

	CATACACCCAAAGTATAGATGATGCAGACAGCATTCAAGAAGTT

	ACCCAAAGGCTAGATTTAAAAACCCCAAATGTGCAATCTGGTGA

	ATTTATAGAAAGAAGTATTGCACCAGGAGGTGCAAATCAAAGAT

	CTGCTCCTCAATGGATGTTGCCTTTACTTTTAGGGTTGTACGGG

	ACTGTAACACCTGCTCTTGAAGCATATGAAGATGGCCCCAACAA

	AAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGTTCCAAAACTT

	CTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAACTGGGGT

	TGACTCAATTACAGAGGTAGAATGCTGCGGCCGC

Screen of JCV siRNAs in Transfected Cells
Cos-7 cells (DSMZ, Braunschweig, Germany, # ACC-60) were seeded at 1.5×10⁴cells/well on white 96-well plates with clear bottoms (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium. Directly after seeding the cells, 50 ng of the corresponding reporter-plasmid per well was transfected with Lipofectamine™ 2000 (Invitrogen GmbH, Karlsruhe, Germany), with the plasmid diluted in Opti-MEM to a final volume of 12.5 μl per well, prepared as a mastermix for the whole plate.
4 h after plasmid transfection, growth medium was removed from cells and replaced by 100 μl/well of fresh medium. siRNA transfections were performed using Lipofectamine™ 2000 (Invitrogen GmbH, Karlsruhe, Germany) as described by the manufacturer. Cells were incubated for 24 h at 37° C. and 5% CO₂in a humidified incubator (Heraeus GmbH, Hanau, Germany). For the primary screen, all siRNAs were screened at a final concentration of 30 nM. Selected sequences were rescreened at a siRNA concentration of 300 pM. Each siRNA was tested in quadruplicate for each concentration.
Cells were lysed by removing growth medium and application of 150 μl of a 1:1 mixture consisting of medium and substrate from the Dual-Glo Luciferase Assay System (Promega, Mannheim, Germany). The luciferase assay was performed according to the manufacturer's protocol for Dual-Glo Luciferase assay and luminescence was measured in a Victor-Light 1420 Luminescence Counter (Perkin Elmer, Rodgau-Jügesheim, Germany). Values obtained with Renilla luciferase were normalized to the respective values obtained with Firefly luciferase in order to correct for transfection efficacy. Renilla/Firefly luciferase activities obtained after transfection with siRNAs directed against a JCV gene were normalized to Renilla/Firefly luciferase activities obtained after transfection of an unrelated control siRNA set to 100%. Tables 1a and b provides the results where the siRNAs, the sequences of which are given in Tables 1a and b, were tested at a single dose of 30 nM. The percentage inhibition±standard deviation, compared to the unrelated control siRNA, is indicated in the column ‘Remaining luciferase activity (% of control)’. A number of JCV siRNAs at 30 nM were effective at reducing levels of the targeted mRNA by more than 70% in Cos-7 cells (i.e. remaining luciferase activity was less than 30%).
Selected JCV siRNAs from the single dose screen were further characterized by dose response curves. Transfections of JCV siRNAs for generation of dose response curves were performed with the following siRNA concentrations according to the above protocol:

- from 33 nM in 3-fold dilutions down to 0.005 nM (for fragment L1)
- from 24 nM in 4-fold dilutions down to 0.001 nM (for fragment E and fragments LA23 1-700 and LA23 701-1438).

IC50 values were determined by parameterized curve fitting using the program XLfit (IDBS, Guildford, Great Britain). Table 2 provides the results from two independent experiments for 32 selected JCV siRNAs. The mean IC50 from these two independent experiments is shown. Several JCV siRNAs (AD-12622, AD-12677, AD-12709, AD-12710, AD-12722, AD-12724, AD-12728, AD-12763, AD-12767, AD-12768, AD-12769, AD-12771, AD-12774, AD-12775, AD-12777, AD-12781, AD-12784, AD-12795, AD-12813, AD-12821, AD-12823, AD-12824, AD-12825, AD-12827, AD-12829, AD-12842) were particularly potent in this experimental paradigm, and exhibited IC50 values between 70 pM and 1 nM.

TABLE 2

IC50s

		Mean IC50
	Duplex name	[nM]

	AD-12599	2.37
	AD-12622	0.57
	AD-12666	3.7
	AD-12677	0.49
	AD-12709	0.19
	AD-12710	0.47
	AD-12712	2.33
	AD-12722	0.12
	AD-12724	0.26
	AD-12728	0.8
	AD-12761	1.2
	AD-12763	0.95
	AD-12767	0.09
	AD-12768	0.19
	AD-12769	0.35
	AD-12771	0.35
	AD-12774	0.13
	AD-12775	0.18
	AD-12777	0.17
	AD-12778	12.65
	AD-12781	0.18
	AD-12784	0.44
	AD-12795	0.65
	AD-12813	0.2
	AD-12818	1.88
	AD-12821	0.07
	AD-12823	0.46
	AD-12824	0.25
	AD-12825	0.52
	AD-12827	0.15
	AD-12829	0.14
	AD-12842	0.44

Screen of JCV siRNAs Against Live JC Virus in SVG-A Cells
Cells and Virus
SVG-A cells (human fetal glial cells transformed by SV40 T antigen) obtained from Walter Atwood at Brown University were cultured in Eagle's Minimum Essential Media (ATCC, Manassas, Va.) supplemented to contain 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, Calif.), Penicillin 100 U/ml, Streptomycin 100 ug/ml (Invitrogen, Carlsbad Calif.) at 37° C. in an atmosphere with 5% CO₂in a humidified incubator (Heraeus HERAcell, Thermo Electron Corporation, Ashville, N.C.). The Mad-1-SVEΔ strain of JCV obtained from Walter Atwood at Brown University was used in all experiments; viral stocks were prepared using SVG-A cells according to standard published methods (Liu and Atwood, Propagation and assay of the JC Virus, Methods Mol Biol. 2001; 165:9-17).
Prophylaxis Assay
SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hours prior to transfection in the media described above minus antibiotics. Cells were transfected with the indicated concentration of siRNA (10 nM, 50 nM, or 100 nM) using Lipofectamine™ 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Twenty-four hours post-transfection cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock (Mad-1-SVEΔ strain) diluted in 2% FBS media. Cells were rocked every 15 minutes by hand several times to get equal virus binding across the entire coverslip for one hour and then additional 10% FBS media was added and the infection was allowed to proceed for 72 hours. Seventy two hours post-infection, cells were fixed in acetone and stained for late viral protein (VP1) by standard immunofluorescence methods using hybridoma supernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood at Brown University) with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad, Calif.). Infected cells were scored by counting VP1-immunoreactive cells using a fluorescence microscope (Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for the control coverslips transfected with Luciferase siRNA. Table 3 shows the results of the prophylaxis assays at different siRNA concentrations (10 nM, 50 nM or 100 nM). The VP1 siRNAs were the most potent as a group, followed by the T antigen siRNAs, with the VP2/3 siRNAs being the least potent. The VP1 siRNAs most effective in reducing virus were consistently AD-12622, AD-12728, AD-12795, and AD-12842. The most potent T antigen siRNA was AD-12813.

TABLE 3

Prophylaxis Assay

			Remaining Virus
	Duplex	Targeted JCV	(% of Luciferase Control)

	Number	Transcript	50 nM	10 nM	100 nM

AD-12599	VP1	79.9	ND	ND
AD-12709	VP1	46.0	ND	ND
AD-12710	VP1	25.9	ND	ND
AD-12784	VP1	30.9	ND	ND
AD-12712	VP1	29.7	ND	ND
AD-12724	VP1	30.5	38.9	25.8
AD-12622	VP1	22.9	28.2	9.1
AD-12728	VP1	21.1	22.2	ND
AD-12795	VP1	13.6	16.9	8.5
AD-12842	VP1	16.0	23.4	12.7
AD-12761	VP1	26.4	52.3	ND
AD-12818	VP1	24.0	50.2	28.0
AD-12666	VP1	54.1	ND	ND
AD-12763	VP1	39.5	ND	ND
AD-12722	T Antigen	43.6	82.1	ND
AD-12813	T Antigen	21.5	48.8	19.4
AD-12767	T Antigen	37.6	52.2	30.9
AD-12821	T Antigen	33.0	51.2	30.8
AD-12774	T Antigen	74.0	89.2	ND
AD-12827	T Antigen	77.0	92.0	ND
AD-12775	T Antigen	81.6	95.4	ND
AD-12777	T Antigen	73.3	93.9	ND
AD-12829	T Antigen	78.6	93.6	ND
AD-12781	T Antigen	38.8	62.6	34.4
AD-12768	VP2/3	73.9	92.4	ND
AD-12771	VP2/3	51.6	83.6	ND
AD-12824	VP2/3	42.1	79.0	43.7
AD-12769	VP2/3	35.2	78.0	39.7
AD-12823	VP2/3	38.1	78.1	42.0
AD-12677	VP2/3	99.1	102.1	ND
AD-12825	VP2/3	100.8	99.1	ND

ND indicates no data.

Post-Infection Treatment Assay
SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hours prior to infection in 10% FBS media. Cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock diluted in 2% FBS media. Cells were rocked by hand approximately 8-10 times to get equal virus binding across the entire coverslip every 15 minutes for one hour and then additional 10% FBS media was added. Twenty-four and forty-eight hours postinfection, cells were washed with 10% FBS media containing no antibiotics and then transfected with 50 nM of the indicated siRNA using Lipofectamine™ 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Seventy-two hours postinfection, cells were fixed in acetone and stained for late viral protein (VP1) by standard immunofluorescence methods using hybridoma supernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood at Brown University) with goat anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes, Eugene, Oreg.). Infected cells were scored by counting VP1-immunoreactive cells using a fluorescence microscope (Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for control coverslips transfected with Luciferase siRNA. Table 4 shows the results of the post-infection treatment experiments. All of the siRNAs tested in the treatment assay showed significant antiviral activity against JCV, such that the remaining virus was significantly less than that in the luciferase siRNA control.

TABLE 4

Treatment Assay

	Targeted JCV	Remaining Virus (% of
Duplex Number	Transcript	Luciferase Control)

AD-12724	VP1	38.9
AD-12622	VP1	28.2
AD-12795	VP1	16.9
AD-12842	VP1	23.4
AD-12818	VP1	ND
AD-12813	T Antigen	48
AD-12767	T Antigen	56.9
AD-12821	T Antigen	75.8
AD-12781	T Antigen	75.8
AD-12824	VP2/3	60.4
AD-12769	VP2/3	70.7
AD-12823	VP2/3	72.4

ND indicates no data.

Prophylaxis Administration of JCV siRNAs Inhibits the Production of Active Progeny JC Virus
SVG-A cells were seeded in 6-well dishes 24 hours prior to transfection in the media described above minus antibiotics. Cells were transfected with 10 nM of the indicated siRNA using Lipofectamine™ 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Twenty-four hours post-transfection cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock (Mad-1-SVEΔ strain) diluted in 2% FBS media. Cells were rocked every 15 minutes by hand several times to get equal virus binding across the entire coverslip for one hour and then additional 10% FBS media was added and the infection was allowed to proceed for 6 days. Six days post-infection, progeny virus was collected either by removal of overlay media from infected cells or by scraping cells and performing virus preparations. The virus preparations consisted of scraping cells into the supernatant media, vortexing, freeze-thawing the re-suspended cells 2 times with vortexing in between, then spinning down the cell debris and taking the supernatant. Fresh SVG-A cells seeded on glass coverslips were infected secondarily with virus collected by either method using the same procedure done with the initial infection to determine the amount of infectious virus produced by cells transfected with the various siRNAs. At 72 hours post-infection of coverslips, cells were fixed in acetone and stained for late viral protein (VP1) by standard immunofluorescence methods using hybridoma supernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood at Brown University) with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad, Calif.). Infected cells were scored by counting VP1-immunoreactive cells using a fluorescence microscope (Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for the control coverslips transfected with Luciferase siRNA. Table 5 shows the results for selected siRNAs, demonstrating the ability of prophylaxis siRNA treatment to inhibit active progeny virus production by either method of virus collection. Transfection with siRNAs targeting VP1 (AD-12622 and AD-12842) had the greatest effect on inhibiting the production of active progeny virus regardless of whether virus was collected from media or from infected cell preparations. The T antigen siRNA AD-12813 had the next strongest inhibitory effect, whereas the VP2/3 siRNAs AD-12824 and AD-12769 still showed some albeit a lesser ability to inhibit active progeny JCV production.

TABLE 5

Prophylaxis administration of JCV siRNAs inhibits
the production of active progeny JC virus capable
of secondary infection

	Remaining Virus (% of
	Luciferase Control)

Duplex	Targeted		Virus
Name	Transcript	Media	Preparation

AD-12622	VP1	30.8	24.9
AD-12842	VP1	33.3	26.9
AD-12813	T Antigen	57.8	38.7
AD-12824	VP2/3	83.6	57.6
AD-12769	VP2/3	79.1	52.2

Stability in Cerebrospinal Fluid (CSF) of Selected siRNAs Targeting JCV
Eleven selected JCV siRNAs were tested for stability at 5 uM over 48 h at 37° C. in human CSF, as well as in PBS for comparison. 30 μl of human cerebrospinal fluid (CSF) was mixed with 3 μl of 50 μM duplex (siRNA) solution (150 pmole/well) in a 96-well plate, sealed to avoid evaporation and incubated for the indicated time at 37° C. Incubation of the siRNA in 30 ul PBS for 48 h served as a control for non-specific degradation. Reactions were stopped by the addition of 4 ul proteinase K (20 mg/ml) and 25 ul of proteinase K buffer, and an incubation for 20′ at 42° C. Samples were then spin filtered through a 0.2 μm 96 well filter plate at 3000 rpm for 20′. Incubation wells were washed with 50 ul Millipore water twice and the combined washing solutions were spin filtered also.
Samples were analyzed by ion exchange HPLC under denaturing conditions. Samples were transferred to single autosampler vials. IEX-HPLC analysis was performed under the following conditions: Dionex DNAPac PA200 (4×250 mm analytical column), temperature of 45° C. (denaturing conditions by pH=11), flow rate of 1 ml/min, injection volume of 50 ul, and detection wavelength of 260 nm with 1 nm bandwidth (reference wavelength 600 nm). In addition, the gradient conditions were as follows with HPLC Eluent A: 20 mM Na₃PO₄in 10% ACN; pH=11 and HPLC Eluent B: 1 M NaBr in HPLC Eluent A:


Time	% A	% B

0.00 min	75	25
1.00 min	75	25
19.0 min	38	62
19.5 min	0	100
21.5 min	0	100
22.0 min	75	25
24.0 min	75	25

Under the above denaturing IEX-HPLC conditions, the duplexes eluted as two separated single strands. All chromatograms were integrated automatically by the Dionex Chromeleon 6.60 HPLC software, and were adjusted manually as necessary. The area under the peak for each strand was calculated and the %-values for each intact full length product (FLP) for each time points were calculated by the following equation:
%-FLP_(s/as;t=x)=(PeakArea_(s/as);t=x/PeakArea_{(s/as);t=0min})*100%
All values were normalized to FLP at t=0 min. Table 6 provides the results after 48 hours of incubation in human CSF at 37° C. At least 75% of both antisense and sense strands of ten JCV siRNAs (AD-12622, AD-12724, AD-12767, AD-12769, AD-12795, AD-12813, AD-12818, AD-12823, AD-12824, AD-12842) were recovered, demonstrating that these siRNAs are highly stable in human CSF at 37° C. For AD-12821, 59% of the antisense and 97% of the sense strand was recovered after 48 h of incubation in human CSF at 37° C., showing that this siRNA has a half-life of greater than 48 h in human CSF at 37° C.

TABLE 6

Stability in human CSF

		% full length
		material after
	Duplex	48 hours

	name	antisense	sense

AD-12622	93	105
AD-12724	90	106
AD-12767	85	104
AD-12769	100	104
AD-12795	86	109
AD-12813	94	98
AD-12818	75	99
AD-12821	59	97
AD-12823	98	98
AD-12824	84	98
AD-12842	87	102

dsRNA Expression Vectors
In another aspect of the invention, JC virus specific dsRNA molecules that modulate JC virus genome expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. NatI. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D 1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single JC virus genome or multiple JC virus genomes over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection. of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
The JC virus specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Tables 1a-1 and 1a-2

TABLE 1a

JCV Gene Walk; siRNAs targeting >95% of all stransins
(>= out of 388); Human specific pan-JCV: 208 siRNAs;
allsiRNAs double overhang design; dTdT, no modifications.
1a-1: sequences; 1a-2: assay results

Table
1a-1	position	SEQ		SEQ
duplex	in	ID	sense strand	ID	antisense strand
name	consensus	NO:	sequence (5′-3′)	NO:	sequence (5′-3′)

AD-14742	1533-1551	515	CUUAUAAGAGGAGGAGUAGTT	516	CUACUCCUCCUCUUAUAAGTT

AD-14743	1703-1721	517	CAUGCUUCCUUGUUACAGUTT	518	ACUGUAACAAGGAAGCAUGTT

AD-14744	1439-1457	519	UACGGGACUGUAACACCUGTT	520	CAGGUGUUACAGUCCCGUATT

AD-14745	1705-1723	521	UGCUUCCUUGUUACAGUGUTT	522	ACACUGUAACAAGGAAGCATT

AD-14746	2064-2082	523	CCUGUUGAAUGUUGGGUUCTT	524	GAACCCAACAUUCAACAGGTT

AD-14747	2067-2085	525	GUUGAAUGUUGGGUUCCUGTT	526	CAGGAACCCAACAUUCAACTT

AD-14748	2071-2089	527	AAUGUUGGGUUCCUGAUCCTT	528	GGAUCAGGAACCCAACAUUTT

AD-14749	2121-2139	529	ACACUAACAGGAGGAGAAATT	530	UUUCUCCUCCUGUUAGUGUTT

AD-14750	1535-1553	531	UAUAAGAGGAGGAGUAGAATT	532	UUCUACUCCUCCUCUUAUATT

AD-14751	1536-1554	533	AUAAGAGGAGGAGUAGAAGTT	534	CUUCUACUCCUCCUCUUAUTT

AD-14752	1445-1463	535	ACUGUAACACCUGCUCUUGTT	536	CAAGAGCAGGUGUUACAGUTT

AD-14753	1700-1718	537	GGACAUGCUUCCUUGUUACTT	538	GUAACAAGGAAGCAUGUCCTT

AD-14754	1702-1720	539	ACAUGCUUCCUUGUUACAGTT	540	CUGUAACAAGGAAGCAUGUTT

AD-14755	1704-1722	541	AUGCUUCCUUGUUACAGUGTT	542	CACUGUAACAAGGAAGCAUTT

AD-14756	2065-2083	543	CUGUUGAAUGUUGGGUUCCTT	544	GGAACCCAACAUUCAACAGTT

AD-14757	2070-2088	545	GAAUGUUGGGUUCCUGAUCTT	546	GAUCAGGAACCCAACAUUCTT

AD-14758	1441-1459	547	CGGGACUGUAACACCUGCUTT	548	AGCAGGUGUUACAGUCCCGTT

AD-14759	1443-1461	549	GGACUGUAACACCUGCUCUTT	550	AGAGCAGGUGUUACAGUCCTT

AD-14760	1444-1462	551	GACUGUAACACCUGCUCUUTT	552	AAGAGCAGGUGUUACAGUCTT

AD-14761	1609-1627	553	CUCCAGAAAUGGGUGACCCTT	554	GGGUCACCCAUUUCUGGAGTT

AD-14762	1537-1555	555	UAAGAGGAGGAGUAGAAGUTT	556	ACUUCUACUCCUCCUCUUATT

AD-14763	629-647	557	GAGGCUGCUGCUACUAUAGTT	558	CUAUAGUAGCAGCAGCCUCTT

AD-14764	656-674	559	AUUGCAUCCCUUGCUACUGTT	560	CAGUAGCAAGGGAUGCAAUTT

AD-14765	658-676	561	UGCAUCCCUUGCUACUGUATT	562	UACAGUAGCAAGGGAUGCATT

AD-14766	517-535	563	UUGUGUUUUCAGGUUCAUGTT	564	CAUGAACCUGAAAACACAATT

AD-14767	559-577	565	GGACCUAGUUGCUACUGUUTT	566	AACAGUAGCAACUAGGUCCTT

AD-14768	591-609	567	CUGCCACAGGAUUUUCAGUTT	568	ACUGAAAAUCCUGUGGCAGTT

AD-14769	638-656	569	GCUACUAUAGAAGUUGAAATT	570	UUUCAACUUCUAUAGUAGCTT

AD-14770	655-673	571	AAUUGCAUCCCUUGCUACUTT	572	AGUAGCAAGGGAUGCAAUUTT

AD-14771	561-579	573	ACCUAGUUGCUACUGUUUCTT	574	GAAACAGUAGCAACUAGGUTT

AD-14772	639-657	575	CUACUAUAGAAGUUGAAAUTT	576	AUUUCAACUUCUAUAGUAGTT

AD-14773	715-733	577	AGGCCUUACUCCUGAAACATT	578	UGUUUCAGGAGUAAGGCCUTT

AD-14774	716-734	579	GGCCUUACUCCUGAAACAUTT	580	AUGUUUCAGGAGUAAGGCCTT

AD-14775	326-344	581	GUAAAACCUGGAGUGGAACTT	582	GUUCCACUCCAGGUUUUACTT

AD-14776	518-536	583	UGUGUUUUCAGGUUCAUGGTT	584	CCAUGAACCUGAAAACACATT

AD-14777	520-538	585	UGUUUUCAGGUUCAUGGGUTT	586	ACCCAUGAACCUGAAAACATT

AD-14778	661-679	587	AUCCCUUGCUACUGUAGAGTT	588	CUCUACAGUAGCAAGGGAUTT

AD-14779	560-578	589	GACCUAGUUGCUACUGUUUTT	590	AAACAGUAGCAACUAGGUCTT

AD-14780	681-699	591	GGAUUACAAGUACCUCUGATT	592	UCAGAGGUACUUGUAAUCCTT

AD-14781	714-732	593	UAGGCCUUACUCCUGAAACTT	594	GUUUCAGGAGUAAGGCCUATT

AD-14782	377-395	595	UGUUAGAAUUUUUGCUGGATT	596	UCCAGCAAAAAUUCUAACATT

AD-14783	589-607	597	UGCUGCCACAGGAUUUUCATT	598	UGAAAAUCCUGUGGCAGCATT

AD-14784	594-612	599	CCACAGGAUUUUCAGUAGCTT	600	GCUACUGAAAAUCCUGUGGTT

AD-14785	648-666	601	AAGUUGAAAUUGCAUCCCUTT	602	AGGGAUGCAAUUUCAACUUTT

AD-14786	649-667	603	AGUUGAAAUUGCAUCCCUUTT	604	AAGGGAUGCAAUUUCAACUTT

AD-14787	587-605	605	GCUGCUGCCACAGGAUUUUTT	606	AAAAUCCUGUGGCAGCAGCTT

AD-14788	325-343	607	AGUAAAACCUGGAGUGGAATT	608	UUCCACUCCAGGUUUUACUTT

AD-14789	515-533	609	UUUUGUGUUUUCAGGUUCATT	610	UGAACCUGAAAACACAAAATT

AD-14790	516-534	611	UUUGUGUUUUCAGGUUCAUTT	612	AUGAACCUGAAAACACAAATT

AD-14791	519-537	613	GUGUUUUCAGGUUCAUGGGTT	614	CCCAUGAACCUGAAAACACTT

AD-14792	521-539	615	GUUUUCAGGUUCAUGGGUGTT	616	CACCCAUGAACCUGAAAACTT

AD-14793	522-540	617	UUUUCAGGUUCAUGGGUGCTT	618	GCACCCAUGAACCUGAAAATT

AD-14794	523-541	619	UUUCAGGUUCAUGGGUGCCTT	620	GGCACCCAUGAACCUGAAATT

AD-14795	616-634	621	AAUUGCUGCUGGAGAGGCUTT	622	AGCCUCUCCAGCAGCAAUUTT

AD-14796	657-675	623	UUGCAUCCCUUGCUACUGUTT	624	ACAGUAGCAAGGGAUGCAATT

AD-14797	761-779	625	GCUGUAGCUGGGUUUGCUGTT	626	CAGCAAACCCAGCUACAGCTT

AD-14798	645-663	627	UAGAAGUUGAAAUUGCAUCTT	628	GAUGCAAUUUCAACUUCUATT

AD-14799	647-665	629	GAAGUUGAAAUUGCAUCCCTT	630	GGGAUGCAAUUUCAACUUCTT

AD-14800	660-678	631	CAUCCCUUGCUACUGUAGATT	632	UCUACAGUAGCAAGGGAUGTT

AD-14801	324-342	633	UAGUAAAACCUGGAGUGGATT	634	UCCACUCCAGGUUUUACUATT

AD-14802	372-390	635	UUUUUUGUUAGAAUUUUUGTT	636	CAAAAAUUCUAACAAAAAATT

AD-14803	640-658	637	UACUAUAGAAGUUGAAAUUTT	638	AAUUUCAACUUCUAUAGUATT

AD-14804	562-580	639	CCUAGUUGCUACUGUUUCUTT	640	AGAAACAGUAGCAACUAGGTT

AD-14805	563-581	641	CUAGUUGCUACUGUUUCUGTT	642	CAGAAACAGUAGCAACUAGTT

AD-14806	566-584	643	GUUGCUACUGUUUCUGAGGTT	644	CCUCAGAAACAGUAGCAACTT

AD-14807	625-643	645	UGGAGAGGCUGCUGCUACUTT	646	AGUAGCAGCAGCCUCUCCATT

AD-14808	627-645	647	GAGAGGCUGCUGCUACUAUTT	648	AUAGUAGCAGCAGCCUCUCTT

AD-14809	628-646	649	AGAGGCUGCUGCUACUAUATT	650	UAUAGUAGCAGCAGCCUCUTT

AD-14810	632-650	651	GCUGCUGCUACUAUAGAAGTT	652	CUUCUAUAGUAGCAGCAGCTT

AD-14811	513-531	653	UUUUUUGUGUUUUCAGGUUTT	654	AACCUGAAAACACAAAAAATT

AD-14812	641-659	655	ACUAUAGAAGUUGAAAUUGTT	656	CAAUUUCAACUUCUAUAGUTT

AD-14813	323-341	657	UUAGUAAAACCUGGAGUGGTT	658	CCACUCCAGGUUUUACUAATT

AD-14814	717-735	659	GCCUUACUCCUGAAACAUATT	660	UAUGUUUCAGGAGUAAGGCTT

AD-14815	646-664	661	AGAAGUUGAAAUUGCAUCCTT	662	GGAUGCAAUUUCAACUUCUTT

AD-14816	592-610	663	UGCCACAGGAUUUUCAGUATT	664	UACUGAAAAUCCUGUGGCATT

AD-14817	590-608	665	GCUGCCACAGGAUUUUCAGTT	666	CUGAAAAUCCUGUGGCAGCTT

AD-14818	526-544	667	CAGGUUCAUGGGUGCCGCATT	668	UGCGGCACCCAUGAACCUGTT

AD-14819	615-633	669	AAAUUGCUGCUGGAGAGGCTT	670	GCCUCUCCAGCAGCAAUUUTT

AD-14820	617-635	671	AUUGCUGCUGGAGAGGCUGTT	672	CAGCCUCUCCAGCAGCAAUTT

AD-14821	652-670	673	UGAAAUUGCAUCCCUUGCUTT	674	AGCAAGGGAUGCAAUUUCATT

AD-14822	374-392	675	UUUUGUUAGAAUUUUUGCUTT	676	AGCAAAAAUUCUAACAAAATT

AD-14823	375-393	677	UUUGUUAGAAUUUUUGCUGTT	678	CAGCAAAAAUUCUAACAAATT

AD-14824	631-649	679	GGCUGCUGCUACUAUAGAATT	680	UUCUAUAGUAGCAGCAGCCTT

AD-14825	376-394	681	UUGUUAGAAUUUUUGCUGGTT	682	CCAGCAAAAAUUCUAACAATT

AD-14826	512-530	683	UUUUUUUGUGUUUUCAGGUTT	684	ACCUGAAAACACAAAAAAATT

AD-14827	1127-1145	685	GAAACUACUUGGGCAAUAGTT	686	CUAUUGCCCAAGUAGUUUCTT

AD-14828	1410-1428	687	AAUGGAUGUUGCCUUUACUTT	688	AGUAAAGGCAACAUCCAUUTT

AD-14829	1406-1424	689	CCUCAAUGGAUGUUGCCUUTT	690	AAGGCAACAUCCAUUGAGGTT

AD-14830	1418-1436	691	UUGCCUUUACUUUUAGGGUTT	692	ACCCUAAAAGUAAAGGCAATT

AD-14831	1126-1144	693	AGAAACUACUUGGGCAAUATT	694	UAUUGCCCAAGUAGUUUCUTT

AD-14832	1125-1143	695	AAGAAACUACUUGGGCAAUTT	696	AUUGCCCAAGUAGUUUCUUTT

AD-14833	1419-1437	697	UGCCUUUACUUUUAGGGUUTT	698	AACCCUAAAAGUAAAGGCATT

AD-14834	1420-1438	699	GCCUUUACUUUUAGGGUUGTT	700	CAACCCUAAAAGUAAAGGCTT

AD-14835	1422-1440	701	CUUUACUUUUAGGGUUGUATT	702	UACAACCCUAAAAGUAAAGTT

AD-14836	1423-1441	703	UUUACUUUUAGGGUUGUACTT	704	GUACAACCCUAAAAGUAAATT

AD-14837	1425-1443	705	UACUUUUAGGGUUGUACGGTT	706	CCGUACAACCCUAAAAGUATT

AD-14838	1123-1141	707	GGAAGAAACUACUUGGGCATT	708	UGCCCAAGUAGUUUCUUCCTT

AD-14839	1409-1427	709	CAAUGGAUGUUGCCUUUACTT	710	GUAAAGGCAACAUCCAUUGTT

AD-14840	1413-1431	711	GGAUGUUGCCUUUACUUUUTT	712	AAAAGUAAAGGCAACAUCCTT

AD-14841	1416-1434	713	UGUUGCCUUUACUUUUAGGTT	714	CCUAAAAGUAAAGGCAACATT

AD-14842	1414-1432	715	GAUGUUGCCUUUACUUUUATT	716	UAAAAGUAAAGGCAACAUCTT

AD-14843	911-929	717	CCAGAAGACUACUAUGAUATT	718	UAUCAUAGUAGUCUUCUGGTT

AD-14844	910-928	719	UCCAGAAGACUACUAUGAUTT	720	AUCAUAGUAGUCUUCUGGATT

AD-14845	1120-1138	721	UUUGGAAGAAACUACUUGGTT	722	CCAAGUAGUUUCUUCCAAATT

AD-14846	1404-1422	723	CUCCUCAAUGGAUGUUGCCTT	724	GGCAACAUCCAUUGAGGAGTT

AD-14847	1337-1355	725	CCAAAUGUGCAAUCUGGUGTT	726	CACCAGAUUGCACAUUUGGTT

AD-14848	1338-1356	727	CAAAUGUGCAAUCUGGUGATT	728	UCACCAGAUUGCACAUUUGTT

AD-14849	1397-1415	729	AGAUCUGCUCCUCAAUGGATT	730	UCCAUUGAGGAGCAGAUCUTT

AD-14850	1407-1425	731	CUCAAUGGAUGUUGCCUUUTT	732	AAAGGCAACAUCCAUUGAGTT

AD-14851	4157-4175	733	GCUCAAAUUUUAUAUAAGATT	734	UCUUAUAUAAAAUUUGAGCTT

AD-14852	4795-4813	735	AGCCUGAUUUUGGUACAUGTT	736	CAUGUACCAAAAUCAGGCUTT

AD-14853	4156-4174	737	CUCAAAUUUUAUAUAAGAATT	738	UUCUUAUAUAAAAUUUGAGTT

AD-14854	5002-5020	739	ACAAAGUGCUGAAUAGGGATT	740	UCCCUAUUCAGCACUUUGUTT

AD-14855	4792-4810	741	CUGAUUUUGGUACAUGGAATT	742	UUCCAUGUACCAAAAUCAGTT

AD-14856	4790-4808	743	GAUUUUGGUACAUGGAAUATT	744	UAUUCCAUGUACCAAAAUCTT

AD-14857	4801-4819	745	CUCAUCAGCCUGAUUUUGGTT	746	CCAAAAUCAGGCUGAUGAGTT

AD-14858	4622-4640	747	AGCCCACUUGUGUGGAUAGTT	748	CUAUCCACACAAGUGGGCUTT

AD-14859	4997-5015	749	GUGCUGAAUAGGGAGGAAUTT	750	AUUCCUCCCUAUUCAGCACTT

AD-14860	5094-5112	751	AGUAAGGGCGUGGAGGCUUTT	752	AAGCCUCCACGCCCUUACUTT

AD-14861	4564-4582	753	GUGACUUAACCCAAGAAGCTT	754	GCUUCUUGGGUUAAGUCACTT

AD-14862	5095-5113	755	UAGUAAGGGCGUGGAGGCUTT	756	AGCCUCCACGCCCUUACUATT

AD-14863	4800-4818	757	UCAUCAGCCUGAUUUUGGUTT	758	ACCAAAAUCAGGCUGAUGATT

AD-14864	4265-4283	759	GUAGAAGACCCUAAAGACUTT	760	AGUCUUUAGGGUCUUCUACTT

AD-14865	4267-4285	761	AGGUAGAAGACCCUAAAGATT	762	UCUUUAGGGUCUUCUACCUTT

AD-14866	4270-4288	763	AAAAGGUAGAAGACCCUAATT	764	UUAGGGUCUUCUACCUUUUTT

AD-14867	4269-4287	765	AAAGGUAGAAGACCCUAAATT	766	UUUAGGGUCUUCUACCUUUTT

AD-14868	2874-2892	767	GAUUGUGCAGUGGAAAGAATT	768	UUCUUUCCACUGCACAAUCTT

AD-14869	2875-2893	769	GGAUUGUGCAGUGGAAAGATT	770	UCUUUCCACUGCACAAUCCTT

AD-14870	3950-3968	771	UGUAGACAGCCAUAUGCAGTT	772	CUGCAUAUGGCUGUCUACATT

AD-14871	3896-3914	773	CAUGACUUUAACCCAGAAGTT	774	CUUCUGGGUUAAAGUCAUGTT

AD-14872	4990-5008	775	AUAGGGAGGAAUCCAUGGATT	776	UCCAUGGAUUCCUCCCUAUTT

AD-14873	4994-5012	777	CUGAAUAGGGAGGAAUCCATT	778	UGGAUUCCUCCCUAUUCAGTT

AD-14874	5000-5018	779	AAAGUGCUGAAUAGGGAGGTT	780	CCUCCCUAUUCAGCACUUUTT

AD-14875	4563-4581	781	UGACUUAACCCAAGAAGCUTT	782	AGCUUCUUGGGUUAAGUCATT

AD-14876	3895-3913	783	AUGACUUUAACCCAGAAGATT	784	UCUUCUGGGUUAAAGUCAUTT

AD-14877	4262-4280	785	GAAGACCCUAAAGACUUUCTT	786	GAAAGUCUUUAGGGUCUUCTT

AD-14878	4162-4180	787	AAAAAGCUCAAAUUUUAUATT	788	UAUAAAAUUUGAGCUUUUUTT

AD-14879	4798-4816	789	AUCAGCCUGAUUUUGGUACTT	790	GUACCAAAAUCAGGCUGAUTT

AD-14880	4799-4817	791	CAUCAGCCUGAUUUUGGUATT	792	UACCAAAAUCAGGCUGAUGTT

AD-14881	5006-5024	793	AUGGACAAAGUGCUGAAUATT	794	UAUUCAGCACUUUGUCCAUTT

AD-14882	4264-4282	795	UAGAAGACCCUAAAGACUUTT	796	AAGUCUUUAGGGUCUUCUATT

AD-14883	4268-4286	797	AAGGUAGAAGACCCUAAAGTT	798	CUUUAGGGUCUUCUACCUUTT

AD-14884	4623-4641	799	CAGCCCACUUGUGUGGAUATT	800	UAUCCACACAAGUGGGCUGTT

AD-14885	4788-4806	801	UUUUGGUACAUGGAAUAGUTT	802	ACUAUUCCAUGUACCAAAATT

AD-14886	4993-5011	803	UGAAUAGGGAGGAAUCCAUTT	804	AUGGAUUCCUCCCUAUUCATT

AD-14887	4995-5013	805	GCUGAAUAGGGAGGAAUCCTT	806	GGAUUCCUCCCUAUUCAGCTT

AD-14888	4996-5014	807	UGCUGAAUAGGGAGGAAUCTT	808	GAUUCCUCCCUAUUCAGCATT

AD-14889	3952-3970	809	UGUGUAGACAGCCAUAUGCTT	810	GCAUAUGGCUGUCUACACATT

AD-14890	4595-4613	811	UGCUUUGAUUGCUUCAGACTT	812	GUCUGAAGCAAUCAAAGCATT

AD-14891	4596-4614	813	UUGCUUUGAUUGCUUCAGATT	814	UCUGAAGCAAUCAAAGCAATT

AD-14892	4597-4615	815	AUUGCUUUGAUUGCUUCAGTT	816	CUGAAGCAAUCAAAGCAAUTT

AD-14893	4599-4617	817	CUAUUGCUUUGAUUGCUUCTT	818	GAAGCAAUCAAAGCAAUAGTT

AD-14894	4726-4744	819	AUUGCAAGGAAUGGCCUAATT	820	UUAGGCCAUUCCUUGCAAUTT

AD-14895	4753-4771	821	AUUUUCCUCCUAAUUCUGATT	822	UCAGAAUUAGGAGGAAAAUTT

AD-14896	4802-4820	823	GCUCAUCAGCCUGAUUUUGTT	824	CAAAAUCAGGCUGAUGAGCTT

AD-14897	4803-4821	825	UGCUCAUCAGCCUGAUUUUTT	826	AAAAUCAGGCUGAUGAGCATT

AD-14898	4806-4824	827	AGUUGCUCAUCAGCCUGAUTT	828	AUCAGGCUGAUGAGCAACUTT

AD-14899	5091-5109	829	AAGGGCGUGGAGGCUUUUUTT	830	AAAAAGCCUCCACGCCCUUTT

AD-14900	5093-5111	831	GUAAGGGCGUGGAGGCUUUTT	832	AAAGCCUCCACGCCCUUACTT

AD-14901	4259-4277	833	GACCCUAAAGACUUUCCUGTT	834	CAGGAAAGUCUUUAGGGUCTT

AD-14902	3901-3919	835	AGGAGCAUGACUUUAACCCTT	836	GGGUUAAAGUCAUGCUCCUTT

AD-14903	4757-4775	837	UGUGAUUUUCCUCCUAAUUTT	838	AAUUAGGAGGAAAAUCACATT

AD-14904	4758-4776	839	UUGUGAUUUUCCUCCUAAUTT	840	AUUAGGAGGAAAAUCACAATT

AD-14905	4562-4580	841	GACUUAACCCAAGAAGCUCTT	842	GAGCUUCUUGGGUUAAGUCTT

AD-14906	4585-4603	843	GCUUCAGACAAUGGUUUGGTT	844	CCAAACCAUUGUCUGAAGCTT

AD-14907	4587-4605	845	UUGCUUCAGACAAUGGUUUTT	846	AAACCAUUGUCUGAAGCAATT

AD-14908	4588-4606	847	AUUGCUUCAGACAAUGGUUTT	848	AACCAUUGUCUGAAGCAAUTT

AD-14909	4591-4609	849	UUGAUUGCUUCAGACAAUGTT	850	CAUUGUCUGAAGCAAUCAATT

AD-14910	5003-5021	851	GACAAAGUGCUGAAUAGGGTT	852	CCCUAUUCAGCACUUUGUCTT

AD-14911	4165-4183	853	AAGAAAAAGCUCAAAUUUUTT	854	AAAAUUUGAGCUUUUUCUUTT

AD-14912	4166-4184	855	AAAGAAAAAGCUCAAAUUUTT	856	AAAUUUGAGCUUUUUCUUUTT

AD-14913	4263-4281	857	AGAAGACCCUAAAGACUUUTT	858	AAAGUCUUUAGGGUCUUCUTT

AD-14914	4274-4292	859	AAAAAAAAGGUAGAAGACCTT	860	GGUCUUCUACCUUUUUUUUTT

AD-14915	4266-4284	861	GGUAGAAGACCCUAAAGACTT	862	GUCUUUAGGGUCUUCUACCTT

AD-14916	4272-4290	863	AAAAAAGGUAGAAGACCCUTT	864	AGGGUCUUCUACCUUUUUUTT

AD-14917	4271-4289	865	AAAAAGGUAGAAGACCCUATT	866	UAGGGUCUUCUACCUUUUUTT

AD-14918	4559-4577	867	UUAACCCAAGAAGCUCUUCTT	868	GAAGAGCUUCUUGGGUUAATT

AD-14919	4789-4807	869	AUUUUGGUACAUGGAAUAGTT	870	CUAUUCCAUGUACCAAAAUTT

AD-14920	4998-5016	871	AGUGCUGAAUAGGGAGGAATT	872	UUCCUCCCUAUUCAGCACUTT

AD-14921	5070-5088	873	GAGGCCAGGGAAAUUCCCUTT	874	AGGGAAUUUCCCUGGCCUCTT

AD-14922	4158-4176	875	AGCUCAAAUUUUAUAUAAGTT	876	CUUAUAUAAAAUUUGAGCUTT

AD-14923	5065-5083	877	CAGGGAAAUUCCCUUGUUUTT	878	AAACAAGGGAAUUUCCCUGTT

AD-14924	2872-2890	879	UUGUGCAGUGGAAAGAAAGTT	880	CUUUCUUUCCACUGCACAATT

AD-14925	4782-4800	881	UACAUGGAAUAGUUCAGAGTT	882	CUCUGAACUAUUCCAUGUATT

AD-14926	4783-4801	883	GUACAUGGAAUAGUUCAGATT	884	UCUGAACUAUUCCAUGUACTT

AD-14927	5064-5082	885	AGGGAAAUUCCCUUGUUUUTT	886	AAAACAAGGGAAUUUCCCUTT

AD-14928	5071-5089	887	GGAGGCCAGGGAAAUUCCCTT	888	GGGAAUUUCCCUGGCCUCCTT

AD-14929	3951-3969	889	GUGUAGACAGCCAUAUGCATT	890	UGCAUAUGGCUGUCUACACTT

AD-14930	3949-3967	891	GUAGACAGCCAUAUGCAGUTT	892	ACUGCAUAUGGCUGUCUACTT

AD-14931	4355-4373	893	GAAGACCUGUUUUGCCAUGTT	894	CAUGGCAAAACAGGUCUUCTT

AD-14932	4363-4381	895	AGUGGGAUGAAGACCUGUUTT	896	AACAGGUCUUCAUCCCACUTT

AD-14933	4356-4374	897	UGAAGACCUGUUUUGCCAUTT	898	AUGGCAAAACAGGUCUUCATT

AD-14934	4361-4379	899	UGGGAUGAAGACCUGUUUUTT	900	AAAACAGGUCUUCAUCCCATT

AD-14935	4560-4578	901	CUUAACCCAAGAAGCUCUUTT	902	AAGAGCUUCUUGGGUUAAGTT

AD-14936	2873-2891	903	AUUGUGCAGUGGAAAGAAATT	904	UUUCUUUCCACUGCACAAUTT

AD-14937	4730-4748	905	CUUUAUUGCAAGGAAUGGCTT	906	GCCAUUCCUUGCAAUAAAGTT

AD-14938	3899-3917	907	GAGCAUGACUUUAACCCAGTT	908	CUGGGUUAAAGUCAUGCUCTT

AD-14939	4756-4774	909	GUGAUUUUCCUCCUAAUUCTT	910	GAAUUAGGAGGAAAAUCACTT

AD-14940	4590-4608	911	UGAUUGCUUCAGACAAUGGTT	912	CCAUUGUCUGAAGCAAUCATT

AD-14941	4159-4177	913	AAGCUCAAAUUUUAUAUAATT	914	UUAUAUAAAAUUUGAGCUUTT

AD-14942	2743-2761	915	CUGGACAUGGAUCAAGCACTT	916	GUGCUUGAUCCAUGUCCAGTT

AD-14943	4155-4173	917	UCAAAUUUUAUAUAAGAAATT	918	UUUCUUAUAUAAAAUUUGATT

AD-14944	2871-2889	919	UGUGCAGUGGAAAGAAAGGTT	920	CCUUUCUUUCCACUGCACATT

AD-14945	4786-4804	921	UUGGUACAUGGAAUAGUUCTT	922	GAACUAUUCCAUGUACCAATT

AD-14946	4364-4382	923	AAGUGGGAUGAAGACCUGUTT	924	ACAGGUCUUCAUCCCACUUTT

AD-14947	4359-4377	925	GGAUGAAGACCUGUUUUGCTT	926	GCAAAACAGGUCUUCAUCCTT

AD-14948	2744-2762	927	UCUGGACAUGGAUCAAGCATT	928	UGCUUGAUCCAUGUCCAGATT

AD-14949	4787-4805	929	UUUGGUACAUGGAAUAGUUTT	930	AACUAUUCCAUGUACCAAATT

TABLE 1a-2

Residual luciferase
activity	SD of	Relative siRNA	SD of

	(relative to	residual	Residual	activity (normalized	relative	Relative
duplex	control siRNA	luciferase	luciferase	to positive control	siRNA	siRNA
name	treated cells)	activity	activity +/− SD	luc-siRNA)	activity	activity +/− SD

AD-14742	40.85	4.38	41 ± 4%	82.24	8.83	82 ± 9%
AD-14743	20.92	4.10	21 ± 4%	109.97	21.56	110 ± 22%
AD-14744	62.20	4.47	62 ± 4%	52.56	3.78	53 ± 4%
AD-14745	43.97	2.60	44 ± 3%	77.91	4.60	78 ± 5%
AD-14746	24.52	1.96	25 ± 2%	104.96	8.38	105 ± 8%
AD-14747	32.67	4.51	33 ± 5%	93.62	12.94	94 ± 13%
AD-14748	93.99	3.23	94 ± 3%	8.36	0.29	8 ± 0%
AD-14749	55.16	2.81	55 ± 3%	62.35	3.18	62 ± 3%
AD-14750	30.86	3.11	31 ± 3%	96.14	9.70	96 ± 10%
AD-14751	54.44	4.03	54 ± 4%	63.35	4.69	63 ± 5%
AD-14752	53.88	7.58	54 ± 8%	64.13	9.02	64 ± 9%
AD-14753	35.24	7.45	35 ± 7%	90.05	19.03	90 ± 19%
AD-14754	70.39	2.80	70 ± 3%	41.17	1.64	41 ± 2%
AD-14755	41.80	1.60	42 ± 2%	80.93	3.10	81 ± 3%
AD-14756	56.69	3.05	57 ± 3%	60.22	3.24	60 ± 3%
AD-14757	39.16	2.16	39 ± 2%	84.60	4.67	85 ± 5%
AD-14758	39.79	2.95	40 ± 3%	83.72	6.22	84 ± 6%
AD-14759	30.62	1.01	31 ± 1%	96.48	3.20	96 ± 3%
AD-14760	28.14	2.74	28 ± 3%	99.93	9.72	100 ± 10%
AD-14761	67.42	2.83	67 ± 3%	45.30	1.90	45 ± 2%
AD-14762	36.10	1.30	36 ± 1%	88.85	3.21	89 ± 3%
AD-14763	49.39	6.77	49 ± 7%	78.14	10.71	78 ± 11%
AD-14764	74.04	5.32	74 ± 5%	40.09	2.88	40 ± 3%
AD-14765	50.84	10.47	51 ± 10%	75.91	15.63	76 ± 16%
AD-14766	72.59	3.55	73 ± 4%	42.32	2.07	42 ± 2%
AD-14767	34.82	7.41	35 ± 7%	100.63	21.40	101 ± 21%
AD-14768	48.68	6.31	49 ± 6%	79.24	10.27	79 ± 10%
AD-14769	39.07	5.53	39 ± 6%	94.08	13.31	94 ± 13%
AD-14770	45.59	5.89	46 ± 6%	84.01	10.85	84 ± 11%
AD-14771	45.57	4.10	46 ± 4%	84.04	7.56	84 ± 8%
AD-14772	33.12	3.64	33 ± 4%	103.26	11.36	103 ± 11%
AD-14773	37.38	5.72	37 ± 6%	96.69	14.78	97 ± 15%
AD-14774	42.38	4.41	42 ± 4%	88.96	9.26	89 ± 9%
AD-14775	46.59	3.00	47 ± 3%	82.47	5.31	82 ± 5%
AD-14776	71.28	8.67	71 ± 9%	44.35	5.40	44 ± 5%
AD-14777	64.55	6.21	65 ± 6%	54.74	5.26	55 ± 5%
AD-14778	60.45	8.91	60 ± 9%	61.07	9.00	61 ± 9%
AD-14779	32.46	0.82	32 ± 1%	104.27	2.63	104 ± 3%
AD-14780	22.96	2.86	23 ± 3%	118.94	14.81	119 ± 15%
AD-14781	56.99	9.43	57 ± 9%	66.41	10.99	66 ± 11%
AD-14782	29.90	8.74	30 ± 9%	108.24	31.65	108 ± 32%
AD-14783	42.63	6.57	43 ± 7%	88.58	13.66	89 ± 14%
AD-14784	67.06	1.35	67 ± 1%	50.86	1.03	51 ± 1%
AD-14785	48.90	3.32	49 ± 3%	78.89	5.35	79 ± 5%
AD-14786	27.74	2.06	28 ± 2%	111.57	8.29	112 ± 8%
AD-14787	38.77	6.24	39 ± 6%	94.53	15.22	95 ± 15%
AD-14788	32.84	8.60	33 ± 9%	103.70	27.17	104 ± 27%
AD-14789	46.96	1.70	47 ± 2%	81.89	2.96	82 ± 3%
AD-14790	43.61	4.90	44 ± 5%	87.06	9.79	87 ± 10%
AD-14791	35.55	4.34	36 ± 4%	99.51	12.15	100 ± 12%
AD-14792	38.22	3.51	38 ± 4%	95.38	8.75	95 ± 9%
AD-14793	90.85	5.92	91 ± 6%	14.13	0.92	14 ± 1%
AD-14794	83.37	3.27	83 ± 3%	25.68	1.01	26 ± 1%
AD-14795	55.06	3.61	55 ± 4%	69.38	4.55	69 ± 5%
AD-14796	30.98	5.78	31 ± 6%	106.56	19.89	107 ± 20%
AD-14797	28.95	3.15	29 ± 3%	109.70	11.95	110 ± 12%
AD-14798	67.39	3.70	67 ± 4%	50.35	2.76	50 ± 3%
AD-14799	66.83	4.72	67 ± 5%	51.21	3.61	51 ± 4%
AD-14800	33.26	5.72	33 ± 6%	103.04	17.71	103 ± 18%
AD-14801	39.15	4.57	39 ± 5%	93.96	10.97	94 ± 11%
AD-14802	91.20	5.35	91 ± 5%	13.58	0.80	14 ± 1%
AD-14803	34.15	7.94	34 ± 8%	101.67	23.64	102 ± 24%
AD-14804	30.08	6.54	30 ± 7%	107.96	23.48	108 ± 23%
AD-14805	32.44	4.27	32 ± 4%	104.31	13.73	104 ± 14%
AD-14806	35.62	3.11	36 ± 3%	99.41	8.67	99 ± 9%
AD-14807	28.27	7.28	28 ± 7%	110.76	28.52	111 ± 29%
AD-14808	30.29	3.96	30 ± 4%	107.63	14.08	108 ± 14%
AD-14809	31.59	4.46	32 ± 4%	105.63	14.91	106 ± 15%
AD-14810	30.11	5.71	30 ± 6%	107.91	20.46	108 ± 20%
AD-14811	55.27	6.82	55 ± 7%	69.06	8.52	69 ± 9%
AD-14812	45.27	5.99	45 ± 6%	84.51	11.19	85 ± 11%
AD-14813	77.97	7.01	78 ± 7%	34.01	3.06	34 ± 3%
AD-14814	29.54	3.56	30 ± 4%	108.78	13.09	109 ± 13%
AD-14815	65.04	3.18	65 ± 3%	53.97	2.64	54 ± 3%
AD-14816	64.03	4.63	64 ± 5%	55.53	4.02	56 ± 4%
AD-14817	37.83	2.89	38 ± 3%	95.99	7.33	96 ± 7%
AD-14818	28.88	5.60	29 ± 6%	109.82	21.30	110 ± 21%
AD-14819	92.90	4.87	93 ± 5%	10.97	0.58	11 ± 1%
AD-14820	75.41	3.69	75 ± 4%	37.97	1.86	38 ± 2%
AD-14821	73.08	6.22	73 ± 6%	41.57	3.54	42 ± 4%
AD-14822	86.39	9.34	86 ± 9%	21.02	2.27	21 ± 2%
AD-14823	96.50	10.46	97 ± 10%	5.40	0.59	5 ± 1%
AD-14824	32.62	3.41	33 ± 3%	104.03	10.89	104 ± 11%
AD-14825	102.71	7.66	103 ± 8%	−4.18	0.31	−4 ± 0%
AD-14826	92.45	5.66	92 ± 6%	11.66	0.71	12 ± 1%
AD-14827	63.46	16.38	63 ± 16%	46.00	11.88	46 ± 12%
AD-14828	45.99	15.21	46 ± 15%	67.99	22.49	68 ± 22%
AD-14829	40.54	16.03	41 ± 16%	74.86	29.60	75 ± 30%
AD-14830	117.10	3.66	117 ± 4%	−21.52	0.67	−22 ± 1%
AD-14831	54.78	21.12	55 ± 21%	56.93	21.95	57 ± 22%
AD-14832	67.07	10.81	67 ± 11%	41.46	6.68	41 ± 7%
AD-14833	71.52	11.90	72 ± 12%	35.85	5.97	36 ± 6%
AD-14834	58.05	16.37	58 ± 16%	52.81	14.89	53 ± 15%
AD-14835	93.36	5.43	93 ± 5%	8.36	0.49	8 ± 0%
AD-14836	108.84	4.85	109 ± 5%	−11.13	0.50	−11 ± 0%
AD-14837	106.68	10.06	107 ± 10%	−8.41	0.79	−8 ± 1%
AD-14838	37.06	6.68	37 ± 7%	79.23	14.28	79 ± 14%
AD-14839	36.03	7.54	36 ± 8%	80.53	16.84	81 ± 17%
AD-14840	38.51	5.90	39 ± 6%	77.40	11.86	77 ± 12%
AD-14841	110.86	8.91	111 ± 9%	−13.67	1.10	−14 ± 1%
AD-14842	34.83	5.51	35 ± 6%	82.04	12.98	82 ± 13%
AD-14843	23.75	6.04	24 ± 6%	95.99	24.41	96 ± 24%
AD-14844	27.47	5.29	27 ± 5%	91.30	17.57	91 ± 18%
AD-14845	93.12	4.70	93 ± 5%	8.67	0.44	9 ± 0%
AD-14846	81.72	8.26	82 ± 8%	23.01	2.33	23 ± 2%
AD-14847	77.89	5.29	78 ± 5%	27.83	1.89	28 ± 2%
AD-14848	44.40	4.95	44 ± 5%	69.99	7.81	70 ± 8%
AD-14849	46.41	5.08	46 ± 5%	67.46	7.38	67 ± 7%
AD-14850	35.52	6.70	36 ± 7%	81.17	15.31	81 ± 15%
AD-14851	36.07	1.13	36 ± 1%	102.63	3.22	103 ± 3%
AD-14852	67.98	6.75	68 ± 7%	51.41	5.11	51 ± 5%
AD-14853	69.44	3.07	69 ± 3%	49.05	2.17	49 ± 2%
AD-14854	29.12	6.88	29 ± 7%	113.79	26.89	114 ± 27%
AD-14855	36.04	7.07	36 ± 7%	102.68	20.14	103 ± 20%
AD-14856	33.61	7.93	34 ± 8%	106.57	25.15	107 ± 25%
AD-14857	50.76	8.76	51 ± 9%	79.04	13.64	79 ± 14%
AD-14858	53.60	7.26	54 ± 7%	74.49	10.09	74 ± 10%
AD-14859	39.07	9.34	39 ± 9%	97.82	23.38	98 ± 23%
AD-14860	62.78	6.85	63 ± 7%	59.75	6.52	60 ± 7%
AD-14861	87.47	1.86	87 ± 2%	20.12	0.43	20 ± 0%
AD-14862	79.95	4.02	80 ± 4%	32.19	1.62	32 ± 2%
AD-14863	30.46	4.49	30 ± 4%	111.64	16.46	112 ± 16%
AD-14864	33.18	5.07	33 ± 5%	107.26	16.38	107 ± 16%
AD-14865	26.25	3.98	26 ± 4%	118.39	17.96	118 ± 18%
AD-14866	36.73	1.24	37 ± 1%	101.57	3.44	102 ± 3%
AD-14867	33.16	3.13	33 ± 3%	107.30	10.12	107 ± 10%
AD-14868	29.91	4.56	30 ± 5%	112.52	17.16	113 ± 17%
AD-14869	28.24	3.66	28 ± 4%	115.20	14.91	115 ± 15%
AD-14870	50.37	3.04	50 ± 3%	79.67	4.81	80 ± 5%
AD-14871	39.37	5.11	39 ± 5%	97.32	12.63	97 ± 13%
AD-14872	34.71	4.12	35 ± 4%	104.82	12.43	105 ± 12%
AD-14873	32.14	1.79	32 ± 2%	108.93	6.07	109 ± 6%
AD-14874	101.77	4.87	102 ± 5%	−2.85	0.14	−3 ± 0%
AD-14875	80.81	4.39	81 ± 4%	30.80	1.67	31 ± 2%
AD-14876	30.74	1.88	31 ± 2%	111.18	6.81	111 ± 7%
AD-14877	57.38	2.84	57 ± 3%	68.42	3.39	68 ± 3%
AD-14878	70.23	3.35	70 ± 3%	47.79	2.28	48 ± 2%
AD-14879	79.03	7.72	79 ± 8%	33.66	3.29	34 ± 3%
AD-14880	21.65	2.46	22 ± 2%	125.78	14.28	126 ± 14%
AD-14881	27.66	1.71	28 ± 2%	116.13	7.17	116 ± 7%
AD-14882	34.01	2.94	34 ± 3%	105.93	9.16	106 ± 9%
AD-14883	40.62	3.22	41 ± 3%	95.33	7.56	95 ± 8%
AD-14884	35.73	5.94	36 ± 6%	103.18	17.14	103 ± 17%
AD-14885	47.40	7.65	47 ± 8%	84.45	13.63	84 ± 14%
AD-14886	37.23	3.94	37 ± 4%	100.76	10.67	101 ± 11%
AD-14887	42.94	7.26	43 ± 7%	91.61	15.50	92 ± 15%
AD-14888	32.58	4.06	33 ± 4%	108.24	13.50	108 ± 14%
AD-14889	83.09	2.98	83 ± 3%	27.15	0.97	27 ± 1%
AD-14890	59.49	2.94	59 ± 3%	65.04	3.22	65 ± 3%
AD-14891	21.93	5.52	22 ± 6%	125.32	31.52	125 ± 32%
AD-14892	72.69	2.19	73 ± 2%	43.84	1.32	44 ± 1%
AD-14893	24.43	7.07	24 ± 7%	121.32	35.11	121 ± 35%
AD-14894	33.84	5.08	34 ± 5%	106.20	15.95	106 ± 16%
AD-14895	21.68	4.46	22 ± 4%	125.73	25.84	126 ± 26%
AD-14896	26.99	5.01	27 ± 5%	117.20	21.73	117 ± 22%
AD-14897	29.04	2.72	29 ± 3%	113.92	10.67	114 ± 11%
AD-14898	32.64	4.87	33 ± 5%	108.14	16.13	108 ± 16%
AD-14899	61.71	4.59	62 ± 5%	61.47	4.57	61 ± 5%
AD-14900	31.01	2.84	31 ± 3%	110.75	10.14	111 ± 10%
AD-14901	31.47	1.57	31 ± 2%	110.01	5.49	110 ± 5%
AD-14902	76.99	0.55	77 ± 1%	36.95	0.26	37 ± 0%
AD-14903	20.55	3.55	21 ± 4%	127.55	22.05	128 ± 22%
AD-14904	22.65	6.87	23 ± 7%	124.18	37.68	124 ± 38%
AD-14905	56.98	4.94	57 ± 5%	69.07	5.99	69 ± 6%
AD-14906	34.20	3.66	34 ± 4%	105.63	11.29	106 ± 11%
AD-14907	28.59	8.12	29 ± 8%	114.64	32.56	115 ± 33%
AD-14908	34.08	3.36	34 ± 3%	105.82	10.44	106 ± 10%
AD-14909	76.57	2.33	77 ± 2%	37.61	1.15	38 ± 1%
AD-14910	46.50	4.14	46 ± 4%	85.89	7.64	86 ± 8%
AD-14911	29.62	2.02	30 ± 2%	112.99	7.69	113 ± 8%
AD-14912	22.27	0.48	22 ± 0%	124.78	2.69	125 ± 3%
AD-14913	59.80	2.85	60 ± 3%	64.53	3.08	65 ± 3%
AD-14914	93.21	5.10	93 ± 5%	10.90	0.60	11 ± 1%
AD-14915	25.99	4.45	26 ± 4%	118.82	20.34	119 ± 20%
AD-14916	48.20	1.46	48 ± 1%	83.16	2.51	83 ± 3%
AD-14917	41.03	3.07	41 ± 3%	94.67	7.08	95 ± 7%
AD-14918	110.62	6.34	111 ± 6%	−17.04	0.98	−17 ± 1%
AD-14919	73.66	3.68	74 ± 4%	42.29	2.11	42 ± 2%
AD-14920	19.80	1.72	20 ± 2%	128.75	11.20	129 ± 11%
AD-14921	33.13	1.14	33 ± 1%	107.34	3.71	107 ± 4%
AD-14922	52.94	6.99	53 ± 7%	63.41	8.37	63 ± 8%
AD-14923	33.77	8.92	34 ± 9%	89.23	23.56	89 ± 24%
AD-14924	64.47	10.96	64 ± 11%	47.86	8.13	48 ± 8%
AD-14925	97.16	7.57	97 ± 8%	3.83	0.30	4 ± 0%
AD-14926	27.29	8.79	27 ± 9%	97.96	31.56	98 ± 32%
AD-14927	27.02	10.01	27 ± 10%	98.33	36.42	98 ± 36%
AD-14928	76.75	4.78	77 ± 5%	31.32	1.95	31 ± 2%
AD-14929	32.92	9.44	33 ± 9%	90.38	25.93	90 ± 26%
AD-14930	31.00	9.40	31 ± 9%	92.97	28.21	93 ± 28%
AD-14931	31.36	8.73	31 ± 9%	92.48	25.74	92 ± 26%
AD-14932	32.42	9.01	32 ± 9%	91.05	25.29	91 ± 25%
AD-14933	39.94	6.96	40 ± 7%	80.92	14.10	81 ± 14%
AD-14934	42.94	7.66	43 ± 8%	76.88	13.71	77 ± 14%
AD-14935	47.74	8.48	48 ± 8%	70.41	12.51	70 ± 13%
AD-14936	35.21	4.02	35 ± 4%	87.29	9.97	87 ± 10%
AD-14937	89.25	3.53	89 ± 4%	14.48	0.57	14 ± 1%
AD-14938	29.38	6.46	29 ± 6%	95.15	20.91	95 ± 21%
AD-14939	26.45	8.33	26 ± 8%	99.09	31.21	99 ± 31%
AD-14940	77.50	6.51	78 ± 7%	30.31	2.55	30 ± 3%
AD-14941	36.40	10.76	36 ± 11%	85.68	25.32	86 ± 25%
AD-14942	65.11	5.84	65 ± 6%	47.01	4.22	47 ± 4%
AD-14943	89.96	4.69	90 ± 5%	13.52	0.71	14 ± 1%
AD-14944	48.98	5.85	49 ± 6%	68.74	8.21	69 ± 8%
AD-14945	43.45	3.29	43 ± 3%	76.18	5.77	76 ± 6%
AD-14946	41.25	1.26	41 ± 1%	79.15	2.43	79 ± 2%
AD-14947	42.10	7.34	42 ± 7%	78.01	13.60	78 ± 14%
AD-14948	42.39	5.75	42 ± 6%	77.62	10.54	78 ± 11%
AD-14949	27.68	5.79	28 ± 6%	97.44	20.39	97 ± 20%

Tables 1b-1 and 1b-2

TABLE 1b

siRNAs targeting JCV transcripts for primary screen;
1b-1: sequences; 1b-2: assay results.
C* column describes chemistries as follows:

Description
of
chemistries:

a	exo/endo-light + 2′-O-methyl in position 2 of antisense

b	exo/endo-light: sense strand: dTsdT + 2′OMe@all Py; antisense
	strand: dTsdT + 2′OMe@ Py in uA, cA

c	exo/endo-light + 2′-O-methyl in position 2 of sense

d	exo/endo-light + 2′-O-methyl in position 2 of sense and antisense

Table 1b-1			SEQ		SEQ
duplex		position in	ID	sense strand	ID	antisense strand
name	C*	consensus	NO:	sequence (5′-3′)	NO:	sequence (5′-3′)

AD-12598	a	1426-1444	1	AcuuuuAGGGuuGuAcGGGTsT	2	CcCGuAcAACCCuAAAAGUTsT

AD-12708	b	1426-1444	3	AcuuuuAGGGuuGuAcGGGTsT	4	CCCGuAcAACCCuAAAAGUTsT

AD-12599	a	1427-1445	5	cuuuuAGGGuuGuAcGGGATsT	6	UcCCGuAcAACCCuAAAAGTsT

AD-12709	b	1427-1445	7	cuuuuAGGGuuGuAcGGGATsT	8	UCCCGuAcAACCCuAAAAGTsT

AD-12600	a	2026-2044	9	cAGAGcAcAAGGcGuAccuTsT	10	AgGuACGCCUUGUGCUCUGTsT

AD-12710	b	2026-2044	11	cAGAGcAcAAGGcGuAccuTsT	12	AGGuACGCCUUGUGCUCUGTsT

AD-12784	c	2026-2044	13	caGAGcAcAAGGcGuAccuTsT	14	AGGuACGCCUUGUGCUCUGTsT

AD-12832	d	2026-2044	15	caGAGcAcAAGGcGuAccuTsT	16	AgGuACGCCUUGUGCUCUGTsT

AD-12601	a	1431-1449	17	uAGGGuuGuAcGGGAcuGuTsT	18	AcAGUCCCGuAcAACCCuATsT

AD-12785	c	1431-1449	19	uaGGGuuGuAcGGGAcuGuTsT	20	AcAGUCCCGuAcAACCCuATsT

AD-12602	a	1432-1450	21	AGGGuuGuAcGGGAcuGuATsT	22	uacAGUCCCGuAcAACCCUTsT

AD-12711	b	1432-1450	23	AGGGuuGuAcGGGAcuGuATsT	24	uAcAGUCCCGuAcAACCCUTsT

AD-12786	c	1432-1450	25	AgGGuuGuAcGGGAcuGuATsT	26	uAcAGUCCCGuAcAACCCUTsT

AD-12833	d	1432-1450	27	AgGGuuGuAcGGGAcuGuATsT	28	uacAGUCCCGuAcAACCCUTsT

AD-12603	a	1436-1454	29	uuGuAcGGGAcuGuAAcAcTsT	30	GuGUuAcAGUCCCGuAcAATsT

AD-12712	b	1436-1454	31	uuGuAcGGGAcuGuAAcAcTsT	32	GUGUuAcAGUCCCGuAcAATsT

AD-12604	a	4794-4812	33	GccuGAuuuuGGuAcAuGGTsT	34	CcAUGuACcAAAAUcAGGCTsT

AD-12605	a	5099-5117	35	GAAGuAGuAAGGGcGuGGATsT	36	UccACGCCCUuACuACUUCTsT

AD-12713	b	5099-5117	37	GAAGuAGuAAGGGcGuGGATsT	38	UCcACGCCCUuACuACUUCTsT

AD-12787	c	5099-5117	39	GaAGuAGuAAGGGcGuGGATsT	40	UCcACGCCCUuACuACUUCTsT

AD-12834	d	5099-5117	41	GaAGuAGuAAGGGcGuGGATsT	42	UccACGCCCUuACuACUUCTsT

AD-12606	a	713-731	43	AuAGGccuuAcuccuGAAATsT	44	UuUcAGGAGuAAGGCCuAUTsT

AD-12714	b	713-731	45	AuAGGccuuAcuccuGAAATsT	46	UUUcAGGAGuAAGGCCuAUTsT

AD-12607	a	3946-3964	47	GAcAGccAuAuGcAGuAGuTsT	48	AcuACUGcAuAUGGCUGUCTsT

AD-12715	b	3946-3964	49	GAcAGccAuAuGcAGuAGuTsT	50	ACuACUGcAuAUGGCUGUCTsT

AD-12788	c	3946-3964	51	GacAGccAuAuGcAGuAGuTsT	52	ACuACUGcAuAUGGCUGUCTsT

AD-12835	d	3946-3964	53	GacAGccAuAuGcAGuAGuTsT	54	AcuACUGcAuAUGGCUGUCTsT

AD-12608	a	1128-1146	55	AAAcuAcuuGGGcAAuAGuTsT	56	AcuAUUGCCcAAGuAGUUUTsT

AD-12716	b	1128-1146	57	AAAcuAcuuGGGcAAuAGuTsT	58	ACuAUUGCCcAAGuAGUUUTsT

AD-12789	c	1128-1146	59	AaAcuAcuuGGGcAAuAGuTsT	60	ACuAUUGCCcAAGuAGUUUTsT

AD-12836	d	1128-1146	61	AaAcuAcuuGGGcAAuAGuTsT	62	AcuAUUGCCcAAGuAGUUUTsT

AD-12609	a	525-543	63	ucAGGuucAuGGGuGccGcTsT	64	GcGGcACCcAUGAACCUGATsT

AD-12717	b	525-543	65	ucAGGuucAuGGGuGccGcTsT	66	GCGGcACCcAUGAACCUGATsT

AD-12610	a	5096-5114	67	GuAGuAAGGGcGuGGAGGcTsT	68	GcCUCcACGCCCUuACuACTsT

AD-12718	b	5096-5114	69	GuAGuAAGGGcGuGGAGGcTsT	70	GCCUCcACGCCCUuACuACTsT

AD-12611	a	4727-4745	71	uAuuGcAAGGAAuGGccuATsT	72	uaGGCcAUUCCUUGcAAuATsT

AD-12719	b	4727-4745	73	uAuuGcAAGGAAuGGccuATsT	74	uAGGCcAUUCCUUGcAAuATsT

AD-12790	c	4727-4745	75	uauuGcAAGGAAuGGccuATsT	76	uAGGCcAUUCCUUGcAAuATsT

AD-12837	d	4727-4745	77	uauuGcAAGGAAuGGccuATsT	78	uaGGCcAUUCCUUGcAAuATsT

AD-12612	a	5097-5115	79	AGuAGuAAGGGcGuGGAGGTsT	80	CcUCcACGCCCUuACuACUTsT

AD-12720	b	5097-5115	81	AGuAGuAAGGGcGuGGAGGTsT	82	CCUCcACGCCCUuACuACUTsT

AD-12791	c	5097-5115	83	AguAGuAAGGGcGuGGAGGTsT	84	CCUCcACGCCCUuACuACUTsT

AD-12838	d	5097-5115	85	AguAGuAAGGGcGuGGAGGTsT	86	CcUCcACGCCCUuACuACUTsT

AD-12613	a	4601-4619	87	uGcuAuuGcuuuGAuuGcuTsT	88	AgcAAUcAAAGcAAuAGcATsT

AD-12721	b	4601-4619	89	uGcuAuuGcuuuGAuuGcuTsT	90	AGcAAUcAAAGcAAuAGcATsT

AD-12792	c	4601-4619	91	ugcuAuuGcuuuGAuuGcuTsT	92	AGcAAUcAAAGcAAuAGcATsT

AD-12839	d	4601-4619	93	ugcuAuuGcuuuGAuuGcuTsT	94	AgcAAUcAAAGcAAuAGcATsT

AD-12614	a	4600-4618	95	GcuAuuGcuuuGAuuGcuuTsT	96	AaGcAAUcAAAGcAAuAGCTsT

AD-12722	b	4600-4618	97	GcuAuuGcuuuGAuuGcuuTsT	98	AAGcAAUcAAAGcAAuAGCTsT

AD-12615	a	1421-1439	99	ccuuuAcuuuuAGGGuuGuTsT	100	AcAACCCuAAAAGuAAAGGTsT

AD-12616	a	1424-1442	101	uuAcuuuuAGGGuuGuAcGTsT	102	CguAcAACCCuAAAAGuAATsT

AD-12723	b	1424-1442	103	uuAcuuuuAGGGuuGuAcGTsT	104	CGuAcAACCCuAAAAGuAATsT

AD-12617	a	1403-1421	105	GcuccucAAuGGAuGuuGcTsT	106	GcAAcAUCcAUUGAGGAGCTsT

AD-12618	a	1534-1552	107	uuAuAAGAGGAGGAGuAGATsT	108	UcuACUCCUCCUCUuAuAATsT

AD-12724	b	1534-1552	109	uuAuAAGAGGAGGAGuAGATsT	110	UCuACUCCUCCUCUuAuAATsT

AD-12619	a	5098-5116	111	AAGuAGuAAGGGcGuGGAGTsT	112	CuCcACGCCCUuACuACUUTsT

AD-12725	b	5098-5116	113	AAGuAGuAAGGGcGuGGAGTsT	114	CUCcACGCCCUuACuACUUTsT

AD-12793	c	5098-5116	115	AaGuAGuAAGGGcGuGGAGTsT	116	CUCcACGCCCUuACuACUUTsT

AD-12840	d	5098-5116	117	AaGuAGuAAGGGcGuGGAGTsT	118	CuCcACGCCCUuACuACUUTsT

AD-12620	a	1430-1448	119	uuAGGGuuGuAcGGGAcuGTsT	120	caGUCCCGuAcAACCCuAATsT

AD-12726	b	1430-1448	121	uuAGGGuuGuAcGGGAcuGTsT	122	cAGUCCCGuAcAACCCuAATsT

AD-12621	a	1701-1719	123	GAcAuGcuuccuuGuuAcATsT	124	UguAAcAAGGAAGcAUGUCTsT

AD-12727	b	1701-1719	125	GAcAuGcuuccuuGuuAcATsT	126	UGuAAcAAGGAAGcAUGUCTsT

AD-12794	c	1701-1719	127	GacAuGcuuccuuGuuAcATsT	128	UGuAAcAAGGAAGcAUGUCTsT

AD-12841	d	1701-1719	129	GacAuGcuuccuuGuuAcATsT	130	UguAAcAAGGAAGcAUGUCTsT

AD-12622	a	2066-2084	131	uGuuGAAuGuuGGGuuccuTsT	132	AgGAACCcAAcAUUcAAcATsT

AD-12728	b	2066-2084	133	uGuuGAAuGuuGGGuuccuTsT	134	AGGAACCcAAcAUUcAAcATsT

AD-12795	c	2066-2084	135	uguuGAAuGuuGGGuuccuTsT	136	AGGAACCcAAcAUUcAAcATsT

AD-12842	d	2066-2084	137	uguuGAAuGuuGGGuuccuTsT	138	AgGAACCcAAcAUUcAAcATsT

AD-12623	a	4561-4579	139	AcuuAAcccAAGAAGcucuTsT	140	AgAGCUUCUUGGGUuAAGUTsT

AD-12729	b	4561-4579	141	AcuuAAcccAAGAAGcucuTsT	142	AGAGCUUCUUGGGUuAAGUTsT

AD-12624	a	4797-4815	143	ucAGccuGAuuuuGGuAcATsT	144	UguACcAAAAUcAGGCUGATsT

AD-12730	b	4797-4815	145	ucAGccuGAuuuuGGuAcATsT	146	UGuACcAAAAUcAGGCUGATsT

AD-12625	a	1428-1446	147	uuuuAGGGuuGuAcGGGAcTsT	148	GuCCCGuAcAACCCuAAAATsT

AD-12731	b	1428-1446	149	uuuuAGGGuuGuAcGGGAcTsT	150	GUCCCGuAcAACCCuAAAATsT

AD-12626	a	1429-1447	151	uuuAGGGuuGuAcGGGAcuTsT	152	AgUCCCGuAcAACCCuAAATsT

AD-12732	b	1429-1447	153	uuuAGGGuuGuAcGGGAcuTsT	154	AGUCCCGuAcAACCCuAAATsT

AD-12627	a	662-680	155	ucccuuGcuAcuGuAGAGGTsT	156	CcUCuAcAGuAGcAAGGGATsT

AD-12733	b	662-680	157	ucccuuGcuAcuGuAGAGGTsT	158	CCUCuAcAGuAGcAAGGGATsT

AD-12628	a	663-681	159	cccuuGcuAcuGuAGAGGGTsT	160	CcCUCuAcAGuAGcAAGGGTsT

AD-12734	b	663-681	161	cccuuGcuAcuGuAGAGGGTsT	162	CCCUCuAcAGuAGcAAGGGTsT

AD-12629	a	1402-1420	163	uGcuccucAAuGGAuGuuGTsT	164	caAcAUCcAUUGAGGAGcATsT

AD-12735	b	1402-1420	165	uGcuccucAAuGGAuGuuGTsT	166	cAAcAUCcAUUGAGGAGcATsT

AD-12796	c	1402-1420	167	ugcuccucAAuGGAuGuuGTsT	168	cAAcAUCcAUUGAGGAGcATsT

AD-12843	d	1402-1420	169	ugcuccucAAuGGAuGuuGTsT	170	caAcAUCcAUUGAGGAGcATsT

AD-12630	a	1398-1416	171	GAucuGcuccucAAuGGAuTsT	172	AuCcAUUGAGGAGcAGAUCTsT

AD-12736	b	1398-1416	173	GAucuGcuccucAAuGGAuTsT	174	AUCcAUUGAGGAGcAGAUCTsT

AD-12797	c	1398-1416	175	GaucuGcuccucAAuGGAuTsT	176	AUCcAUUGAGGAGcAGAUCTsT

AD-12844	d	1398-1416	177	GaucuGcuccucAAuGGAuTsT	178	AuCcAUUGAGGAGcAGAUCTsT

AD-12631	a	1399-1417	179	AucuGcuccucAAuGGAuGTsT	180	caUCcAUUGAGGAGcAGAUTsT

AD-12737	b	1399-1417	181	AucuGcuccucAAuGGAuGTsT	182	cAUCcAUUGAGGAGcAGAUTsT

AD-12632	a	1400-1418	183	ucuGcuccucAAuGGAuGuTsT	184	AcAUCcAUUGAGGAGcAGATsT

AD-12633	a	1401-1419	185	cuGcuccucAAuGGAuGuuTsT	186	AacAUCcAUUGAGGAGcAGTsT

AD-12738	b	1401-1419	187	cuGcuccucAAuGGAuGuuTsT	188	AAcAUCcAUUGAGGAGcAGTsT

AD-12634	a	1435-1453	189	GuuGuAcGGGAcuGuAAcATsT	190	UgUuAcAGUCCCGuAcAACTsT

AD-12739	b	1435-1453	191	GuuGuAcGGGAcuGuAAcATsT	192	UGUuAcAGUCCCGuAcAACTsT

AD-12635	a	1437-1455	193	uGuAcGGGAcuGuAAcAccTsT	194	GgUGUuAcAGUCCCGuAcATsT

AD-12740	b	1437-1455	195	uGuAcGGGAcuGuAAcAccTsT	196	GGUGUuAcAGUCCCGuAcATsT

AD-12798	c	1437-1455	197	uguAcGGGAcuGuAAcAccTsT	198	GGUGUuAcAGUCCCGuAcATsT

AD-12845	d	1437-1455	199	uguAcGGGAcuGuAAcAccTsT	200	GgUGUuAcAGUCCCGuAcATsT

AD-12636	a	1438-1456	201	GuAcGGGAcuGuAAcAccuTsT	202	AgGUGUuAcAGUCCCGuACTsT

AD-12741	b	1438-1456	203	GuAcGGGAcuGuAAcAccuTsT	204	AGGUGUuAcAGUCCCGuACTsT

AD-12637	a	4796-4814	205	cAGccuGAuuuuGGuAcAuTsT	206	AuGuACcAAAAUcAGGCUGTsT

AD-12742	b	4796-4814	207	cAGccuGAuuuuGGuAcAuTsT	208	AUGuACcAAAAUcAGGCUGTsT

AD-12799	c	4796-4814	209	caGccuGAuuuuGGuAcAuTsT	210	AUGuACcAAAAUcAGGCUGTsT

AD-12846	d	4796-4814	211	caGccuGAuuuuGGuAcAuTsT	212	AuGuACcAAAAUcAGGCUGTsT

AD-12638	a	4992-5010	213	GAAuAGGGAGGAAuccAuGTsT	214	caUGGAUUCCUCCCuAUUCTsT

AD-12743	b	4992-5010	215	GAAuAGGGAGGAAuccAuGTsT	216	cAUGGAUUCCUCCCuAUUCTsT

AD-12800	c	4992-5010	217	GaAuAGGGAGGAAuccAuGTsT	218	cAUGGAUUCCUCCCuAUUCTsT

AD-12847	d	4992-5010	219	GaAuAGGGAGGAAuccAuGTsT	220	caUGGAUUCCUCCCuAUUCTsT

AD-12639	a	4999-5017	221	AAGuGcuGAAuAGGGAGGATsT	222	UcCUCCCuAUUcAGcACUUTsT

AD-12744	b	4999-5017	223	AAGuGcuGAAuAGGGAGGATsT	224	UCCUCCCuAUUcAGcACUUTsT

AD-12801	c	4999-5017	225	AaGuGcuGAAuAGGGAGGATsT	226	UCCUCCCuAUUcAGcACUUTsT

AD-12848	d	4999-5017	227	AaGuGcuGAAuAGGGAGGATsT	228	UcCUCCCuAUUcAGcACUUTsT

AD-12640	a	630-648	229	AGGcuGcuGcuAcuAuAGATsT	230	UcuAuAGuAGcAGcAGCCUTsT

AD-12745	b	630-648	231	AGGcuGcuGcuAcuAuAGATsT	232	UCuAuAGuAGcAGcAGCCUTsT

AD-12802	c	630-648	233	AgGcuGcuGcuAcuAuAGATsT	234	UCuAuAGuAGcAGcAGCCUTsT

AD-12849	d	630-648	235	AgGcuGcuGcuAcuAuAGATsT	236	UcuAuAGuAGcAGcAGCCUTsT

AD-12641	a	3947-3965	237	AGAcAGccAuAuGcAGuAGTsT	238	CuACUGcAuAUGGCUGUCUTsT

AD-12803	c	3947-3965	239	AgAcAGccAuAuGcAGuAGTsT	240	CuACUGcAuAUGGCUGUCUTsT

AD-12642	a	524-542	241	uucAGGuucAuGGGuGccGTsT	242	CgGcACCcAUGAACCUGAATsT

AD-12746	b	524-542	243	uucAGGuucAuGGGuGccGTsT	244	CGGcACCcAUGAACCUGAATsT

AD-12643	a	3948-3966	245	uAGAcAGccAuAuGcAGuATsT	246	uaCUGcAuAUGGCUGUCuATsT

AD-12747	b	3948-3966	247	uAGAcAGccAuAuGcAGuATsT	248	uACUGcAuAUGGCUGUCuATsT

AD-12804	c	3948-3966	249	uaGAcAGccAuAuGcAGuATsT	250	uACUGcAuAUGGCUGUCuATsT

AD-12850	d	3948-3966	251	uaGAcAGccAuAuGcAGuATsT	252	uaCUGcAuAUGGCUGUCuATsT

AD-12644	a	3900-3918	253	GGAGcAuGAcuuuAAcccATsT	254	UgGGUuAAAGUcAUGCUCCTsT

AD-12748	b	3900-3918	255	GGAGcAuGAcuuuAAcccATsT	256	UGGGUuAAAGUcAUGCUCCTsT

AD-12805	c	3900-3918	257	GgAGcAuGAcuuuAAcccATsT	258	UGGGUuAAAGUcAUGCUCCTsT

AD-12851	d	3900-3918	259	GgAGcAuGAcuuuAAcccATsT	260	UgGGUuAAAGUcAUGCUCCTsT

AD-12645	a	1417-1435	261	GuuGccuuuAcuuuuAGGGTsT	262	CcCuAAAAGuAAAGGcAACTsT

AD-12749	b	1417-1435	263	GuuGccuuuAcuuuuAGGGTsT	264	CCCuAAAAGuAAAGGcAACTsT

AD-12646	a	4565-4583	265	uGuGAcuuAAcccAAGAAGTsT	266	CuUCUUGGGUuAAGUcAcATsT

AD-12750	b	4565-4583	267	uGuGAcuuAAcccAAGAAGTsT	268	CUUCUUGGGUuAAGUcAcATsT

AD-12806	c	4565-4583	269	uguGAcuuAAcccAAGAAGTsT	270	CUUCUUGGGUuAAGUcAcATsT

AD-12852	d	4565-4583	271	uguGAcuuAAcccAAGAAGTsT	272	CuUCUUGGGUuAAGUcAcATsT

AD-12647	a	4598-4616	273	uAuuGcuuuGAuuGcuucATsT	274	UgAAGcAAUcAAAGcAAuATsT

AD-12751	b	4598-4616	275	uAuuGcuuuGAuuGcuucATsT	276	UGAAGcAAUcAAAGcAAuATsT

AD-12807	c	4598-4616	277	uauuGcuuuGAuuGcuucATsT	278	UGAAGcAAUcAAAGcAAuATsT

AD-12853	d	4598-4616	279	uauuGcuuuGAuuGcuucATsT	280	UgAAGcAAUcAAAGcAAuATsT

AD-12648	a	2060-2078	281	AuAuccuGuuGAAuGuuGGTsT	282	CcAAcAUUcAAcAGGAuAUTsT

AD-12649	a	4729-4747	283	uuuAuuGcAAGGAAuGGccTsT	284	GgCcAUUCCUUGcAAuAAATsT

AD-12752	b	4729-4747	285	uuuAuuGcAAGGAAuGGccTsT	286	GGCcAUUCCUUGcAAuAAATsT

AD-12650	a	1122-1140	287	uGGAAGAAAcuAcuuGGGcTsT	288	GcCcAAGuAGUUUCUUCcATsT

AD-12753	b	1122-1140	289	uGGAAGAAAcuAcuuGGGcTsT	290	GCCcAAGuAGUUUCUUCcATsT

AD-12808	c	1122-1140	291	ugGAAGAAAcuAcuuGGGcTsT	292	GCCcAAGuAGUUUCUUCcATsT

AD-12854	d	1122-1140	293	ugGAAGAAAcuAcuuGGGcTsT	294	GcCcAAGuAGUUUCUUCcATsT

AD-12651	a	4261-4279	295	AAGAcccuAAAGAcuuuccTsT	296	GgAAAGUCUUuAGGGUCUUTsT

AD-12754	b	4261-4279	297	AAGAcccuAAAGAcuuuccTsT	298	GGAAAGUCUUuAGGGUCUUTsT

AD-12809	c	4261-4279	299	AaGAcccuAAAGAcuuuccTsT	300	GGAAAGUCUUuAGGGUCUUTsT

AD-12855	d	4261-4279	301	AaGAcccuAAAGAcuuuccTsT	302	GgAAAGUCUUuAGGGUCUUTsT

AD-12652	a	1412-1430	303	uGGAuGuuGccuuuAcuuuTsT	304	AaAGuAAAGGcAAcAUCcATsT

AD-12755	b	1412-1430	305	uGGAuGuuGccuuuAcuuuTsT	306	AAAGuAAAGGcAAcAUCcATsT

AD-12810	c	1412-1430	307	ugGAuGuuGccuuuAcuuuTsT	308	AAAGuAAAGGcAAcAUCcATsT

AD-12856	d	1412-1430	309	ugGAuGuuGccuuuAcuuuTsT	310	AaAGuAAAGGcAAcAUCcATsT

AD-12653	a	4592-4610	311	uuuGAuuGcuucAGAcAAuTsT	312	AuUGUCUGAAGcAAUcAAATsT

AD-12756	b	4592-4610	313	uuuGAuuGcuucAGAcAAuTsT	314	AUUGUCUGAAGcAAUcAAATsT

AD-12654	a	4991-5009	315	AAuAGGGAGGAAuccAuGGTsT	316	CcAUGGAUUCCUCCCuAUUTsT

AD-12811	c	4991-5009	317	AauAGGGAGGAAuccAuGGTsT	318	CcAUGGAUUCCUCCCuAUUTsT

AD-12655	a	5004-5022	319	GGAcAAAGuGcuGAAuAGGTsT	320	CcuAUUcAGcACUUUGUCCTsT

AD-12757	b	5004-5022	321	GGAcAAAGuGcuGAAuAGGTsT	322	CCuAUUcAGcACUUUGUCCTsT

AD-12812	c	5004-5022	323	GgAcAAAGuGcuGAAuAGGTsT	324	CCuAUUcAGcACUUUGUCCTsT

AD-12857	d	5004-5022	325	GgAcAAAGuGcuGAAuAGGTsT	326	CcuAUUcAGcACUUUGUCCTsT

AD-12656	a	5005-5023	327	uGGAcAAAGuGcuGAAuAGTsT	328	CuAUUcAGcACUUUGUCcATsT

AD-12813	c	5005-5023	329	ugGAcAAAGuGcuGAAuAGTsT	330	CuAUUcAGcACUUUGUCcATsT

AD-12657	a	654-672	331	AAAuuGcAucccuuGcuAcTsT	332	GuAGcAAGGGAUGcAAUUUTsT

AD-12814	c	654-672	333	AaAuuGcAucccuuGcuAcTsT	334	GuAGcAAGGGAUGcAAUUUTsT

AD-12658	a	659-677	335	GcAucccuuGcuAcuGuAGTsT	336	CuAcAGuAGcAAGGGAUGCTsT

AD-12659	a	4273-4291	337	AAAAAAAGGuAGAAGAcccTsT	338	GgGUCUUCuACCUUUUUUUTsT

AD-12758	b	4273-4291	339	AAAAAAAGGuAGAAGAcccTsT	340	GGGUCUUCuACCUUUUUUUTsT

AD-12815	c	4273-4291	341	AaAAAAAGGuAGAAGAcccTsT	342	GGGUCUUCuACCUUUUUUUTsT

AD-12858	d	4273-4291	343	AaAAAAAGGuAGAAGAcccTsT	344	GgGUCUUCuACCUUUUUUUTsT

AD-12660	a	2025-2043	345	AcAGAGcAcAAGGcGuAccTsT	346	GguACGCCUUGUGCUCUGUTsT

AD-12759	b	2025-2043	347	AcAGAGcAcAAGGcGuAccTsT	348	GGuACGCCUUGUGCUCUGUTsT

AD-12661	a	4791-4809	349	uGAuuuuGGuAcAuGGAAuTsT	350	AuUCcAUGuACcAAAAUcATsT

AD-12760	b	4791-4809	351	uGAuuuuGGuAcAuGGAAuTsT	352	AUUCcAUGuACcAAAAUcATsT

AD-12816	c	4791-4809	353	ugAuuuuGGuAcAuGGAAuTsT	354	AUUCcAUGuACcAAAAUcATsT

AD-12859	d	4791-4809	355	ugAuuuuGGuAcAuGGAAuTsT	356	AuUCcAUGuACcAAAAUcATsT

AD-12662	a	1433-1451	357	GGGuuGuAcGGGAcuGuAATsT	358	UuAcAGUCCCGuAcAACCCTsT

AD-12817	c	1433-1451	359	GgGuuGuAcGGGAcuGuAATsT	360	UuAcAGUCCCGuAcAACCCTsT

AD-12663	a	1434-1452	361	GGuuGuAcGGGAcuGuAAcTsT	362	GuuAcAGUCCCGuAcAACCTsT

AD-12761	b	1434-1452	363	GGuuGuAcGGGAcuGuAAcTsT	364	GUuAcAGUCCCGuAcAACCTsT

AD-12818	c	1434-1452	365	GguuGuAcGGGAcuGuAAcTsT	366	GUuAcAGUCCCGuAcAACCTsT

AD-12860	d	1434-1452	367	GguuGuAcGGGAcuGuAAcTsT	368	GuuAcAGUCCCGuAcAACCTsT

AD-12664	a	1440-1458	369	AcGGGAcuGuAAcAccuGcTsT	370	GcAGGUGUuAcAGUCCCGUTsT

AD-12665	a	1442-1460	371	GGGAcuGuAAcAccuGcucTsT	372	GaGcAGGUGUuAcAGUCCCTsT

AD-12762	b	1442-1460	373	GGGAcuGuAAcAccuGcucTsT	374	GAGcAGGUGUuAcAGUCCCTsT

AD-12819	c	1442-1460	375	GgGAcuGuAAcAccuGcucTsT	376	GAGcAGGUGUuAcAGUCCCTsT

AD-12861	d	1442-1460	377	GgGAcuGuAAcAccuGcucTsT	378	GaGcAGGUGUuAcAGUCCCTsT

AD-12666	a	1608-1626	379	AcuccAGAAAuGGGuGAccTsT	380	GgUcACCcAUUUCUGGAGUTsT

AD-12763	b	1608-1626	381	AcuccAGAAAuGGGuGAccTsT	382	GGUcACCcAUUUCUGGAGUTsT

AD-12667	a	4793-4811	383	ccuGAuuuuGGuAcAuGGATsT	384	UccAUGuACcAAAAUcAGGTsT

AD-12764	b	4793-4811	385	ccuGAuuuuGGuAcAuGGATsT	386	UCcAUGuACcAAAAUcAGGTsT

AD-12668	a	5001-5019	387	cAAAGuGcuGAAuAGGGAGTsT	388	CuCCCuAUUcAGcACUUUGTsT

AD-12765	b	5001-5019	389	cAAAGuGcuGAAuAGGGAGTsT	390	CUCCCuAUUcAGcACUUUGTsT

AD-12820	c	5001-5019	391	caAAGuGcuGAAuAGGGAGTsT	392	CUCCCuAUUcAGcACUUUGTsT

AD-12862	d	5001-5019	393	caAAGuGcuGAAuAGGGAGTsT	394	CuCCCuAUUcAGcACUUUGTsT

AD-12669	a	5066-5084	395	ccAGGGAAAuucccuuGuuTsT	396	AacAAGGGAAUUUCCCUGGTsT

AD-12766	b	5066-5084	397	ccAGGGAAAuucccuuGuuTsT	398	AAcAAGGGAAUUUCCCUGGTsT

AD-12670	a	5069-5087	399	AGGccAGGGAAAuucccuuTsT	400	AaGGGAAUUUCCCUGGCCUTsT

AD-12767	b	5069-5087	401	AGGccAGGGAAAuucccuuTsT	402	AAGGGAAUUUCCCUGGCCUTsT

AD-12821	c	5069-5087	403	AgGccAGGGAAAuucccuuTsT	404	AAGGGAAUUUCCCUGGCCUTsT

AD-12863	d	5069-5087	405	AgGccAGGGAAAuucccuuTsT	406	AaGGGAAUUUCCCUGGCCUTsT

AD-12671	a	564-582	407	uAGuuGcuAcuGuuucuGATsT	408	UcAGAAAcAGuAGcAACuATsT

AD-12822	c	564-582	409	uaGuuGcuAcuGuuucuGATsT	410	UcAGAAAcAGuAGcAACuATsT

AD-12672	a	633-651	411	cuGcuGcuAcuAuAGAAGuTsT	412	AcUUCuAuAGuAGcAGcAGTsT

AD-12768	b	633-651	413	cuGcuGcuAcuAuAGAAGuTsT	414	ACUUCuAuAGuAGcAGcAGTsT

AD-12673	a	634-652	415	uGcuGcuAcuAuAGAAGuuTsT	416	AaCUUCuAuAGuAGcAGcATsT

AD-12769	b	634-652	417	uGcuGcuAcuAuAGAAGuuTsT	418	AACUUCuAuAGuAGcAGcATsT

AD-12823	c	634-652	419	ugcuGcuAcuAuAGAAGuuTsT	420	AACUUCuAuAGuAGcAGcATsT

AD-12864	d	634-652	421	ugcuGcuAcuAuAGAAGuuTsT	422	AaCUUCuAuAGuAGcAGcATsT

AD-12674	a	635-653	423	GcuGcuAcuAuAGAAGuuGTsT	424	caACUUCuAuAGuAGcAGCTsT

AD-12770	b	635-653	425	GcuGcuAcuAuAGAAGuuGTsT	426	cAACUUCuAuAGuAGcAGCTsT

AD-12675	a	636-654	427	cuGcuAcuAuAGAAGuuGATsT	428	UcAACUUCuAuAGuAGcAGTsT

AD-12676	a	637-655	429	uGcuAcuAuAGAAGuuGAATsT	430	UucAACUUCuAuAGuAGcATsT

AD-12771	b	637-655	431	uGcuAcuAuAGAAGuuGAATsT	432	UUcAACUUCuAuAGuAGcATsT

AD-12824	c	637-655	433	ugcuAcuAuAGAAGuuGAATsT	434	UUcAACUUCuAuAGuAGcATsT

AD-12865	d	637-655	435	ugcuAcuAuAGAAGuuGAATsT	436	UucAACUUCuAuAGuAGcATsT

AD-12677	a	912-930	437	cAGAAGAcuAcuAuGAuAuTsT	438	AuAUcAuAGuAGUCUUCUGTsT

AD-12825	c	912-930	439	caGAAGAcuAcuAuGAuAuTsT	440	AuAUcAuAGuAGUCUUCUGTsT

AD-12678	a	4153-4171	441	AAAuuuuAuAuAAGAAAcuTsT	442	AgUUUCUuAuAuAAAAUUUTsT

AD-12772	b	4153-4171	443	AAAuuuuAuAuAAGAAAcuTsT	444	AGUUUCUuAuAuAAAAUUUTsT

AD-12826	c	4153-4171	445	AaAuuuuAuAuAAGAAAcuTsT	446	AGUUUCUuAuAuAAAAUUUTsT

AD-12866	d	4153-4171	447	AaAuuuuAuAuAAGAAAcuTsT	448	AgUUUCUuAuAuAAAAUUUTsT

AD-12679	a	4779-4797	449	AuGGAAuAGuucAGAGGuuTsT	450	AaCCUCUGAACuAUUCcAUTsT

AD-12773	b	4779-4797	451	AuGGAAuAGuucAGAGGuuTsT	452	AACCUCUGAACuAUUCcAUTsT

AD-12680	a	4780-4798	453	cAuGGAAuAGuucAGAGGuTsT	454	AcCUCUGAACuAUUCcAUGTsT

AD-12774	b	4780-4798	455	cAuGGAAuAGuucAGAGGuTsT	456	ACCUCUGAACuAUUCcAUGTsT

AD-12827	c	4780-4798	457	cauGGAAuAGuucAGAGGuTsT	458	ACCUCUGAACuAUUCcAUGTsT

AD-12867	d	4780-4798	459	cauGGAAuAGuucAGAGGuTsT	460	AcCUCUGAACuAUUCcAUGTsT

AD-12681	a	4781-4799	461	AcAuGGAAuAGuucAGAGGTsT	462	CcUCUGAACuAUUCcAUGUTsT

AD-12775	b	4781-4799	463	AcAuGGAAuAGuucAGAGGTsT	464	CCUCUGAACuAUUCcAUGUTsT

AD-12682	a	4784-4802	465	GGuAcAuGGAAuAGuucAGTsT	466	CuGAACuAUUCcAUGuACCTsT

AD-12776	b	4784-4802	467	GGuAcAuGGAAuAGuucAGTsT	468	CUGAACuAUUCcAUGuACCTsT

AD-12828	c	4784-4802	469	GguAcAuGGAAuAGuucAGTsT	470	CUGAACuAUUCcAUGuACCTsT

AD-12868	d	4784-4802	471	GguAcAuGGAAuAGuucAGTsT	472	CuGAACuAUUCcAUGuACCTsT

AD-12683	a	4785-4803	473	uGGuAcAuGGAAuAGuucATsT	474	UgAACuAUUCcAUGuACcATsT

AD-12777	b	4785-4803	475	uGGuAcAuGGAAuAGuucATsT	476	UGAACuAUUCcAUGuACcATsT

AD-12829	c	4785-4803	477	ugGuAcAuGGAAuAGuucATsT	478	UGAACuAUUCcAUGuACcATsT

AD-12869	d	4785-4803	479	ugGuAcAuGGAAuAGuucATsT	480	UgAACuAUUCcAUGuACcATsT

AD-12684	a	719-737	481	cuuAcuccuGAAAcAuAuGTsT	482	cauAUGUUUcAGGAGuAAGTsT

AD-12778	b	719-737	483	cuuAcuccuGAAAcAuAuGTsT	484	cAuAUGUUUcAGGAGuAAGTsT

AD-12685	a	909-927	485	AuccAGAAGAcuAcuAuGATsT	486	UcAuAGuAGUCUUCUGGAUTsT

AD-12686	a	1119-1137	487	uuuuGGAAGAAAcuAcuuGTsT	488	caAGuAGUUUCUUCcAAAATsT

AD-12779	b	1119-1137	489	uuuuGGAAGAAAcuAcuuGTsT	490	cAAGuAGUUUCUUCcAAAATsT

AD-12687	a	1121-1139	491	uuGGAAGAAAcuAcuuGGGTsT	492	CccAAGuAGUUUCUUCcAATsT

AD-12780	b	1121-1139	493	uuGGAAGAAAcuAcuuGGGTsT	494	CCcAAGuAGUUUCUUCcAATsT

AD-12688	a	4357-4375	495	AuGAAGAccuGuuuuGccATsT	496	UgGcAAAAcAGGUCUUcAUTsT

AD-12781	b	4357-4375	497	AuGAAGAccuGuuuuGccATsT	498	UGGcAAAAcAGGUCUUcAUTsT

AD-12689	a	4358-4376	499	GAuGAAGAccuGuuuuGccTsT	500	GgcAAAAcAGGUCUUcAUCTsT

AD-12782	b	4358-4376	501	GAuGAAGAccuGuuuuGccTsT	502	GGcAAAAcAGGUCUUcAUCTsT

AD-12830	c	4358-4376	503	GauGAAGAccuGuuuuGccTsT	504	GGcAAAAcAGGUCUUcAUCTsT

AD-12870	d	4358-4376	505	GauGAAGAccuGuuuuGccTsT	506	GgcAAAAcAGGUCUUcAUCTsT

AD-12690	a	4360-4378	507	GGGAuGAAGAccuGuuuuGTsT	508	caAAAcAGGUCUUcAUCCCTsT

AD-12783	b	4360-4378	509	GGGAuGAAGAccuGuuuuGTsT	510	cAAAAcAGGUCUUcAUCCCTsT

AD-12831	c	4360-4378	511	GgGAuGAAGAccuGuuuuGTsT	512	cAAAAcAGGUCUUcAUCCCTsT

AD-12871	d	4360-4378	513	GgGAuGAAGAccuGuuuuGTsT	514	caAAAcAGGUCUUcAUCCCTsT

TABLE 1b-2

Residual luciferase
activity	SD of	Relative siRNA	SD of

AD-12598	91	11	91 ± 11%	9	2	9 ± 2%
AD-12708	32	5	32 ± 5%	76	17	76 ± 17%
AD-12599	25	6	25 ± 6%	79	13	79 ± 13%
AD-12709	16	4	16 ± 4%	97	26	97 ± 26%
AD-12600	79	9	79 ± 9%	21	3	21 ± 3%
AD-12710	25	4	25 ± 4%	85	24	85 ± 24%
AD-12784	23	2	23 ± 2%	87	14	87 ± 14%
AD-12832	84	11	84 ± 11%	18	4	18 ± 4%
AD-12601	102	8	102 ± 8%	−6	1	−6 ± 1%
AD-12785	95	10	95 ± 10%	6	1	6 ± 1%
AD-12602	107	9	107 ± 9%	−11	2	−11 ± 2%
AD-12711	70	4	70 ± 4%	34	3	34 ± 3%
AD-12786	69	8	69 ± 8%	35	7	35 ± 7%
AD-12833	94	8	94 ± 8%	7	1	7 ± 1%
AD-12603	100	9	100 ± 9%	−4	1	−4 ± 1%
AD-12712	27	5	27 ± 5%	82	16	82 ± 16%
AD-12604	15	2	15 ± 2%	94	13	94 ± 13%
AD-12605	94	5	94 ± 5%	7	0	7 ± 0%
AD-12713	61	10	61 ± 10%	41	8	41 ± 8%
AD-12787	55	6	55 ± 6%	47	6	47 ± 6%
AD-12834	92	16	92 ± 16%	8	2	8 ± 2%
AD-12606	78	3	78 ± 3%	25	1	25 ± 1%
AD-12714	63	6	63 ± 6%	42	5	42 ± 5%
AD-12607	101	9	101 ± 9%	−1	0	−1 ± 0%
AD-12715	101	5	101 ± 5%	−1	0	−1 ± 0%
AD-12788	85	18	85 ± 18%	15	4	15 ± 4%
AD-12835	95	9	95 ± 9%	6	1	6 ± 1%
AD-12608	103	13	103 ± 13%	−3	0	−3 ± 0%
AD-12716	81	9	81 ± 9%	22	3	22 ± 3%
AD-12789	61	4	61 ± 4%	44	4	44 ± 4%
AD-12836	103	11	103 ± 11%	−3	0	−3 ± 0%
AD-12609	108	19	108 ± 19%	−9	2	−9 ± 2%
AD-12717	94	17	94 ± 17%	7	1	7 ± 1%
AD-12610	88	9	88 ± 9%	14	2	14 ± 2%
AD-12718	39	4	39 ± 4%	64	8	64 ± 8%
AD-12611	38	6	38 ± 6%	69	12	69 ± 12%
AD-12719	26	4	26 ± 4%	78	13	78 ± 13%
AD-12790	17	3	17 ± 3%	87	18	87 ± 18%
AD-12837	22	4	22 ± 4%	81	16	81 ± 16%
AD-12612	100	6	100 ± 6%	0	0	0 ± 0%
AD-12720	73	6	73 ± 6%	28	3	28 ± 3%
AD-12791	46	9	46 ± 9%	57	12	57 ± 12%
AD-12838	97	15	97 ± 15%	3	1	3 ± 1%
AD-12613	26	4	26 ± 4%	82	15	82 ± 15%
AD-12721	10	1	10 ± 1%	94	12	94 ± 12%
AD-12792	10	3	10 ± 3%	94	40	94 ± 40%
AD-12839	22	3	22 ± 3%	81	12	81 ± 12%
AD-12614	15	5	15 ± 5%	94	38	94 ± 38%
AD-12722	6	1	6 ± 1%	98	26	98 ± 26%
AD-12615	93	4	93 ± 4%	8	0	8 ± 0%
AD-12616	95	4	95 ± 4%	5	0	5 ± 0%
AD-12723	73	7	73 ± 7%	30	3	30 ± 3%
AD-12617	88	10	88 ± 10%	13	2	13 ± 2%
AD-12618	42	7	42 ± 7%	60	7	60 ± 7%
AD-12724	21	5	21 ± 5%	89	32	89 ± 32%
AD-12619	95	7	95 ± 7%	6	1	6 ± 1%
AD-12725	71	2	71 ± 2%	30	1	30 ± 1%
AD-12793	54	7	54 ± 7%	48	7	48 ± 7%
AD-12840	94	9	94 ± 9%	7	1	7 ± 1%
AD-12620	106	7	106 ± 7%	−8	1	−8 ± 1%
AD-12726	100	7	100 ± 7%	0	0	0 ± 0%
AD-12621	107	9	107 ± 9%	−7	1	−7 ± 1%
AD-12727	47	4	47 ± 4%	60	8	60 ± 8%
AD-12794	40	8	40 ± 8%	67	20	67 ± 20%
AD-12841	78	13	78 ± 13%	25	8	25 ± 8%
AD-12622	16	4	16 ± 4%	92	29	92 ± 29%
AD-12728	25	6	25 ± 6%	84	29	84 ± 29%
AD-12795	23	3	23 ± 3%	86	20	86 ± 20%
AD-12842	19	4	19 ± 4%	91	20	91 ± 20%
AD-12623	103	9	103 ± 9%	−3	0	−3 ± 0%
AD-12729	84	8	84 ± 8%	17	2	17 ± 2%
AD-12624	31	4	31 ± 4%	77	12	77 ± 12%
AD-12730	18	1	18 ± 1%	85	3	85 ± 3%
AD-12625	94	10	94 ± 10%	5	1	5 ± 1%
AD-12731	57	4	57 ± 4%	48	4	48 ± 4%
AD-12626	99	7	99 ± 7%	0	0	0 ± 0%
AD-12732	82	5	82 ± 5%	20	1	20 ± 1%
AD-12627	80	6	80 ± 6%	22	2	22 ± 2%
AD-12733	65	6	65 ± 6%	39	4	39 ± 4%
AD-12628	81	6	81 ± 6%	21	2	21 ± 2%
AD-12734	82	7	82 ± 7%	21	2	21 ± 2%
AD-12629	113	11	113 ± 11%	−14	2	−14 ± 2%
AD-12735	90	9	90 ± 9%	11	1	11 ± 1%
AD-12796	92	8	92 ± 8%	9	1	9 ± 1%
AD-12843	117	7	117 ± 7%	−19	1	−19 ± 1%
AD-12630	124	3	124 ± 3%	−27	1	−27 ± 1%
AD-12736	85	4	85 ± 4%	16	1	16 ± 1%
AD-12797	52	1	52 ± 1%	53	1	53 ± 1%
AD-12844	96	4	96 ± 4%	5	0	5 ± 0%
AD-12631	110	11	110 ± 11%	−12	1	−12 ± 1%
AD-12737	115	13	115 ± 13%	−17	2	−17 ± 2%
AD-12632	106	2	106 ± 2%	−7	0	−7 ± 0%
AD-12633	107	12	107 ± 12%	−8	1	−8 ± 1%
AD-12738	88	5	88 ± 5%	14	1	14 ± 1%
AD-12634	79	5	79 ± 5%	24	1	24 ± 1%
AD-12739	69	8	69 ± 8%	35	6	35 ± 6%
AD-12635	75	8	75 ± 8%	25	6	25 ± 6%
AD-12740	65	8	65 ± 8%	40	8	40 ± 8%
AD-12798	56	4	56 ± 4%	50	6	50 ± 6%
AD-12845	74	6	74 ± 6%	30	3	30 ± 3%
AD-12636	89	8	89 ± 8%	9	1	9 ± 1%
AD-12741	31	4	31 ± 4%	78	14	78 ± 14%
AD-12637	16	2	16 ± 2%	93	14	93 ± 14%
AD-12742	18	3	18 ± 3%	85	14	85 ± 14%
AD-12799	18	4	18 ± 4%	86	22	86 ± 22%
AD-12846	15	2	15 ± 2%	89	14	89 ± 14%
AD-12638	95	6	95 ± 6%	5	0	5 ± 0%
AD-12743	23	4	23 ± 4%	81	15	81 ± 15%
AD-12800	14	1	14 ± 1%	90	10	90 ± 10%
AD-12847	90	12	90 ± 12%	10	2	10 ± 2%
AD-12639	113	11	113 ± 11%	−15	2	−15 ± 2%
AD-12744	42	4	42 ± 4%	60	7	60 ± 7%
AD-12801	34	3	34 ± 3%	68	8	68 ± 8%
AD-12848	114	3	114 ± 3%	−14	0	−14 ± 0%
AD-12640	96	11	96 ± 11%	4	1	4 ± 1%
AD-12745	52	7	52 ± 7%	53	8	53 ± 8%
AD-12802	74	9	74 ± 9%	29	4	29 ± 4%
AD-12849	111	5	111 ± 5%	−12	1	−12 ± 1%
AD-12641	103	8	103 ± 8%	−3	0	−3 ± 0%
AD-12803	94	13	94 ± 13%	6	1	6 ± 1%
AD-12642	105	3	105 ± 3%	−6	0	−6 ± 0%
AD-12746	100	9	100 ± 9%	0	0	0 ± 0%
AD-12643	33	4	33 ± 4%	74	10	74 ± 10%
AD-12747	21	3	21 ± 3%	83	13	83 ± 13%
AD-12804	25	4	25 ± 4%	78	14	78 ± 14%
AD-12850	28	4	28 ± 4%	75	11	75 ± 11%
AD-12644	82	7	82 ± 7%	20	2	20 ± 2%
AD-12748	25	4	25 ± 4%	78	14	78 ± 14%
AD-12805	23	7	23 ± 7%	80	30	80 ± 30%
AD-12851	61	7	61 ± 7%	41	5	41 ± 5%
AD-12645	112	6	112 ± 6%	−14	1	−14 ± 1%
AD-12749	86	10	86 ± 10%	16	2	16 ± 2%
AD-12646	94	10	94 ± 10%	6	1	6 ± 1%
AD-12750	93	11	93 ± 11%	7	1	7 ± 1%
AD-12806	77	8	77 ± 8%	24	3	24 ± 3%
AD-12852	96	4	96 ± 4%	5	0	5 ± 0%
AD-12647	27	3	27 ± 3%	81	11	81 ± 11%
AD-12751	29	6	29 ± 6%	74	19	74 ± 19%
AD-12807	31	2	31 ± 2%	72	6	72 ± 6%
AD-12853	26	3	26 ± 3%	78	11	78 ± 11%
AD-12648	81	9	81 ± 9%	17	3	17 ± 3%
AD-12649	92	9	92 ± 9%	8	1	8 ± 1%
AD-12752	71	9	71 ± 9%	30	5	30 ± 5%
AD-12650	81	2	81 ± 2%	21	1	21 ± 1%
AD-12753	57	1	57 ± 1%	48	1	48 ± 1%
AD-12808	52	4	52 ± 4%	54	5	54 ± 5%
AD-12854	77	5	77 ± 5%	26	2	26 ± 2%
AD-12651	89	6	89 ± 6%	13	1	13 ± 1%
AD-12754	88	7	88 ± 7%	12	1	12 ± 1%
AD-12809	67	6	67 ± 6%	35	4	35 ± 4%
AD-12855	88	10	88 ± 10%	12	2	12 ± 2%
AD-12652	91	2	91 ± 2%	10	0	10 ± 0%
AD-12755	40	3	40 ± 3%	67	6	67 ± 6%
AD-12810	35	1	35 ± 1%	72	3	72 ± 3%
AD-12856	75	8	75 ± 8%	28	4	28 ± 4%
AD-12653	79	8	79 ± 8%	23	3	23 ± 3%
AD-12756	17	5	17 ± 5%	86	27	86 ± 27%
AD-12654	97	6	97 ± 6%	3	0	3 ± 0%
AD-12811	74	5	74 ± 5%	27	2	27 ± 2%
AD-12655	46	6	46 ± 6%	59	9	59 ± 9%
AD-12757	14	0	14 ± 0%	89	2	89 ± 2%
AD-12812	12	3	12 ± 3%	92	28	92 ± 28%
AD-12857	35	7	35 ± 7%	70	17	70 ± 17%
AD-12656	10	3	10 ± 3%	99	33	99 ± 33%
AD-12813	9	1	9 ± 1%	95	18	95 ± 18%
AD-12657	108	1	108 ± 1%	−9	0	−9 ± 0%
AD-12814	101	4	101 ± 4%	−1	0	−1 ± 0%
AD-12658	98	9	98 ± 9%	2	0	2 ± 0%
AD-12659	83	4	83 ± 4%	18	1	18 ± 1%
AD-12758	80	14	80 ± 14%	21	4	21 ± 4%
AD-12815	25	3	25 ± 3%	79	11	79 ± 11%
AD-12858	67	4	67 ± 4%	35	2	35 ± 2%
AD-12660	95	11	95 ± 11%	8	3	8 ± 3%
AD-12759	66	7	66 ± 7%	39	6	39 ± 6%
AD-12661	34	2	34 ± 2%	73	5	73 ± 5%
AD-12760	10	3	10 ± 3%	94	30	94 ± 30%
AD-12816	12	4	12 ± 4%	92	37	92 ± 37%
AD-12859	33	1	33 ± 1%	72	2	72 ± 2%
AD-12662	92	7	92 ± 7%	7	1	7 ± 1%
AD-12817	91	11	91 ± 11%	10	2	10 ± 2%
AD-12663	99	10	99 ± 10%	3	1	3 ± 1%
AD-12761	20	5	20 ± 5%	89	22	89 ± 22%
AD-12818	20	4	20 ± 4%	90	20	90 ± 20%
AD-12860	93	11	93 ± 11%	8	1	8 ± 1%
AD-12664	93	9	93 ± 9%	6	2	6 ± 2%
AD-12665	94	8	94 ± 8%	10	1	10 ± 1%
AD-12762	58	8	58 ± 8%	47	10	47 ± 10%
AD-12819	49	6	49 ± 6%	58	9	58 ± 9%
AD-12861	93	8	93 ± 8%	8	1	8 ± 1%
AD-12666	30	5	30 ± 5%	76	18	76 ± 18%
AD-12763	25	2	25 ± 2%	84	9	84 ± 9%
AD-12667	65	10	65 ± 10%	38	7	38 ± 7%
AD-12764	34	7	34 ± 7%	69	17	69 ± 17%
AD-12668	34	4	34 ± 4%	73	10	73 ± 10%
AD-12765	13	3	13 ± 3%	91	22	91 ± 22%
AD-12820	11	2	11 ± 2%	93	17	93 ± 17%
AD-12862	19	4	19 ± 4%	87	22	87 ± 22%
AD-12669	22	3	22 ± 3%	87	12	87 ± 12%
AD-12766	11	4	11 ± 4%	93	39	93 ± 39%
AD-12670	45	3	45 ± 3%	61	5	61 ± 5%
AD-12767	10	3	10 ± 3%	94	31	94 ± 31%
AD-12821	12	1	12 ± 1%	92	13	92 ± 13%
AD-12863	41	4	41 ± 4%	64	8	64 ± 8%
AD-12671	83	9	83 ± 9%	19	2	19 ± 2%
AD-12822	74	7	74 ± 7%	29	3	29 ± 3%
AD-12672	52	7	52 ± 7%	54	9	54 ± 9%
AD-12768	28	3	28 ± 3%	81	12	81 ± 12%
AD-12673	56	5	56 ± 5%	49	5	49 ± 5%
AD-12769	36	2	36 ± 2%	72	5	72 ± 5%
AD-12823	33	2	33 ± 2%	75	5	75 ± 5%
AD-12864	49	7	49 ± 7%	57	10	57 ± 10%
AD-12674	90	9	90 ± 9%	11	1	11 ± 1%
AD-12770	45	6	45 ± 6%	61	9	61 ± 9%
AD-12675	45	5	45 ± 5%	62	8	62 ± 8%
AD-12676	47	6	47 ± 6%	59	9	59 ± 9%
AD-12771	31	4	31 ± 4%	77	11	77 ± 11%
AD-12824	31	3	31 ± 3%	77	10	77 ± 10%
AD-12865	43	7	43 ± 7%	64	12	64 ± 12%
AD-12677	23	4	23 ± 4%	86	16	86 ± 16%
AD-12825	22	4	22 ± 4%	87	16	87 ± 16%
AD-12678	102	8	102 ± 8%	−2	0	−2 ± 0%
AD-12772	101	13	101 ± 13%	−1	0	−1 ± 0%
AD-12826	99	1	99 ± 1%	1	0	1 ± 0%
AD-12866	91	7	91 ± 7%	10	1	10 ± 1%
AD-12679	81	8	81 ± 8%	21	2	21 ± 2%
AD-12773	11	2	11 ± 2%	93	19	93 ± 19%
AD-12680	17	3	17 ± 3%	92	17	92 ± 17%
AD-12774	15	2	15 ± 2%	89	17	89 ± 17%
AD-12827	11	2	11 ± 2%	93	18	93 ± 18%
AD-12867	15	3	15 ± 3%	91	22	91 ± 22%
AD-12681	28	3	28 ± 3%	79	10	79 ± 10%
AD-12775	8	1	8 ± 1%	95	19	95 ± 19%
AD-12682	43	6	43 ± 6%	63	9	63 ± 9%
AD-12776	23	5	23 ± 5%	80	19	80 ± 19%
AD-12828	23	5	23 ± 5%	80	20	80 ± 20%
AD-12868	25	4	25 ± 4%	81	16	81 ± 16%
AD-12683	17	2	17 ± 2%	91	15	91 ± 15%
AD-12777	11	2	11 ± 2%	92	22	92 ± 22%
AD-12829	12	1	12 ± 1%	92	11	92 ± 11%
AD-12869	19	3	19 ± 3%	87	16	87 ± 16%
AD-12684	87	12	87 ± 12%	14	2	14 ± 2%
AD-12778	41	4	41 ± 4%	66	8	66 ± 8%
AD-12685	35	1	35 ± 1%	72	1	72 ± 1%
AD-12686	68	5	68 ± 5%	36	3	36 ± 3%
AD-12779	58	5	58 ± 5%	47	5	47 ± 5%
AD-12687	73	8	73 ± 8%	30	4	30 ± 4%
AD-12780	62	8	62 ± 8%	42	7	42 ± 7%
AD-12688	18	1	18 ± 1%	91	4	91 ± 4%
AD-12781	11	3	11 ± 3%	93	33	93 ± 33%
AD-12689	96	4	96 ± 4%	4	0	4 ± 0%
AD-12782	45	7	45 ± 7%	58	10	58 ± 10%
AD-12830	15	3	15 ± 3%	89	19	89 ± 19%
AD-12870	51	3	51 ± 3%	52	4	52 ± 4%
AD-12690	93	6	93 ± 6%	8	1	8 ± 1%
AD-12783	36	3	36 ± 3%	66	7	66 ± 7%
AD-12831	27	2	27 ± 2%	76	7	76 ± 7%
AD-12871	81	18	81 ± 18%	21	5	21 ± 5%

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human JC virus genome in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of an mRNA encoding the JC virus, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing the JC virus, inhibits expression of the JC virus genome.

2. The dsRNA of claim 1, wherein the first sequence is selected from the group consisting of Tables 1a and b and the second sequence is selected from the group consisting of Tables 1a and b.

3. The dsRNA of claim 1, wherein the dsRNA comprises at least one modified nucleotide.

4. The dsRNA of claim 2, wherein the dsRNA comprises at least one modified nucleotide.

5. The dsRNA of claim 3, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

6. The dsRNA of claim 3, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

7. The dsRNA of claim 3, wherein the first sequence is selected from the group consisting of Tables 1a and b and said second sequence is selected from the group consisting of Tables 1a and b.

8. The dsRNA of claim 6, wherein the first sequence is selected from the group consisting of Tables 1a and b and the second sequence is selected from the group consisting of Tables 1a and b.

9. A cell comprising the dsRNA of claim 1.

10. A pharmaceutical composition for inhibiting the expression of a gene from the JC Virus in an organism, comprising the dsRNA of claim 1 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the first sequence of said dsRNA is selected from the group consisting of Tables 1a and b and the second sequence of the dsRNA is selected from the group consisting of Tables 1a and b.

12. The pharmaceutical composition of claim 10, wherein said first sequence of the dsRNA is selected from the group consisting of Tables 1a and b and the second sequence of said dsRNA is selected from the group consisting of Tables 1a and b.

13. A method for inhibiting the expression of a gene from a JC Virus in a cell, the method comprising:

(a) introducing into the cell the dsRNA of claim 1; and

(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a gene from the JC Virus, thereby inhibiting expression of a gene from the JC Virus in the cell.

14. A method of treating or preventing pathological processes mediated by JC virus expression comprising administering to a patient in need of such treatment or prevention a therapeutically or prophylactically effective amount of the dsRNA of claim 1.

15. A vector for inhibiting the expression of a gene from the JC Virus in a cell, the vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of claim 1.

16. A cell comprising the vector of claim 15.