Compositions And Methods For Inhibiting Expression Of A Gene From The Jc Virus

  *US07691824B2*
  US007691824B2                                 
(12)United States Patent(10)Patent No.: US 7,691,824 B2
 Tan et al. (45) Date of Patent:Apr.  6, 2010

(54)Compositions and methods for inhibiting expression of a gene from the JC virus 
    
(75)Inventors: Pamela Tan,  Kulmbach (DE); 
  Dinah Sah,  Boston, MA (US); 
  Birgit Bramlage,  Kulmbach (DE) 
(73)Assignee:Alnylam Pharmaceuticals, Inc.,  Cambridge, MA (US), Type: US Company 
(*)Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 181 days. 
(21)Appl. No.: 11/741,205 
(22)Filed: Apr.  27, 2007 
(65)Prior Publication Data 
 US 2009/0062225 A1 Mar.  5, 2009 
 Related U.S. Patent Documents 
(60)Provisional application No. 60/795,765, filed on Apr.  28, 2006.
 
(51)Int. Cl. A61K 031/70 (20060101); C07H 021/04 (20060101); C12N 021/00 (20060101); C12N 015/00 (20060101); C12N 005/00 (20060101)
(52)U.S. Cl. 514/44; 536/24.5; 435/455; 435/320.1; 435/325
(58)Field of Search  None

 
(56)References Cited
 
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     * cited by examiner
 
     Primary Examiner —Richard Schnizer
     Art Unit — 1635
     Exemplary claim number — 1
 
(74)Attorney, Agent, or Firm — Fenwick & West LLP

(57)

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.
15 Claims, Drawing Sheets, and Figures
 
 

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/795,765, filed Apr. 28, 2006, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] 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

[0003] 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).
[0004] 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 antobody, 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).
[0005] 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).
[0006] 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).
[0007] 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.
[0008] 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

[0009] 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.
[0010] 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%.
[0011] 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 b.
[0012] 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.
[0013] 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.
[0014] 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:
[0015] (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
[0016] (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.
[0017] 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.
[0018] 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.
[0019] 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

[0020] No Figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

[0021] 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).
[0022] 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 relication and/or maintenance in a cell.
[0023] 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.
[0024] 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

[0025] 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.
[0026] “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.
[0027] As used herein, “JC virus” refers to the latent polyomavirus JC Virus that has a reference sequence NC001699. 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, NC001699.1, U61771.1, U73500.1-U73502.1.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] “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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] “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.
[0040] 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
[0041]  [see pdf for image]
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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)

[0049] 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%.
[0050] 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).
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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
[0059] 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.
[0060] 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.
[0061] 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.
[0062] Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH.sub.2-NH—CH.sub.2-, —CH.sub.2-N(CH.sub.3)-O—CH.sub.2-[known as a methylene (methylimino) or MMI backbone], —CH.sub.2-O—N(CH.sub.3)-CH.sub.2-, —CH.sub.2-N(CH.sub.3)-—N(CH.sub.3)-CH.sub.2- and —N(CH.sub.3)-CH.sub.2-CH.sub.2-[wherein the native phosphodiester backbone is represented as —O—P—O—CH.sub.2-] 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.
[0063] 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 C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C.sub. 1 to C.sub. 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, 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—CH.sub.2CH.sub.2OCH.sub.3, 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(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH.sub.2-O—CH.sub.2-N(CH.sub.2).sub.2, also described in examples hereinbelow.
[0064] Other preferred modifications include 2′-methoxy(2′-OCH.sub.3), 2′-aminopropoxy(2′-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) 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.
[0065] 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.
[0066] 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.
[0067] 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).
[0068] 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.
[0069] 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.
[0070] 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-5-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.
[0071] Vector Encoded RNAi Agents
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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

[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] Liposomes
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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
[0098] 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).
[0099] 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).
[0100] 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.
[0101] 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).
[0102] 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).
[0103] 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.sub.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).
[0104] 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.sub.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.sub.M1 or 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).
[0105] 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.sub.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.
[0106] 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.
[0107] 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.
[0108] 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).
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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.
[0116] Carriers
[0117] 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.
[0118] Excipients
[0119] 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).
[0120] 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.
[0121] 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.
[0122] Other Components
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] Methods for Treating Diseases Caused by Expression of a Gene from the JC Virus
[0130] 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.
[0131] 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.
[0132] 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.
[0133] Methods for Inhibiting Expression of a Gene from the JC Virus
[0134] 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.
[0135] 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.
[0136] 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

[0137] 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; NC001699.1; U61771.1; U73500.1-U73502.1). NC001699 was defined as reference sequence.
[0138] 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.
[0139] All conserved 19mer 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 19mer 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.
[0140] 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.
[0141] For predicting siRNA-specific off-target potential, the following assumptions were made:
[0142] 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)
[0143] 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
[0144] 3) an off-target score can be calculated for each hit, based on identity to siRNA sequence and position of mismatches
[0145] 4) assuming potential abortion of sense strand activity by internal modifications introduced, only off-target potential of antisense strand will be relevant
[0146] To identify potential off-target genes, 19mer input sequences were subjected to a homology search against publicly available human mRNA sequences.
[0147] To this purpose, fastA (version 3.4) searches were performed with all 19mer 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 19mer 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.
[0148] 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 19mer input sequence were extracted for each off-target to calculate the off-target score:
[0149] Number of mismatches in non-seed region
[0150] Number of mismatches in seed region
[0151] Number of mismatches in cleavage site region
[0152] 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
[0153] The most relevant off-target gene for input each 19mer 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.
[0154] 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).
[0155] 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).
[0156] 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

[0157] All siRNAs were synthesized in 0.2 μmole synthesis scale on an ABI3900 DNA synthesizer according to standard procedures.
[0158] For the initial screening set (93 different siRNA sequences), 4 different strategies of chemical modification were used:
[0159] 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)
[0160] —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)
[0161] 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)
[0162] c) EEL plus 2′-O-methyl in position 2 of sense strand (only if no pyrimidine in position 2, otherwise already covered by EEL)
[0163] 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)
[0164] 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

[0165] Construction of Reporter-Systems Encoding JCV Transcripts
[0166] 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.
[0167] 
[00001] [TABLE-US-00001]
  L1  
  (SEQ ID NO:931)  
CTCGAGACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCAT  
 
  ATGAAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCA
 
  AGTTCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAA
 
  CTGGGGTTGACTCAATTACAGAGGTAGAATGCTTTTTAACTCCAGAAATG
 
  GGTGACCCAGATGAGCATCTTAGGGGTTTTAGTAAGTCAATTTCTATATC
 
  AGATACATTTGAAAGTGACTCCCCAAATAAGGACATGCTTCCTTGTTACA
 
  GTGTGGCCAGAATTCCACTACCCAATCTAAATGAGGATCTAACCTGTGGA
 
  AATATACTAATGTGGGAGGCTGTGACCTTAAAAACTGAGGTTCTAGGGGT
 
  GACAACTTTGATGAATGTGCACTCTAATGGTCAAGCAACTCATGACAATG
 
  GTGCAGGAAAGCCAGTGCAGGGCACCAGCTTTCATTTTTTTTCTGTTGGC
 
  GGGGAGGCTTTAGAATTACAGGGGGTGGTTTTTAATTACAGAACAAAGTA
 
  CCCAGATGGAACAATTTTTCCAAAGAATGCAACAGTGCAATCTCAAGTAA
 
  TGAACACAGAGCACAAGGCGTACCTAGATAAGAACAAAGCATATCCTGTT
 
  GAATGTTGGGTTCCTGATCCCACCAGAAATGAAAACACAAGATATTTTGG
 
  GACACTAACAGGAGGAGAAAATGTTCCTCCAGTTCTTCATATAACAAACA
 
  CTGCCACAACAGTGCTGCTTGATGAATTTGGTGTTGGGCCACTTTGCAAA
 
  GGTGACAACTTGTATTTGTCAGCTGTTGATGTTTGTGGAATGTTTACTAA
 
  CAGATCTGGTTCCCAGCAGTGGAGAGGACTGTCCAGATATTTTAAGGTTC
 
  AGCTCAGAAAAAGGAGGGTTAAAAACCCCTACCCAATTTCTTTCCTTCTT
 
  ACTGATTTGATTAACAGAAGGACCCCTAGAGTTGATGGGCAACCTATGTA
 
  TGGTATGGATGCTCAGGTAGAGGAGGTTAGAGTTTTTGAGGGGACAGAGG
 
  AACTTCCAGGGGACCCAGACATGATGAGATATGTTGACAGATATGGACAG
 
  TTGCAAACAAAGATGCTGTAATCAAAATCCTTTATTGTAATATGCAGTAC
 
ATTTTAATAAAGTATAACCAGCTTTACTTTACAGTTGCAGTCATGCGGCC
 
GC
 
  E
  (SEQ ID NO:932)  
CTCGAGCCGCCTCCAAGCTTACTCAGAAGTAGTAAGGGCGTGGAGGCTTT  
 
  TTAGGAGGCCAGGGAAATTCCCTTGTTTTTCCCTTTTTTGCAGTAATTTT
 
  TTGCTGCAAAAAGCTAAAATGGACAAAGTGCTGAATAGGGAGGAATCCAT
 
  GGAGCTTATGGATTTATTAGGCCTTGATAGGTCTGCATGGGGGAACATTC
 
  CTGTCATGAGAAAAGCTTATCTGAAAAAATGCAAAGAACTCCACCCTGAT
 
  AAAGGTGGGGACGAAGACAAGATGAAGAGAATGAATTTTTTATATAAAAA
 
  AATGGAACAAGGTGTAAAAGTTGCTCATCAGCCTGATTTTGGTACATGGA
 
  ATAGTTCAGAGGTTGGTTGTGATTTTCCTCCTAATTCTGATACCCTTTAT
 
  TGCAAGGAATGGCCTAACTGTGCCACTAATCCTTCAGTGCATTGCCCCTG
 
  TTTAATGTGCATGCTAAAATTAAGGCATAGAAACAGAAAATTTTTAAGAA
 
  GCAGCCCACTTGTGTGGATAGATTGCTATTGCTTTGATTGCTTCAGACAA
 
  TGGTTTGGGTGTGACTTAACCCAAGAAGCTCTTCATTGCTGGGAGAAAGT
 
  TCTTGGAGACACCCCCTACAGGGATCTAAAGCTTTAAGTGCCAACCTATG
 
  GAACAGATGAATGGGAATCCTGGTGGAATACATTTAATGAGAAGTGGGAT
 
  GAAGACCTGTTTTGCCATGAAGAAATGTTTGCCAGTGATGATGAAAACAC
 
  AGGATCCCAACACTCTACCCCACCTAAAAAGAAAAAAAAGGTAGAAGACC
 
  CTAAAGACTTTCCTGTAGATCTGCATGCATTCCTCAGTCAAGCTGTGTTT
 
  AGTAATAGAACTGTTGCTTCTTTTGCTGTGTATACCACTAAAGAAAAAGC
 
  TCAAATTTTATATAAGAAACTTATGGAAAAATATTCTGTAACTTTTATAA
 
  GTAGACATGGTTTTGGGGGTCATAATATTTTGTTTTTCTTAACACCACAT
 
  AGACATAGAGTGTCAGCAATTAATAACTACTGTCAAAAACTATGTACCTT
 
  TAGTTTTTTAATTTGTAAAGGTGTGAATAAGGAATACTTGTTTTATAGTG
 
  CCCTGTGTAGACAGCCATATGCAGTAGTGGAAGAAAGTATTCAGGGGGGC
 
  CTTAAGGAGCATGACTTTAACCCAGAAGAACCAGAAGAAACTAAGCAGGT
 
  TTCATGGAAATTAGTTACACAGTATGCCTTGGAAACCAAGTGTGAGGATG
 
  TTTTTTTGCTTATGGGCATGTACTTAGACTTTCAGGAAAACCCACAGCAA
 
  TGCAAAAAATGTGAAAAAAAGGATCAGCCAAATCACTTTAACCATCATGA
 
  AAAACACTATTATAATGCCCAAATTTTTGCAGATAGCAAAAATCAAAAAA
 
  GCATTTGCCAGCAGGCTGTTGATACTGTAGCAGCCAAACAAAGGGTTGAC
 
  AGCATCCACATGACCAGAGAAGAAATGTTAGTTGAAAGGTTTAATTTCTT
 
  GCTTGATAAAATGGACTTAATTTTTGGGGCACATGGCAATGCTGTTTTAG
 
  AGCAATATATGGCTGGGGTGGCCTGGATTCATTGCTTGCTGCCTCAAATG
 
  GACACTGTTATTTATGACTTTCTAAAATGCATTGTATTAAACATTCCAAA
 
  AAAAAGGTACTGGCTATTCAAGGGGCCAATAGACAGTGGCAAAACTACTT
 
  TAGCTGCAGCTTTACTTGATCTCTGTGGGGGAAAGTCATTAAATGTTAAT
 
  ATGCCATTAGAAAGATTAAACTTTGAATTAGGAGTGGGTATAGATCAGTT
 
  TATGGTTGTATTTGAGGATGTAAAAGGCACTGGTGCAGAGTCAAGGGATT
 
  TACCTTCAGGGCATGGCATAAGCAACCTTGATTGCTTAAGAGATTACTTA
 
  GATGGAAGTGTAAAAGTTAATTTAGAGAGAAAACACCAAAACAAAAGAAC
 
  ACAGGTGTTTCCACCTGGAATTGTAACCATGAATGAATATTCAGTGCCTA
 
  GAACTTTACAGGCCAGATTTGTAAGGCAGATAGATTTTAGACCAAAGGCC
 
  TACCTGAGAAAATCACTAAGCTGCTCTGAGTATTTGCTAGAAAAAAGGAT
 
  TTTGCAAAGTGGTATGACTTTGCTTTTGCTTTTAATCTGGTTTAGGCCAG
 
  TTGCTGACTTTGCAGCTGCCATTCATGAGAGGATTGTGCAGTGGAAAGAA
 
  AGGCTGGATTTAGAAATAAGCATGTATACATTTTCTACTATGAAAGCTAA
 
  TGTTGGTATGGGGAGACCCATTCTTGACTTTCCTAGAGAGGAAGATTCTG
 
  AAGCAGAAGACTCTGGACATGGATCAAGCACTGAATCACAATCACAATGC
 
  TTTTCCCAGGTCTCAGAAGCCTCTGGTGCAGACACACAGGAAAACTGCAC
 
  TTTTCACATCTGTAAAGGCTTTCAATGTTTCAAAAAACCAAAGACCCCTC
 
CCCCAAAATAACTGCAACTGTGCGGCCGC
 
  LA23 1-700
  (SEQ ID NO:933)  
CTCGAGCAGCTAACAGCCAGTAAACAAAGCACAAGGGGAAGTGGAAAGCA  
 
  GCCAAGGGAACATGTTTTGCGAGCCAGAGCTGTTTTGGCTTGTCACCAGC
 
  TGGCCATGGTTCTTCGCCAGCTGTCACGTAAGGCTTCTGTGAAAGTTAGT
 
  AAAACCTGGAGTGGAACTAAAAAAAGAGCTCAAAGGATTTTAATTTTTTT
 
  GTTAGAATTTTTGCTGGACTTTTGCACAGGTGAAGACAGTGTAGACGGGA
 
  AAAAAAGACAGAGACACAGTGGTTTGACTGAGCAGACATACAGTGCTTTG
 
  CCTGAACCAAAAGCTACATAGGTAAGTAATGTTTTTTTTTGTGTTTTCAG
 
  GTTCATGGGTGCCGCACTTGCACTTTTGGGGGACCTAGTTGCTACTGTTT
 
  CTGAGGCTGCTGCTGCCACAGGATTTTCAGTAGCTGAAATTGCTGCTGGA
 
  GAGGCTGCTGCTACTATAGAAGTTGAAATTGCATCCCTTGCTACTGTAGA
 
  GGGGATTACAAGTACCTCTGAGGCTATAGCTGCTATAGGCCTTACTCCTG
 
  AAACATATGCTGTAATAACTGGAGCTCCGGGGGCTGTAGCTGGGTTTGCT
 
  GCATTGGTTCAAACTGTAACTGGTGGTAGTGCTATTGCTCAGTTGGGATA
 
  TAGATTTTTTGCTGACTGGGATCATAAAGTTTCAACAGTTGGGCTTTTTC
 
GCGGCCGC
 
  LA23 701-1438
  (SEQ ID NO:934)  
CTCGAGAGCAGCCAGCTATGGCTTTACAATTATTTAATCCAGAAGACTAC  
 
  TATGATATTTTATTTCCTGGAGTGAATGCCTTTGTTAACAATATTCACTA
 
  TTTAGATCCTAGACATTGGGGCCCGTCCTTGTTCTCCACAATCTCCCAGG
 
  CTTTTTGGAATCTTGTTAGAGATGATTTGCCAGCCTTAACCTCTCAGGAA
 
  ATTCAGAGAAGAACCCAAAAACTATTTGTTGAAAGTTTAGCAAGGTTTTT
 
  GGAAGAAACTACTTGGGCAATAGTTAATTCACCAGCTAACTTATATAATT
 
  ATATTTCAGACTATTATTCTAGATTGTCTCCAGTTAGGCCCTCTATGGTA
 
  AGGCAAGTTGCCCAAAGGGAGGGAACCTATATTTCTTTTGGCCACTCATA
 
  CACCCAAAGTATAGATGATGCAGACAGCATTCAAGAAGTTACCCAAAGGC
 
  TAGATTTAAAAACCCCAAATGTGCAATCTGGTGAATTTATAGAAAGAAGT
 
  ATTGCACCAGGAGGTGCAAATCAAAGATCTGCTCCTCAATGGATGTTGCC
 
  TTTACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCATATG
 
  AAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGT
 
  TCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAACTG
 
GGGTTGACTCAATTACAGAGGTAGAATGCTGCGGCCGC
[0168] Screen of JCV siRNAs in Transfected Cells
[0169] Cos-7 cells (DSMZ, Braunschweig, Germany, # ACC-60) were seeded at 1.5×104 cells/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.
[0170] 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% CO2 in 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.
[0171] 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-Jüigesheim, 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%).
[0172] 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:
[0173] from 33 nM in 3-fold dilutions down to 0.005 nM (for fragment L1)
[0174] from 24 nM in 4-fold dilutions down to 0.001 nM (for fragment E and fragments LA23 1-700 and LA23 701-1438).
[0175] 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 μM and 1 nM.
[0176] 
[00002] [TABLE-US-00002]
  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
[0177] Cells and Virus
[0178] 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% CO2 in 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).
[0179] Prophylaxis Assay
[0180] 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 immunofluoresence 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.
[0181] 
[00003] [TABLE-US-00003]
  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.
[0182] Post-Infection Treatment Assay
[0183] 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 immunofluoresence 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.Z 1, 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.
[0184] 
[00004] [TABLE-US-00004]
  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
[0185] 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 immunofluoresence 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.
[0186] 
[00005] [TABLE-US-00005]
  TABLE 5
 
  Prophylaxis administration of JCV siRNAs inhibits the production
  of active progeny JC virus capable of secondary infection
    Remaining Virus (% of  
    Luciferase Control)
      Targeted     Virus
    Duplex 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
[0187] 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.
[0188] 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 Na3PO4 in 10% ACN; pH=11 and HPLC Eluent B: 1 M NaBr in HPLC Eluent A:
[0189] 
[00006] [TABLE-US-00006]
 
  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
 
[0190] 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.
[0191] 
[00007] [TABLE-US-00007]
  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
   
[0192] dsRNA Expression Vectors
[0193] 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).
[0194] 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.
[0195] 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. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. 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.
[0196] 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)).
[0197] 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-D1-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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 
[00008] [TABLE-US-00008]
 
  JCV Gene Walk  
  siRNAs targeting >95% of all strains (>=369 out of 388)
  Human specific pan-JCV: 208 siRNAs
  all siRNAs double overhang design, dTdT, no modifications
          Residual  
          luciferase
          activity
          (relative to
      sense strand   antisense strand   control
      SEQ     SEQ     siRNA  
  position in   duplex   ID     ID     treated
  consensus   name   NO:   sequence (5′-3′)   NO:   sequence (5′-3′)   cells)
 
  1533-1551   AD-14742   515   CUUAUAAGAGGAGGAGUAGTT   516   CUACUCCUCCUCUUAUAAGTT   40.85  
 
  1703-1721   AD-14743   517   CAUGCUUCCUUGUUACAGUTT   518   ACUGUAACAAGGAAGCAUGTT   20.92
 
  1439-1457   AD-14744   519   UACGGGACUGUAACACCUGTT   520   CAGGUGUUACAGUCCCGUATT   62.20
 
  1705-1723   AD-14745   521   UGCUUCCUUGUUACAGUGUTT   522   ACACUGUAACAAGGAAGCATT   43.97
 
  2064-2082   AD-14746   523   CCUGUUGAAUGUUGGGUUCTT   524   GAACCCAACAUUCAACAGGTT   24.52
 
  2067-2085   AD-14747   525   GUUGAAUGUUGGGUUCCUGTT   526   CAGGAACCCAACAUUCAACTT   32.67
 
  2071-2089   AD-14748   527   AAUGUUGGGUUCCUGAUCCTT   528   GGAUCAGGAACCCAACAUUTT   93.99
 
  2121-2139   AD-14749   529   ACACUAACAGGAGGAGAAATT   530   UUUCUCCUCCUGUUAGUGUTT   55.16
 
  1535-1553   AD-14750   531   UAUAAGAGGAGGAGUAGAATT   532   UUCUACUCCUCCUCUUAUATT   30.86
 
  1536-1554   AD-14751   533   AUAAGAGGAGGAGUAGAAGTT   534   CUUCUACUCCUCCUCUUAUTT   54.44
 
  1445-1463   AD-14752   535   ACUGUAACACCUGCUCUUGTT   536   CAAGAGCAGGUGUUACAGUTT   53.88
 
  1700-1718   AD-14753   537   GGACAUGCUUCCUUGUUACTT   538   GUAACAAGGAAGCAUGUCCTT   35.24
 
  1702-1720   AD-14754   539   ACAUGCUUCCUUGUUACAGTT   540   CUGUAACAAGGAAGCAUGUTT   70.39
 
  1704-1722   AD-14755   541   AUGCUUCCUUGUUACAGUGTT   542   CACUGUAACAAGGAAGCAUTT   41.80
 
  2065-2083   AD-14756   543   CUGUUGAAUGUUGGGUUCCTT   544   GGAACCCAACAUUCAACAGTT   56.69
 
  2070-2088   AD-14757   545   GAAUGUUGGGUUCCUGAUCTT   546   GAUCAGGAACCCAACAUUCTT   39.16
 
  1441-1459   AD-14758   547   CGGGACUGUAACACCUGCUTT   548   AGCAGGUGUUACAGUCCCGTT   39.79
 
  1443-1461   AD-14759   549   GGACUGUAACACCUGCUCUTT   550   AGAGCAGGUGUUACAGUCCTT   30.62
 
  1444-1462   AD-14760   551   GACUGUAACACCUGCUCUUTT   552   AAGAGCAGGUGUUACAGUCTT   28.14
 
  1609-1627   AD-14761   553   CUCCAGAAAUGGGUGACCCTT   554   GGGUCACCCAUUUCUGGAGTT   67.42
 
  1537-1555   AD-14762   555   UAAGAGGAGGAGUAGAAGUTT   556   ACUUCUACUCCUCCUCUUATT   36.10
 
  629-647   AD-14763   557   GAGGCUGCUGCUACUAUAGTT   558   CUAUAGUAGCAGCAGCCUCTT   49.39
 
  656-674   AD-14764   559   AUUGCAUCCCUUGCUACUGTT   560   CAGUAGCAAGGGAUGCAAUTT   74.04
 
  658-676   AD-14765   561   UGCAUCCCUUGCUACUGUATT   562   UACAGUAGCAAGGGAUGCATT   50.84
 
  517-535   AD-14766   563   UUGUGUUUUCAGGUUCAUGTT   564   CAUGAACCUGAAAACACAATT   72.59
 
  559-577   AD-14767   565   GGACCUAGUUGCUACUGUUTT   566   AACAGUAGCAACUAGGUCCTT   34.82
 
  591-609   AD-14768   567   CUGCCACAGGAUUUUCAGUTT   568   ACUGAAAAUCCUGUGGCAGTT   48.68
 
  638-656   AD-14769   569   GCUACUAUAGAAGUUGAAATT   570   UUUCAACUUCUAUAGUAGCTT   39.07
 
  655-673   AD-14770   571   AAUUGCAUCCCUUGCUACUTT   572   AGUAGCAAGGGAUGCAAUUTT   45.59
 
  561-579   AD-14771   573   ACCUAGUUGCUACUGUUUCTT   574   GAAACAGUAGCAACUAGGUTT   45.57
 
  639-657   AD-14772   575   CUACUAUAGAAGUUGAAAUTT   576   AUUUCAACUUCUAUAGUAGTT   33.12
 
  715-733   AD-14773   577   AGGCCUUACUCCUGAAACATT   578   UGUUUCAGGAGUAAGGCCUTT   37.38
 
  716-734   AD-14774   579   GGCCUUACUCCUGAAACAUTT   580   AUGUUUCAGGAGUAAGGCCTT   42.38
 
  326-344   AD-14775   581   GUAAAACCUGGAGUGGAACTT   582   GUUCCACUCCAGGUUUUACTT   46.59
 
  518-536   AD-14776   583   UGUGUUUUCAGGUUCAUGGTT   584   CCAUGAACCUGAAAACACATT   71.28
 
  520-538   AD-14777   585   UGUUUUCAGGUUCAUGGGUTT   586   ACCCAUGAACCUGAAAACATT   64.55
 
  661-679   AD-14778   587   AUCCCUUGCUACUGUAGAGTT   588   CUCUACAGUAGCAAGGGAUTT   60.45
 
  560-578   AD-14779   589   GACCUAGUUGCUACUGUUUTT   590   AAACAGUAGCAACUAGGUCTT   32.46
 
  681-699   AD-14780   591   GGAUUACAAGUACCUCUGATT   592   UCAGAGGUACUUGUAAUCCTT   22.96
 
  714-732   AD-14781   593   UAGGCCUUACUCCUGAAACTT   594   GUUUCAGGAGUAAGGCCUATT   56.99
 
  377-395   AD-14782   595   UGUUAGAAUUUUUGCUGGATT   596   UCCAGCAAAAAUUCUAACATT   29.90
 
  589-607   AD-14783   597   UGCUGCCACAGGAUUUUCATT   598   UGAAAAUCCUGUGGCAGCATT   42.63
 
  594-612   AD-14784   599   CCACAGGAUUUUCAGUAGCTT   600   GCUACUGAAAAUCCUGUGGTT   67.06
 
  648-666   AD-14785   601   AAGUUGAAAUUGCAUCCCUTT   602   AGGGAUGCAAUUUCAACUUTT   48.90
 
  649-667   AD-14786   603   AGUUGAAAUUGCAUCCCUUTT   604   AAGGGAUGCAAUUUCAACUTT   27.74
 
  587-605   AD-14787   605   GCUGCUGCCACAGGAUUUUTT   606   AAAAUCCUGUGGCAGCAGCTT   38.77
 
  325-343   AD-14788   607   AGUAAAACCUGGAGUGGAATT   608   UUCCACUCCAGGUUUUACUTT   32.84
 
  515-533   AD-14789   609   UUUUGUGUUUUCAGGUUCATT   610   UGAACCUGAAAACACAAAATT   46.96
 
  516-534   AD-14790   611   UUUGUGUUUUCAGGUUCAUTT   612   AUGAACCUGAAAACACAAATT   43.61
 
  519-537   AD-14791   613   GUGUUUUCAGGUUCAUGGGTT   614   CCCAUGAACCUGAAAACACTT   35.55
 
  521-539   AD-14792   615   GUUUUCAGGUUCAUGGGUGTT   616   CACCCAUGAACCUGAAAACTT   38.22
 
  522-540   AD-14793   617   UUUUCAGGUUCAUGGGUGCTT   618   GCACCCAUGAACCUGAAAATT   90.85
 
  523-541   AD-14794   619   UUUCAGGUUCAUGGGUGCCTT   620   GGCACCCAUGAACCUGAAATT   83.37
 
  616-634   AD-14795   621   UUGCAUCCCUUGCUACUGUTT   622   AGCCUCUCCAGCAGCAAUUTT   55.06
 
  657-675   AD-14796   623   UUGCAUCCCUUGCUACUGUTT   624   ACAGUAGCAAGGGAUGCAATT   30.98
 
  761-779   AD-14797   625   GCUGUAGCUGGGUUUGCUGTT   626   CAGCAAACCCAGCUACAGCTT   28.95
 
  645-663   AD-14798   627   UAGAAGUUGAAAUUGCAUCTT   628   GAUGCAAUUUCAACUUCUATT   67.39
 
  647-665   AD-14799   629   GAAGUUGAAAUUGCAUCCCTT   630   GGGAUGCAAUUUCAACUUCTT   66.83
 
  660-678   AD-14800   631   CAUCCCUUGCUACUGUAGATT   632   UCUACAGUAGCAAGGGAUGTT   33.26
 
  324-342   AD-14801   633   UAGUAAAACCUGGAGUGGATT   634   UCCACUCCAGGUUUUACUATT   39.15
 
  372-390   AD-14802   635   UUUUUUGUUAGAAUUUUUGTT   636   CAAAAAUUCUAACAAAAAATT   91.20
 
  640-658   AD-14803   637   UACUAUAGAAGUUGAAAUUTT   638   AAUUUCAACUUCUAUAGUATT   34.15
 
  562-580   AD-14804   639   CCUAGUUGCUACUGUUUCUTT   640   AGAAACAGUAGCAACUAGGTT   30.08
 
  563-581   AD-14805   641   CUAGUUGCUACUGUUUCUGTT   642   CAGAAACAGUAGCAACUAGTT   32.44
 
  566-584   AD-14806   643   GUUGCUACUGUUUCUGAGGTT   644   CCUCAGAAACAGUAGCAACTT   35.62
 
  625-643   AD-14807   645   UGGAGAGGCUGCUGCUACUTT   646   AGUAGCAGCAGCCUCUCCATT   28.27
 
  627-645   AD-14808   647   GAGAGGCUGCUGCUACUAUTT   648   AUAGUAGCAGCAGCCUCUCTT   30.29
 
  628-646   AD-14809   649   AGAGGCUGCUGCUACUAUATT   650   UAUAGUAGCAGCAGCCUCUTT   31.59
 
  632-650   AD-14810   651   GCUGCUGCUACUAUAGAAGTT   652   CUUCUAUAGUAGCAGCAGCTT   30.11
 
  513-531   AD-14811   653   UUUUUUGUGUUUUCAGGUUTT   654   AACCUGAAAACACAAAAAATT   55.27
 
  641-659   AD-14812   655   ACUAUAGAAGUUGAAAUUGTT   656   CAAUUUCAACUUCUAUAGUTT   45.27
 
  323-341   AD-14813   657   UUAGUAAAACCUGGAGUGGTT   658   CCACUCCAGGUUUUACUAATT   77.97
 
  717-735   AD-14814   659   GCCUUACUCCUGAAACAUATT   660   UAUGUUUCAGGAGUAAGGCTT   29.54
 
  646-664   AD-14815   661   AGAAGUUGAAAUUGCAUCCTT   662   GGAUGCAAUUUCAACUUCUTT   65.04
 
  592-610   AD-14816   663   UGCCACAGGAUUUUCAGUATT   664   UACUGAAAAUCCUGUGGCATT   64.03
 
  590-608   AD-14817   665   GCUGCCACAGGAUUUUCAGTT   666   CUGAAAAUCCUGUGGCAGCTT   37.83
 
  526-544   AD-14818   667   CAGGUUCAUGGGUGCCGCATT   668   UGCGGCACCCAUGAACCUGTT   28.88
 
  615-633   AD-14819   669   AAAUUGCUGCUGGAGAGGCTT   670   GCCUCUCCAGCAGCAAUUUTT   92.90
 
  617-635   AD-14820   671   AUUGCUGCUGGAGAGGCUGTT   672   CAGCCUCUCCAGCAGCAAUTT   75.41
 
  652-670   AD-14821   673   UGAAAUUGCAUCCCUUGCUTT   674   AGCAAGGGAUGCAAUUUCATT   73.08
 
  374-392   AD-14822   675   UUUUGUUAGAAUUUUUGCUTT   676   AGCAAAAAUUCUAACAAAATT   86.39
 
  375-393   AD-14823   677   UUUGUUAGAAUUUUUGCUGTT   678   CAGCAAAAAUUCUAACAAATT   96.50
 
  631-649   AD-14824   679   GGCUGCUGCUACUAUAGAATT   680   UUCUAUAGUAGCAGCAGCCTT   32.62
 
  376-394   AD-14825   681   UUGUUAGAAUUUUUGCUGGTT   682   CCAGCAAAAAUUCUAACAATT   102.71
 
  512-530   AD-14826   683   UUUUUUUGUGUUUUCAGGUTT   684   ACCUGAAAACACAAAAAAATT   92.45
 
  1127-1145   AD-14827   685   GAAACUACUUGGGCAAUAGTT   686   CUAUUGCCCAAGUAGUUUCTT   63.46
 
  1410-1428   AD-14828   687   AAUGGAUGUUGCCUUUACUTT   688   AGUAAAGGCAACAUCCAUUTT   45.99
 
  1406-1424   AD-14829   689   CCUCAAUGGAUGUUGCCUUTT   690   AAGGCAACAUCCAUUGAGGTT   40.54
 
  1418-1436   AD-14830   691   UUGCCUUUACUUUUAGGGUTT   692   ACCCUAAAAGUAAAGGCAATT   117.10
 
  1126-1144   AD-14831   693   AGAAACUACUUGGGCAAUATT   694   UAUUGCCCAAGUAGUUUCUTT   54.78
 
  1125-1143   AD-14832   695   AAGAAACUACUUGGGCAAUTT   696   AUUGCCCAAGUAGUUUCUUTT   67.07
 
  1419-1437   AD-14833   697   UGCCUUUACUUUUAGGGUUTT   698   AACCCUAAAAGUAAAGGCATT   71.52
 
  1420-1438   AD-14834   699   GCCUUUACUUUUAGGGUUGTT   700   CAACCCUAAAAGUAAAGGCTT   58.05
 
  1422-1440   AD-14835   701   CUUUACUUUUAGGGUUGUATT   702   UACAACCCUAAAAGUAAAGTT   93.36
 
  1423-1441   AD-14836   703   UUUACUUUUAGGGUUGUACTT   704   GUACAACCCUAAAAGUAAATT   108.84
 
  1425-1443   AD-14837   705   UACUUUUAGGGUUGUACGGTT   706   CCGUACAACCCUAAAAGUATT   106.68
 
  1123-1141   AD-14838   707   GGAAGAAACUACUUGGGCATT   708   UGCCCAAGUAGUUUCUUCCTT   37.06
 
  1409-1427   AD-14839   709   CAAUGGAUGUUGCCUUUACTT   710   GUAAAGGCAACAUCCAUUGTT   36.03
 
  1413-1431   AD-14840   711   GGAUGUUGCCUUUACUUUUTT   712   AAAAGUAAAGGCAACAUCCTT   38.51
 
  1416-1434   AD-14841   713   UGUUGCCUUUACUUUUAGGTT   714   CCUAAAAGUAAAGGCAACATT   110.86
 
  1414-1432   AD-14842   715   GAUGUUGCCUUUACUUUUATT   716   UAAAAGUAAAGGCAACAUCTT   34.83
 
  911-929   AD-14843   717   CCAGAAGACUACUAUGAUATT   718   UAUCAUAGUAGUCUUCUGGTT   23.75
 
  910-928   AD-14844   719   UCCAGAAGACUACUAUGAUTT   720   AUCAUAGUAGUCUUCUGGATT   27.47
 
  1120-1138   AD-14845   721   UUUGGAAGAAACUACUUGGTT   722   CCAAGUAGUUUCUUCCAAATT   93.12
 
  1404-1422   AD-14846   723   CUCCUCAAUGGAUGUUGCCTT   724   GGCAACAUCCAUUGAGGAGTT   81.72
 
  1337-1355   AD-14847   725   CCAAAUGUGCAAUCUGGUGTT   726   CACCAGAUUGCACAUUUGGTT   77.89
 
  1338-1356   AD-14848   727   CAAAUGUGCAAUCUGGUGATT   728   UCACCAGAUUGCACAUUUGTT   44.40
 
  1397-1415   AD-14849   729   AGAUCUGCUCCUCAAUGGATT   730   UCCAUUGAGGAGCAGAUCUTT   46.41
 
  1407-1425   AD-14850   731   CUCAAUGGAUGUUGCCUUUTT   732   AAAGGCAACAUCCAUUGAGTT   35.52
 
  4157-4175   AD-14851   733   GCUCAAAUUUUAUAUAAGATT   734   UCUUAUAUAAAAUUUGAGCTT   36.07
 
  4795-4813   AD-14852   735   AGCCUGAUUUUGGUACAUGTT   736   CAUGUACCAAAAUCAGGCUTT   67.98
 
  4156-4174   AD-14853   737   CUCAAAUUUUAUAUAAGAATT   738   UUCUUAUAUAAAAUUUGAGTT   69.44
 
  5002-5020   AD-14854   739   ACAAAGUGCUGAAUAGGGATT   740   UCCCUAUUCAGCACUUUGUTT   29.12
 
  4792-4810   AD-14855   741   CUGAUUUUGGUACAUGGAATT   742   UUCCAUGUACCAAAAUCAGTT   36.04
 
  4790-4808   AD-14856   743   GAUUUUGGUACAUGGAAUATT   744   UAUUCCAUGUACCAAAAUCTT   33.61
 
  4801-4819   AD-14857   745   CUCAUCAGCCUGAUUUUGGTT   746   CCAAAAUCAGGCUGAUGAGTT   50.76
 
  4622-4640   AD-14858   747   AGCCCACUUGUGUGGAUAGTT   748   CUAUCCACACAAGUGGGCUTT   53.60
 
  4997-5015   AD-14859   749   GUGCUGAAUAGGGAGGAAUTT   750   AUUCCUCCCUAUUCAGCACTT   39.07
 
  5094-5112   AD-14860   751   AGUAAGGGCGUGGAGGCUUTT   752   AAGCCUCCACGCCCUUACUTT   62.78
 
  4564-4582   AD-14861   753   GUGACUUAACCCAAGAAGCTT   754   GCUUCUUGGGUUAAGUCACTT   87.47
 
  5095-5113   AD-14862   755   UAGUAAGGGCGUGGAGGCUTT   756   AGCCUCCACGCCCUUACUATT   79.95
 
  4800-4818   AD-14863   757   UCAUCAGCCUGAUUUUGGUTT   758   ACCAAAAUCAGGCUGAUGATT   30.46
 
  4265-4283   AD-14864   759   GUAGAAGACCCUAAAGACUTT   760   AGUCUUUAGGGUCUUCUACTT   33.18
 
  4267-4285   AD-14865   761   AGGUAGAAGACCCUAAAGATT   762   UCUUUAGGGUCUUCUACCUTT   26.25
 
  4270-4288   AD-14866   763   AAAAGGUAGAAGACCCUAATT   764   UUAGGGUCUUCUACCUUUUTT   36.73
 
  4269-4287   AD-14867   765   AAAGGUAGAAGACCCUAAATT   766   UUUAGGGUCUUCUACCUUUTT   33.16
 
  2874-2892   AD-14868   767   GAUUGUGCAGUGGAAAGAATT   768   UUCUUUCCACUGCACAAUCTT   29.91
 
  2875-2893   AD-14869   769   GGAUUGUGCAGUGGAAAGATT   770   UCUUUCCACUGCACAAUCCTT   28.24
 
  3950-3968   AD-14870   771   UGUAGACAGCCAUAUGCAGTT   772   CUGCAUAUGGCUGUCUACATT   50.37
 
  3896-3914   AD-14871   773   CAUGACUUUAACCCAGAAGTT   774   CUUCUGGGUUAAAGUCAUGTT   39.37
 
  4990-5008   AD-14872   775   AUAGGGAGGAAUCCAUGGATT   776   UCCAUGGAUUCCUCCCUAUTT   34.71
 
  4994-5012   AD-14873   777   CUGAAUAGGGAGGAAUCCATT   778   CCUCCCUAUUCAGCACUUUTT   32.14
 
  5000-5018   AD-14874   779   AAAGUGCUGAAUAGGGAGGTT   780   CCUCCCUAUUCAGCACUUUTT   101.77
 
  4563-4581   AD-14875   781   UGACUUAACCCAAGAAGCUTT   782   AGCUUCUUGGGUUAAGUCATT   80.81
 
  3895-3913   AD-14876   783   AUGACUUUAACCCAGAAGATT   784   UCUUCUGGGUUAAAGUCAUTT   30.74
 
  4262-4280   AD-14877   785   GAAGACCCUAAAGACUUUCTT   786   GAAAGUCUUUAGGGUCUUCTT   57.38
 
  4162-4180   AD-14878   787   AAAAAGCUCAAAUUUUAUATT   788   UAUAAAAUUUGAGCUUUUUTT   70.23
 
  4798-4816   AD-14879   789   AUCAGCCUGAUUUUGGUACTT   790   GUACCAAAAUCAGGCUGAUTT   79.03
 
  4799-4817   AD-14880   791   CAUCAGCCUGAUUUUGGUATT   792   UACCAAAAUCAGGCUGAUGTT   21.65
 
  5006-5024   AD-14881   793   AUGGACAAAGUGCUGAAUATT   794   UAUUCAGCACUUUGUCCAUTT   27.66
 
  4264-4282   AD-14882   795   UAGAAGACCCUAAAGACUUTT   796   AAGUCUUUAGGGUCUUCUATT   34.01
 
  4268-4286   AD-14883   797   AAGGUAGAAGACCCUAAAGTT   798   CUUUAGGGUCUUCUACCUUTT   40.62
 
  4623-4641   AD-14884   799   CAGCCCACUUGUGUGGAUATT   800   UAUCCACACAAGUGGGCUGTT   35.73
 
  4788-4806   AD-14885   801   UUUUGGUACAUGGAAUAGUTT   802   ACUAUUCCAUGUACCAAAATT   47.40
 
  4993-5011   AD-14886   803   UGAAUAGGGAGGAAUCCAUTT   804   AUGGAUUCCUCCCUAUUCATT   37.23
 
  4995-5013   AD-14887   805   GCUGAAUAGGGAGGAAUCCTT   806   GGAUUCCUCCCUAUUCAGCTT   42.94
 
  4996-5014   AD-14888   807   UGCUGAAUAGGGAGGAAUCTT   808   GAUUCCUCCCUAUUCAGCATT   32.58
 
  3952-3970   AD-14889   809   UGUGUAGACAGCCAUAUGCTT   810   GCAUAUGGCUGUCUACACATT   83.09
 
  4595-4613   AD-14890   811   UGCUUUGAUUGCUUCAGACTT   812   GUCUGAAGCAAUCAAAGCATT   59.49
 
  4596-4614   AD-14891   813   UUGCUUUGAUUGCUUCAGATT   814   UCUGAAGCAAUCAAAGCAATT   21.93
 
  4597-4615   AD-14892   815   AUUGCUUUGAUUGCUUCAGTT   816   CUGAAGCAAUCAAAGCAAUTT   72.69
 
  4599-4617   AD-14893   817   CUAUUGCUUUGAUUGCUUCTT   818   GAAGCAAUCAAAGCAAUAGTT   24.43
 
  4726-4744   AD-14894   819   AUUGCAAGGAAUGGCCUAATT   820   UUAGGCCAUUCCUUGCAAUTT   33.84
 
  4753-4771   AD-14895   821   AUUUUCCUCCUAAUUCUGATT   822   UCAGAAUUAGGAGGAAAAUTT   21.68
 
  4802-4820   AD-14896   823   GCUCAUCAGCCUGAUUUUGTT   824   CAAAAUCAGGCUGAUGAGCTT   26.99
 
  4803-4821   AD-14897   825   UGCUCAUCAGCCUGAUUUUTT   826   AAAAUCAGGCUGAUGAGCATT   29.04
 
  4806-4824   AD-14898   827   AGUUGCUCAUCAGCCUGAUTT   828   AUCAGGCUGAUGAGCAACUTT   32.64
 
  5091-5109   AD-14899   829   AAGGGCGUGGAGGCUUUUUTT   830   AAAAAGCCUCCACGCCCUUTT   61.71
 
  5093-5111   AD-14900   831   GUAAGGGCGUGGAGGCUUUTT   832   AAAGCCUCCACGCCCUUACTT   31.01
 
  4259-4277   AD-14901   833   GACCCUAAAGACUUUCCUGTT   834   CAGGAAAGUCUUUAGGGUCTT   31.47
 
  3901-3919   AD-14902   835   AGGAGCAUGACUUUAACCCTT   836   GGGUUAAAGUCAUGCUCCUTT   76.99
 
  4757-4775   AD-14903   837   UGUGAUUUUCCUCCUAAUUTT   838   AAUUAGGAGGAAAAUCACATT   20.55
 
  4758-4776   AD-14904   839   UUGUGAUUUUCCUCCUAAUTT   840   AUUAGGAGGAAAAUCACAATT   22.65
 
  4562-4580   AD-14905   841   GACUUAACCCAAGAAGCUCTT   842   GAGCUUCUUGGGUUAAGUCTT   56.98
 
  4585-4603   AD-14906   843   GCUUCAGACAAUGGUUUGGTT   844   CCAAACCAUUGUCUGAAGCTT   34.20
 
  4587-4605   AD-14907   845   UUGCUUCAGACAAUGGUUUTT   846   AAACCAUUGUCUGAAGCAATT   28.59
 
  4588-4606   AD-14908   847   AUUGCUUCAGACAAUGGUUTT   848   AACCAUUGUCUGAAGCAAUTT   34.08
 
  4591-4609   AD-14909   849   UUGAUUGCUUCAGACAAUGTT   850   CAUUGUCUGAAGCAAUCAATT   76.57
 
  5003-5021   AD-14910   851   GACAAAGUGCUGAAUAGGGTT   852   CCCUAUUCAGCACUUUGCCTT   46.50
 
  4165-4183   AD-14911   853   AAGAAAAAGCUCAAAUUUUTT   854   AAAAUUUGAGCUUUUUCUUTT   29.62
 
  4166-4184   AD-14912   855   AAAGAAAAAGCUCAAAUUUTT   856   AAAUUUGAGCUUUUUCUUUTT   22.27
 
  4263-4281   AD-14913   857   AGAAGACCCUAAAGACUUUTT   858   AAAGUCUUUAGGGUCUUCUTT   59.80
 
  4274-4292   AD-14914   859   AAAAAAAAGGUAGAAGACCTT   860   GGUCUUCUACCUUUUUUUUTT   93.21
 
  4266-4284   AD-14915   861   GGUAGAAGACCCUAAAGACTT   862   GUCUUUAGGGUCUUCUACCTT   25.99
 
  4272-4290   AD-14916   863   AAAAAAGGUAGAAGACCCUTT   864   AGGGUCUUCUACCUUUUUUTT   48.20
 
  4271-4289   AD-14917   865   AAAAAGGUAGAAGACCCUATT   866   UAGGGUCUUCUACCUUUUUTT   41.03
 
  4559-4577   AD-14918   867   UUAACCCAAGAAGCUCUUCTT   868   GAAGAGCUUCUUGGGUUAATT   110.62
 
  4789-4807   AD-14919   869   AUUUUGGUACAUGGAAUAGTT   870   CUAUUCCAUGUACCAAAAUTT   73.66
 
  4998-5016   AD-14920   871   AGUGCUGAAUAGGGAGGAATT   872   UUCCUCCCUAUUCAGCACUTT   19.80
 
  5070-5088   AD-14921   873   GAGGCCAGGGAAAUUCCCUTT   874   AGGGAAUUUCCCUGGCCUCTT   33.13
 
  4158-4176   AD-14922   875   AGCUCAAAUUUUAUAUAAGTT   876   CUUAUAUAAAAUUUGAGCUTT   52.94
 
  5065-5083   AD-14923   877   CAGGGAAAUUCCCUUGUUUTT   878   AAACAAGGGAAUUUCCCUGTT   33.77
 
  2872-2890   AD-14924   879   UUGUGCAGUGGAAAGAAAGTT   880   CUUUCUUUCCACUGCACAATT   64.47
 
  4782-4800   AD-14925   881   UACAUGGAAUAGUUCAGAGTT   882   CUCUGAACUAUUCCAUGUATT   97.16
 
  4783-4801   AD-14926   883   GUACAUGGAAUAGUUCAGATT   884   UCUGAACUAUUCCAUGUACTT   27.29
 
  5064-5082   AD-14927   885   AGGGAAAUUCCCUUGUUUUTT   886   AAAACAAGGGAAUUUCCCUTT   27.02
 
  5071-5089   AD-14928   887   GGAGGCCAGGGAAAUUCCCTT   888   GGGAAUUUCCCUGGCCUCCTT   76.75
 
  3951-3969   AD-14929   889   GUGUAGACAGCCAUAUGCATT   890   UGCAUAUGGCUGUCUACACTT   32.92
 
  3949-3967   AD-14930   891   GUAGACAGCCAUAUGCAGUTT   892   ACUGCAUAUGGCUGUCUACTT   31.00
 
  4355-4373   AD-14931   893   GAAGACCUGUUUUGCCAUGTT   894   CAUGGCAAAACAGGUCUUCTT   31.36
 
  4363-4381   AD-14932   895   AGUGGGAUGAAGACCUGUUTT   896   AACAGGUCUUCAUCCCACUTT   32.42
 
  4356-4374   AD-14933   897   UGAAGACCUGUUUUGCCAUTT   898   AUGGCAAAACAGGUCUUCATT   39.94
 
  4361-4379   AD-14934   899   UGGGAUGAAGACCUGUUUUTT   900   AAAACAGGUCUUCAUCCCATT   42.94
 
  4560-4578   AD-14935   901   CUUAACCCAAGAAGCUCUUTT   902   AAGAGCUUCUUGGGUUAAGTT   47.74
 
  2873-2891   AD-14936   903   AUUGUGCAGUGGAAAGAAATT   904   UUUCUUUCCACUGCACAAUTT   35.21
 
  4730-4748   AD-14937   905   CUUUAUUGCAAGGAAUGGCTT   906   GCCAUUCCUUGCAAUAAAGTT   89.25
 
  3899-3917   AD-14938   907   GAGCAUGACUUUAACCCAGTT   908   CUGGGUUAAAGUCAUGCUCTT   29.38
 
  4756-4774   AD-14939   909   GUGAUUUUCCUCCUAAUUCTT   910   GAAUUAGGAGGAAAAUCACTT   26.45
 
  4590-4608   AD-14940   911   UGAUUGCUUCAGACAAUGGTT   912   CCAUUGUCUGAAGCAAUCATT   77.50
 
  4159-4177   AD-14941   913   AAGCUCAAAUUUUAUAUAATT   914   UUAUAUAAAAUUUGAGCUUTT   36.40
 
  2743-2761   AD-14942   915   CUGGACAUGGAUCAAGCACTT   916   GUGCUUGAUCCAUGUCCAGTT   65.11
 
  4155-4173   AD-14943   917   UCAAAUUUUAUAUAAGAAATT   918   UUUCUUAUAUAAAAUUUGATT   89.96
 
  2871-2889   AD-14944   919   UGUGCAGUGGAAAGAAAGGTT   920   CCUUUCUUUCCACUGCACATT   48.98
 
  4786-4804   AD-14945   921   UUGGUACAUGGAAUAGUUCTT   922   GAACUAUUCCAUGUACCAATT   43.45
 
  4364-4382   AD-14946   923   AAGUGGGAUGAAGACCUGUTT   924   ACAGGUCUUCAUCCCACUUTT   41.25
 
  4359-4377   AD-14947   925   GGAUGAAGACCUGUUUUGCTT   926   GCAAAACAGGUCUUCAUCCTT   42.10
 
  2744-2762   AD-14948   927   UCUGGACAUGGAUCAAGCATT   928   UGCUUGAUCCAUGUCCAGATT   42.39
 
  4787-4805   AD-14949   929   UUUGGUACAUGGAAUAGUUTT   930   AACUAUUCCAUGUACCAAATT   27.68
 
[0202] 
[00009] [TABLE-US-00009]
 
      Relative    
      siRNA
      activity
  SD of   Residual   (normalized   SD of   Relative
  residual   luciferase   to positive   relative   siRNA
  luciferase   activity +/−   control luc-   siRNA   activity +/−
  activity   SD   siRNA)   activity   SD
 
 
  4.38   41 ± 4%   82.24   8.83   82 ± 9%
  4.10   21 ± 4%   109.97   21.56   110 ± 22%
  4.47   62 ± 4%   52.56   3.78   53 ± 4%
  2.60   44 ± 3%   77.91   4.60   78 ± 5%
  1.96   25 ± 2%   104.96   8.38   105 ± 8% 
  4.51   33 ± 5%   93.62   12.94    94 ± 13%
  3.23   94 ± 3%   8.36   0.29    8 ± 0%
  2.81   55 ± 3%   62.35   3.18   62 ± 3%
  3.11   31 ± 3%   96.14   9.70    96 ± 10%
  4.03   54 ± 4%   63.35   4.69   63 ± 5%
  7.58   54 ± 8%   64.13   9.02   64 ± 9%
  7.45   35 ± 7%   90.05   19.03    90 ± 19%
  2.80   70 ± 3%   41.17   1.64   41 ± 2%
  1.60   42 ± 2%   80.93   3.10   81 ± 3%
  3.05   57 ± 3%   60.22   3.24   60 ± 3%
  2.16   39 ± 2%   84.60   4.67   85 ± 5%
  2.95   40 ± 3%   83.72   6.22   84 ± 6%
  1.01   31 ± 1%   96.48   3.20   96 ± 3%
  2.74   28 ± 3%   99.93   9.72   100 ± 10%
  2.83   67 ± 3%   45.30   1.90   45 ± 2%
  1.30   36 ± 1%   88.85   3.21   89 ± 3%
  6.77   49 ± 7%   78.14   10.71    78 ± 11%
  5.32   74 ± 5%   40.09   2.88   40 ± 3%
  10.47    51 ± 10%   75.91   15.63    76 ± 16%
  3.55   73 ± 4%   42.32   2.07   42 ± 2%
  7.41   35 ± 7%   100.63   21.40   101 ± 21%
  6.31   49 ± 6%   79.24   10.27    79 ± 10%
  5.53   39 ± 6%   94.08   13.31    94 ± 13%
  5.89   46 ± 6%   84.01   10.85    84 ± 11%
  4.10   46 ± 4%   84.04   7.56   84 ± 8%
  3.64   33 ± 4%   103.26   11.36   103 ± 11%
  5.72   37 ± 6%   96.69   14.78    97 ± 15%
  4.41   42 ± 4%   88.96   9.26   89 ± 9%
  3.00   47 ± 3%   82.47   5.31   82 ± 5%
  8.67   71 ± 9%   44.35   5.40   44 ± 5%
  6.21   65 ± 6%   54.74   5.26   55 ± 5%
  8.91   60 ± 9%   61.07   9.00   61 ± 9%
  0.82   32 ± 1%   104.27   2.63   104 ± 3% 
  2.86   23 ± 3%   118.94   14.81   119 ± 15%
  9.43   57 ± 9%   66.41   10.99    66 ± 11%
  8.74   30 ± 9%   108.24   31.65   108 ± 32%
  6.57   43 ± 7%   88.58   13.66    89 ± 14%
  1.35   67 ± 1%   50.86   1.03   51 ± 1%
  3.32   49 ± 3%   78.89   5.35   79 ± 5%
  2.06   28 ± 2%   111.57   8.29   112 ± 8% 
  6.24   39 ± 6%   94.53   15.22    95 ± 15%
  8.60   33 ± 9%   103.70   27.17   104 ± 27%
  1.70   47 ± 2%   81.89   2.96   82 ± 3%
  4.90   44 ± 5%   87.06   9.79    87 ± 10%
  4.34   36 ± 4%   99.51   12.15   100 ± 12%
  3.51   38 ± 4%   95.38   8.75   95 ± 9%
  5.92   91 ± 6%   14.13   0.92   14 ± 1%
  3.27   83 ± 3%   25.68   1.01   26 ± 1%
  3.61   55 ± 4%   69.38   4.55   69 ± 5%
  5.78   31 ± 6%   106.56   19.89   107 ± 20%
  3.15   29 ± 3%   109.70   11.95   110 ± 12%
  3.70   67 ± 4%   50.35   2.76   50 ± 3%
  4.72   67 ± 5%   51.21   3.61   51 ± 4%
  5.72   33 ± 6%   103.04   17.71   103 ± 18%
  4.57   39 ± 5%   93.96   10.97    94 ± 11%
  5.35   91 ± 5%   13.58   0.80   14 ± 1%
  7.94   34 ± 8%   101.67   23.64   102 ± 24%
  6.54   30 ± 7%   107.96   23.48   108 ± 23%
  4.27   32 ± 4%   104.31   13.73   104 ± 14%
  3.11   36 ± 3%   99.41   8.67   99 ± 9%
  7.28   28 ± 7%   110.76   28.52   111 ± 29%
  3.96   30 ± 4%   107.63   14.08   108 ± 14%
  4.46   32 ± 4%   105.63   14.91   106 ± 15%
  5.71   30 ± 6%   107.91   20.46   108 ± 20%
  6.82   55 ± 7%   69.06   8.52   69 ± 9%
  5.99   45 ± 6%   84.51   11.19    85 ± 11%
  7.01   78 ± 7%   34.01   3.06   34 ± 3%
  3.56   30 ± 4%   108.78   13.09   109 ± 13%
  3.18   65 ± 3%   53.97   2.64   54 ± 3%
  4.63   64 ± 5%   55.53   4.02   56 ± 4%
  2.89   38 ± 3%   95.99   7.33   96 ± 7%
  5.60   29 ± 6%   109.82   21.30   110 ± 21%
  4.87   93 ± 5%   10.97   0.58   11 ± 1%
  3.69   75 ± 4%   37.97   1.86   38 ± 2%
  6.22   73 ± 6%   41.57   3.54   42 ± 4%
  9.34   86 ± 9%   21.02   2.27   21 ± 2%
  10.46    97 ± 10%   5.40   0.59    5 ± 1%
  3.41   33 ± 3%   104.03   10.89   104 ± 11%
  7.66   103 ± 8%    −4.18   0.31   −4 ± 0%
  5.66   92 ± 6%   11.66   0.71   12 ± 1%
  16.38    63 ± 16%   46.00   11.88    46 ± 12%
  15.21    46 ± 15%   67.99   22.49    68 ± 22%
  16.03    41 ± 16%   74.86   29.60    75 ± 30%
  3.66   117 ± 4%    −21.52   0.67   −22 ± 1% 
  21.12    55 ± 21%   56.93   21.95    57 ± 22%
  10.81    67 ± 11%   41.46   6.68   41 ± 7%
  11.90    72 ± 12%   35.85   5.97   36 ± 6%
  16.37    58 ± 16%   52.81   14.89    53 ± 15%
  5.43   93 ± 5%   8.36   0.49    8 ± 0%
  4.85   109 ± 5%    −11.13   0.50   −11 ± 0% 
  10.06   107 ± 10%   −8.41   0.79   −8 ± 1%
  6.68   37 ± 7%   79.23   14.28    79 ± 14%
  7.54   36 ± 8%   80.53   16.84    81 ± 17%
  5.90   39 ± 6%   77.40   11.86    77 ± 12%
  8.91   111 ± 9%    −13.67   1.10   −14 ± 1% 
  5.51   35 ± 6%   82.04   12.98    82 ± 13%
  6.04   24 ± 6%   95.99   24.41    96 ± 24%
  5.29   27 ± 5%   91.30   17.57    91 ± 18%
  4.70   93 ± 5%   8.67   0.44    9 ± 0%
  8.26   82 ± 8%   23.01   2.33   23 ± 2%
  5.29   78 ± 5%   27.83   1.89   28 ± 2%
  4.95   44 ± 5%   69.99   7.81   70 ± 8%
  5.08   46 ± 5%   67.46   7.38   67 ± 7%
  6.70   36 ± 7%   81.17   15.31    81 ± 15%
  1.13   36 ± 1%   102.63   3.22   103 ± 3% 
  6.75   68 ± 7%   51.41   5.11   51 ± 5%
  3.07   69 ± 3%   49.05   2.17   49 ± 2%
  6.88   29 ± 7%   113.79   26.89   114 ± 27%
  7.07   36 ± 7%   102.68   20.14   103 ± 20%
  7.93   34 ± 8%   106.57   25.15   107 ± 25%
  8.76   51 ± 9%   79.04   13.64    79 ± 14%
  7.26   54 ± 7%   74.49   10.09    74 ± 10%
  9.34   39 ± 9%   97.82   23.38    98 ± 23%
  6.85   63 ± 7%   59.75   6.52   60 ± 7%
  1.86   87 ± 2%   20.12   0.43   20 ± 0%
  4.02   80 ± 4%   32.19   1.62   32 ± 2%
  4.49   30 ± 4%   111.64   16.46   112 ± 16%
  5.07   33 ± 5%   107.26   16.38   107 ± 16%
  3.98   26 ± 4%   118.39   17.96   118 ± 18%
  1.24   37 ± 1%   101.57   3.44   102 ± 3% 
  3.13   33 ± 3%   107.30   10.12   107 ± 10%
  4.56   30 ± 5%   112.52   17.16   113 ± 17%
  3.66   28 ± 4%   115.20   14.91   115 ± 15%
  3.04   50 ± 3%   79.67   4.81   80 ± 5%
  5.11   39 ± 5%   97.32   12.63    97 ± 13%
  4.12   35 ± 4%   104.82   12.43   105 ± 12%
  1.79   32 ± 2%   108.93   6.07   109 ± 6% 
  4.87   102 ± 5%    −2.85   0.14   −3 ± 0%
  4.39   81 ± 4%   30.80   1.67   31 ± 2%
  1.88   31 ± 2%   111.18   6.81   111 ± 7% 
  2.84   57 ± 3%   68.42   3.39   68 ± 3%
  3.35   70 ± 3%   47.79   2.28   48 ± 2%
  7.72   79 ± 8%   33.66   3.29   34 ± 3%
  2.46   22 ± 2%   125.78   14.28   126 ± 14%
  1.71   28 ± 2%   116.13   7.17   116 ± 7% 
  2.94   34 ± 3%   105.93   9.16   106 ± 9% 
  3.22   41 ± 3%   95.33   7.56   95 ± 8%
  5.94   36 ± 6%   103.18   17.14   103 ± 17%
  7.65   47 ± 8%   84.45   13.63    84 ± 14%
  3.94   37 ± 4%   100.76   10.67   101 ± 11%
  7.26   43 ± 7%   91.61   15.50    92 ± 15%
  4.06   33 ± 4%   108.24   13.50   108 ± 14%
  2.98   83 ± 3%   27.15   0.97   27 ± 1%
  2.94   59 ± 3%   65.04   3.22   65 ± 3%
  5.52   22 ± 6%   125.32   31.52   125 ± 32%
  2.19   73 ± 2%   43.84   1.32   44 ± 1%
  7.07   24 ± 7%   121.32   35.11   121 ± 35%
  5.08   34 ± 5%   106.20   15.95   106 ± 16%
  4.46   22 ± 4%   125.73   25.84   126 ± 26%
  5.01   27 ± 5%   117.20   21.73   117 ± 22%
  2.72   29 ± 3%   113.92   10.67   114 ± 11%
  4.87   33 ± 5%   108.14   16.13   108 ± 16%
  4.59   62 ± 5%   61.47   4.57   61 ± 5%
  2.84   31 ± 3%   110.75   10.14   111 ± 10%
  1.57   31 ± 2%   110.01   5.49   110 ± 5% 
  0.55   77 ± 1%   36.95   0.26   37 ± 0%
  3.55   21 ± 4%   127.55   22.05   128 ± 22%
  6.87   23 ± 7%   124.18   37.68   124 ± 38%
  4.94   57 ± 5%   69.07   5.99   69 ± 6%
  3.66   34 ± 4%   105.63   11.29   106 ± 11%
  8.12   29 ± 8%   114.64   32.56   115 ± 33%
  3.36   34 ± 3%   105.82   10.44   106 ± 10%
  2.33   77 ± 2%   37.61   1.15   38 ± 1%
  4.14   46 ± 4%   85.89   7.64   86 ± 8%
  2.02   30 ± 2%   112.99   7.69   113 ± 8% 
  0.48   22 ± 0%   124.78   2.69   125 ± 3% 
  2.85   60 ± 3%   64.53   3.08   65 ± 3%
  5.10   93 ± 5%   10.90   0.60   11 ± 1%
  4.45   26 ± 4%   118.82   20.34   119 ± 20%
  1.46   48 ± 1%   83.16   2.51   83 ± 3%
  3.07   41 ± 3%   94.67   7.08   95 ± 7%
  6.34   111 ± 6%    −17.04   0.98   −17 ± 1% 
  3.68   74 ± 4%   42.29   2.11   42 ± 2%
  1.72   20 ± 2%   128.75   11.20   129 ± 11%
  1.14   33 ± 1%   107.34   3.71   107 ± 4% 
  6.99   53 ± 7%   63.41   8.37   63 ± 8%
  8.92   34 ± 9%   89.23   23.56    89 ± 24%
  10.96    64 ± 11%   47.86   8.13   48 ± 8%
  7.57   97 ± 8%   3.83   0.30    4 ± 0%
  8.79   27 ± 9%   97.96   31.56    98 ± 32%
  10.01    27 ± 10%   98.33   36.42    98 ± 36%
  4.78   77 ± 5%   31.32   1.95   31 ± 2%
  9.44   33 ± 9%   90.38   25.93    90 ± 26%
  9.40   31 ± 9%   92.97   28.21    93 ± 28%
  8.73   31 ± 9%   92.48   25.74    92 ± 26%
  9.01   32 ± 9%   91.05   25.29    91 ± 25%
  6.96   40 ± 7%   80.92   14.10    81 ± 14%
  7.66   43 ± 8%   76.88   13.71    77 ± 14%
  8.48   48 ± 8%   70.41   12.51    70 ± 13%
  4.02   35 ± 4%   87.29   9.97    87 ± 10%
  3.53   89 ± 4%   14.48   0.57   14 ± 1%
  6.46   29 ± 6%   95.15   20.91    95 ± 21%
  8.33   26 ± 8%   99.09   31.21    99 ± 31%
  6.51   78 ± 7%   30.31   2.55   30 ± 3%
  10.76    36 ± 11%   85.68   25.32    86 ± 25%
  5.84   65 ± 6%   47.01   4.22   47 ± 4%
  4.69   90 ± 5%   13.52   0.71   14 ± 1%
  5.85   49 ± 6%   68.74   8.21   69 ± 8%
  3.29   43 ± 3%   76.18   5.77   76 ± 6%
  1.26   41 ± 1%   79.15   2.43   79 ± 2%
  7.34   42 ± 7%   78.01   13.60    78 ± 14%
  5.75   42 ± 6%   77.62   10.54    78 ± 11%
  5.79   28 ± 6%   97.44   20.39    97 ± 20%
 
[0203] 
[00010] [TABLE-US-00010]
 
  siRNAs targeting JCV transcripts for primary screen   Description of chemistries:  
    a b c d
        sense strand   antisense strand
      position   SEQ     SEQ    
  duplex     in   ID     ID
  name   chemistry   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   GaauGAuuuuGGuAaAuGGTsT   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   AuAGGaauuAauaauGAAATsT   44   UuUCAGGAGuAAGGCCuAUTsT
 
  AD-12714   b   713-131   45   AuAGGccuuAcuccuGAAATsT   46   UUUcAGGAGuAAGGCCuAUTsT
 
  AD-12607   a   3946-3964   47   GAcAGccAuAuGcAGuAGuTsT   48   AcuACUGcAuAUGGCUGUCTsT
 
  AD-12715   b   3946-3964   49   GAcAGccAuAuGcAGuGuTsT   50   ACuACUGcAuAUGGCUGUCTsT
 
  AD-12788   c   3946-3964   51   GacAGccAuAuGcAGuAGuTsT   52   ACuACUGcAuAUGGCUGUCTsT
 
  AD-12835   d   3946-3964   53   GacAGccAuAuGcAGUGUTsT   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   AaAcuAcuGGGcAAoAGuTsT   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   GcCUCcAcGCCCUuACuATsT
 
  AD-12718   b   5096-5114   69   GuAGuAAGGGcGuGGaGGcTsT   70   GcCUCcAcGCCCUuACuATsT
 
  AD-12611   a   4727-4745   71   uAuuGcAAGGAAuGGccuATsT   72   uaGGCcAUUCCUUUGcAAuATsT
 
  AD-12719   b   4727-4745   73   uAuuGcAAGGAAuGGccuATsT   74   uAGGCcAUUCCUUUGcAAuATsT
 
  AD-12790   c   4727-4745   75   uAuuGcAAGGAAuGGccuATsT   76   uAGGCcAUUCCUUUGcAAuATsT
 
  AD-12837   d   4727-4745   77   uAuuGcAAGGAAuGGccuATsT   78   uaGGCcAUUCCUUUGcAAuATsT
 
  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   AAGUCCCGuAcAACCCuAAATsT
 
  AD-12627   a   662-680   155   uccccuuGcuAcuGuAGAGGTsT   156   CcUCuAcAGuAGcAAGGGATsT
 
  AD-12733   b   662-6   157   uccccuuGcuAcuGuAGAGGTsT   158   CCUCuAcAGuAGcAAGGGATsT
 
  AD-12628   a   663-681   159   cccuuGcuAcuGuAGAGGGTsT   160   CcUCuAcAGuAGcAAGGGATsT
 
  AD-12734   b   663-681   161   cccuuGcuAcuGuAGAGGGTsT   162   CCUCuAcAGuAGcAAGGGATsT
 
  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   UcCUCCCuAUUcAGcACUUUTsT
 
  AD-12744   b   4999-5017   223   AAGuGcuGAAuAGGGAGGATsT   224   UcCUCCCuAUUcAGcACUUUTsT
 
  AD-12801   c   4999-5017   225   AaGuGcuGAAuAGGGAGGATsT   226   UcCUCCCuAUUcAGcACUUUTsT
 
  AD-12848   d   4999-5017   227   AaGuGcuGAAuAGGGAGGATsT   228   UcCUCCCuAUUcAGcACUUUTsT
 
  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   234   AgGcuGcuGcuAcuAuAGATsT   236   UcuAuAGuAGcAGcAGCCUTsT
 
  AD-12641   a   3947-3965   237   AGAcAGccAuAuGcAGuAGTsT   238   CuACUGcAuAUGGUGUCUTsT
 
  AD-12803   c   3947-3965   239   AGAcAGccAuAuGcAGuAGTsT   240   CuACUGcAuAUGGUGUCUTsT
 
  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   3099-3918   255   GGAGcAuGAcuuuAAcccATsT   256   UGGGUuAAAGUcAUGCUCCTsT
 
  AD-12805   c   3099-3918   257   GgAGcAuGAcuuuAAcccATsT   258   UGGGUuAAAGUcAUGCUCCTsT
 
  AD-12851   d   3099-3918   259   GgAGcAuGAcuuuAAcccATsT   260   UgGGUuAAAGUcAUGCUCCTsT
 
  AD-12645   a   1417-1435   261   GuuGccuuuAcuuuuAGGGTsT   262   CcCuAAAGuAAAGGcAACCTsT
 
  AD-12749   b   1417-1435   263   GuuGccuuuAcuuuuAGGGTsT   264   CCCuAAAGuAAAGGcAACCTsT
 
  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   4779-4797   453   cAuGGAAuAGuucAGAGGuTsT   454   AcCUCUGAACuAUUCcAUGTsT
 
  AD-12774   b   4779-4797   455   cAuGGAAuAGuucAGAGGuTsT   456   ACCUCUGAACuAUUCcAUGTsT
 
  AD-12827   c   4779-4797   457   cauGGAAuAGuucAGAGGuTsT   458   ACCUCUGAACuAUUCcAUGTsT
 
  AD-12867   d   4779-4797   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   2465   GGuAcAuGGAAuAGuucAGTsT   466   CuGAACuAUUCcAUGuACCTsT
 
  AD-12776   b   4784-4802   2467   GGuAcAuGGAAuAGuucAGTsT   468   CUGAACuAUUCcAUGuACCTsT
 
  AD-12828   c   4784-4802   2469   GguAcAuGGAAuAGuucAGTsT   470   CUGAACuAUUCcAUGuACCTsT
 
  AD-12868   d   4784-4802   2471   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   CccAAGuAGUUUCUUUCcAATsT
 
  AD-12780   b   1121-1139   493   uuGGAAGAAAcuAcuuGGGTsT   494   CCcAAGuAGUUUCUUUCcAATsT
 
  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
 
  exo/endo-light + 2′-O-methyl in position 2 of antisense
  exo/endo-light: sense strand: dTsdT + 2′OME@all Py; antinsense strand: dTsdT + 2′OMe@ Py in uA, cA
  exo/endo-light + 2′O-methyl in position 2 of sense
  exo/endo-light + 2′O-methyl in position 2 of sense and antisense
[0204] 
[00011] [TABLE-US-00011]
 
  Residual          
  luciferase       Relative
  activity       siRNA
  (relative to   SD of     activity
  control   residual   Residual   (normalized   SD of   Relative
  siRNA   luci-   luciferase   to positive   relative   siRNA
  treated   ferase   activity +/−   control luc-   siRNA   activity +/−
  cells)   activity   SD   siRNA)   activity   SD
 
 
  91   11    91 ± 11%   9   2   9 ± 2%
  32   5   32 ± 5%   76   17   76 ± 17%
  25   6   25 ± 6%   79   13   79 ± 13%
  16   4   16 ± 4%   97   26   97 ± 26%
  79   9   79 ± 9%   21   3   21 ± 3% 
  25   4   25 ± 4%   85   24   85 ± 24%
  23   2   23 ± 2%   87   14   87 ± 14%
  84   11    84 ± 11%   18   4   18 ± 4% 
  102   8   102 ± 8%    −6   1   −6 ± 1% 
  95   10    95 ± 10%   6   1   6 ± 1%
  107   9   107 ± 9%    −11   2   −11 ± 2% 
  70   4   70 ± 4%   34   3   34 ± 3% 
  69   8   69 ± 8%   35   7   35 ± 7% 
  94   8   94 ± 8%   7   1   7 ± 1%
  100   9   100 ± 9%    −4   1   −4 ± 1% 
  27   5   27 ± 5%   82   16   82 ± 16%
  15   2   15 ± 2%   94   13   94 ± 13%
  94   5   94 ± 5%   7   0   7 ± 0%
  61   10    61 ± 10%   41   8   41 ± 8% 
  55   6   55 ± 6%   47   6   47 ± 6% 
  92   16    92 ± 16%   8   2   8 ± 2%
  78   3   78 ± 3%   25   1   25 ± 1% 
  63   6   63 ± 6%   42   5   42 ± 5% 
  101   9   101 ± 9%    −1   0   −1 ± 0% 
  101   5   101 ± 5%    −1   0   −1 ± 0% 
  85   18    85 ± 18%   15   4   15 ± 4% 
  95   9   95 ± 9%   6   1   6 ± 1%
  103   13   103 ± 13%   −3   0   −3 ± 0% 
  81   9   81 ± 9%   22   3   22 ± 3% 
  61   4   61 ± 4%   44   4   44 ± 4% 
  103   11   103 ± 11%   −3   0   −3 ± 0% 
  108   19   108 ± 19%   −9   2   −9 ± 2% 
  94   17    94 ± 17%   7   1   7 ± 1%
  88   9   88 ± 9%   14   2   14 ± 2% 
  39   4   39 ± 4%   64   8   64 ± 8% 
  38   6   38 ± 6%   69   12   69 ± 12%
  26   4   26 ± 4%   78   13   78 ± 13%
  17   3   17 ± 3%   87   18   87 ± 18%
  22   4   22 ± 4%   81   16   81 ± 16%
  100   6   100 ± 6%    0   0   0 ± 0%
  73   6   73 ± 6%   28   3   28 ± 3% 
  46   9   46 ± 9%   57   12   57 ± 12%
  97   15    97 ± 15%   3   1   3 ± 1%
  26   4   26 ± 4%   82   15   82 ± 15%
  10   1   10 ± 1%   94   12   94 ± 12%
  10   3   10 ± 3%   94   40   94 ± 40%
  22   3   22 ± 3%   81   12   81 ± 12%
  15   5   15 ± 5%   94   38   94 ± 38%
  6   1    6 ± 1%   98   26   98 ± 26%
  93   4   93 ± 4%   8   0   8 ± 0%
  95   4   95 ± 4%   5   0   5 ± 0%
  73   7   73 ± 7%   30   3   30 ± 3% 
  88   10    88 ± 10%   13   2   13 ± 2% 
  42   7   42 ± 7%   60   7   60 ± 7% 
  21   5   21 ± 5%   89   32   89 ± 32%
  95   7   95 ± 7%   6   1   6 ± 1%
  71   2   71 ± 2%   30   1   30 ± 1% 
  54   7   54 ± 7%   48   7   48 ± 7% 
  94   9   94 ± 9%   7   1   7 ± 1%
  106   7   106 ± 7%    −8   1   −8 ± 1% 
  100   7   100 ± 7%    0   0   0 ± 0%
  107   9   107 ± 9%    −7   1   −7 ± 1% 
  47   4   47 ± 4%   60   8   60 ± 8% 
  40   8   40 ± 8%   67   20   67 ± 20%
  78   13    78 ± 13%   25   8   25 ± 8% 
  16   4   16 ± 4%   92   29   92 ± 29%
  25   6   25 ± 6%   84   29   84 ± 29%
  23   3   23 ± 3%   86   20   86 ± 20%
  19   4   19 ± 4%   91   20   91 ± 20%
  103   9   103 ± 9%    −3   0   −3 ± 0% 
  84   8   84 ± 8%   17   2   17 ± 2% 
  31   4   31 ± 4%   77   12   77 ± 12%
  18   1   18 ± 1%   85   3   85 ± 3% 
  94   10    94 ± 10%   5   1   5 ± 1%
  57   4   57 ± 4%   48   4   48 ± 4% 
  99   7   99 ± 7%   0   0   0 ± 0%
  82   5   82 ± 5%   20   1   20 ± 1% 
  80   6   80 ± 6%   22   2   22 ± 2% 
  65   6   65 ± 6%   39   4   39 ± 4% 
  81   6   81 ± 6%   21   2   21 ± 2% 
  82   7   82 ± 7%   21   2   21 ± 2% 
  113   11   113 ± 11%   −14   2   −14 ± 2% 
  90   9   90 ± 9%   11   1   11 ± 1% 
  92   8   92 ± 8%   9   1   9 ± 1%
  117   7   117 ± 7%    −19   1   −19 ± 1% 
  124   3   124 ± 3%    −27   1   −27 ± 1% 
  85   4   85 ± 4%   16   1   16 ± 1% 
  52   1   52 ± 1%   53   1   53 ± 1% 
  96   4   96 ± 4%   5   0   5 ± 0%
  110   11   110 ± 11%   −12   1   −12 ± 1% 
  115   13   115 ± 13%   −17   2   −17 ± 2% 
  106   2   106 ± 2%    −7   0   −7 ± 0% 
  107   12   107 ± 12%   −8   1   −8 ± 1% 
  88   5   88 ± 5%   14   1   14 ± 1% 
  79   5   79 ± 5%   24   1   24 ± 1% 
  69   8   69 ± 8%   35   6   35 ± 6% 
  75   8   75 ± 8%   25   6   25 ± 6% 
  65   8   65 ± 8%   40   8   40 ± 8% 
  56   4   56 ± 4%   50   6   50 ± 6% 
  74   6   74 ± 6%   30   3   30 ± 3% 
  89   8   89 ± 8%   9   1   9 ± 1%
  31   4   31 ± 4%   78   14   78 ± 14%
  16   2   16 ± 2%   93   14   93 ± 14%
  18   3   18 ± 3%   85   14   85 ± 14%
  18   4   18 ± 4%   86   22   86 ± 22%
  15   2   15 ± 2%   89   14   89 ± 14%
  95   6   95 ± 6%   5   0   5 ± 0%
  23   4   23 ± 4%   81   15   81 ± 15%
  14   1   14 ± 1%   90   10   90 ± 10%
  90   12    90 ± 12%   10   2   10 ± 2% 
  113   11   113 ± 11%   −15   2   −15 ± 2% 
  42   4   42 ± 4%   60   7   60 ± 7% 
  34   3   34 ± 3%   68   8   68 ± 8% 
  114   3   114 ± 3%    −14   0   −14 ± 0% 
  96   11    96 ± 11%   4   1   4 ± 1%
  52   7   52 ± 7%   53   8   53 ± 8% 
  74   9   74 ± 9%   29   4   29 ± 4% 
  111   5   111 ± 5%    −12   1   −12 ± 1% 
  103   8   103 ± 8%    −3   0   −3 ± 0% 
  94   13    94 ± 13%   6   1   6 ± 1%
  105   3   105 ± 3%    −6   0   −6 ± 0% 
  100   9   100 ± 9%    0   0   0 ± 0%
  33   4   33 ± 4%   74   10   74 ± 10%
  21   3   21 ± 3%   83   13   83 ± 13%
  25   4   25 ± 4%   78   14   78 ± 14%
  28   4   28 ± 4%   75   11   75 ± 11%
  82   7   82 ± 7%   20   2   20 ± 2% 
  25   4   25 ± 4%   78   14   78 ± 14%
  23   7   23 ± 7%   80   30   80 ± 30%
  61   7   61 ± 7%   41   5   41 ± 5% 
  112   6   112 ± 6%    −14   1   −14 ± 1% 
  86   10    86 ± 10%   16   2   16 ± 2% 
  94   10    94 ± 10%   6   1   6 ± 1%
  93   11    93 ± 11%   7   1   7 ± 1%
  77   8   77 ± 8%   24   3   24 ± 3% 
  96   4   96 ± 4%   5   0   5 ± 0%
  27   3   27 ± 3%   81   11   81 ± 11%
  29   6   29 ± 6%   74   19   74 ± 19%
  31   2   31 ± 2%   72   6   72 ± 6% 
  26   3   26 ± 3%   78   11   78 ± 11%
  81   9   81 ± 9%   17   3   17 ± 3% 
  92   9   92 ± 9%   8   1   8 ± 1%
  71   9   71 ± 9%   30   5   30 ± 5% 
  81   2   81 ± 2%   21   1   21 ± 1% 
  57   1   57 ± 1%   48   1   48 ± 1% 
  52   4   52 ± 4%   54   5   54 ± 5% 
  77   5   77 ± 5%   26   2   26 ± 2% 
  89   6   89 ± 6%   13   1   13 ± 1% 
  88   7   88 ± 7%   12   1   12 ± 1% 
  67   6   67 ± 6%   35   4   35 ± 4% 
  88   10    88 ± 10%   12   2   12 ± 2% 
  91   2   91 ± 2%   10   0   10 ± 0% 
  40   3   40 ± 3%   67   6   67 ± 6% 
  35   1   35 ± 1%   72   3   72 ± 3% 
  75   8   75 ± 8%   28   4   28 ± 4% 
  79   8   79 ± 8%   23   3   23 ± 3% 
  17   5   17 ± 5%   86   27   86 ± 27%
  97   6   97 ± 6%   3   0   3 ± 0%
  74   5   74 ± 5%   27   2   27 ± 2% 
  46   6   46 ± 6%   59   9   59 ± 9% 
  14   0   14 ± 0%   89   2   89 ± 2% 
  12   3   12 ± 3%   92   28   92 ± 28%
  35   7   35 ± 7%   70   17   70 ± 17%
  10   3   10 ± 3%   99   33   99 ± 33%
  9   1    9 ± 1%   95   18   95 ± 18%
  108   1   108 ± 1%    −9   0   −9 ± 0% 
  101   4   101 ± 4%    −1   0   −1 ± 0% 
  98   9   98 ± 9%   2   0   2 ± 0%
  83   4   83 ± 4%   18   1   18 ± 1% 
  80   14    80 ± 14%   21   4   21 ± 4% 
  25   3   25 ± 3%   79   11   79 ± 11%
  67   4   67 ± 4%   35   2   35 ± 2% 
  95   11    95 ± 11%   8   3   8 ± 3%
  66   7   66 ± 7%   39   6   39 ± 6% 
  34   2   34 ± 2%   73   5   73 ± 5% 
  10   3   10 ± 3%   94   30   94 ± 30%
  12   4   12 ± 4%   92   37   92 ± 37%
  33   1   33 ± 1%   72   2   72 ± 2% 
  92   7   92 ± 7%   7   1   7 ± 1%
  91   11    91 ± 11%   10   2   10 ± 2% 
  99   10    99 ± 10%   3   1   3 ± 1%
  20   5   20 ± 5%   89   22   89 ± 22%
  20   4   20 ± 4%   90   20   90 ± 20%
  93   11    93 ± 11%   8   1   8 ± 1%
  93   9   93 ± 9%   6   2   6 ± 2%
  94   8   94 ± 8%   10   1   10 ± 1% 
  58   8   58 ± 8%   47   10   47 ± 10%
  49   6   49 ± 6%   58   9   58 ± 9% 
  93   8   93 ± 8%   8   1   8 ± 1%
  30   5   30 ± 5%   76   18   76 ± 18%
  25   2   25 ± 2%   84   9   84 ± 9% 
  65   10    65 ± 10%   38   7   38 ± 7% 
  34   7   34 ± 7%   69   17   69 ± 17%
  34   4   34 ± 4%   73   10   73 ± 10%
  13   3   13 ± 3%   91   22   91 ± 22%
  11   2   11 ± 2%   93   17   93 ± 17%
  19   4   19 ± 4%   87   22   87 ± 22%
  22   3   22 ± 3%   87   12   87 ± 12%
  11   4   11 ± 4%   93   39   93 ± 39%
  45   3   45 ± 3%   61   5   61 ± 5% 
  10   3   10 ± 3%   94   31   94 ± 31%
  12   1   12 ± 1%   92   13   92 ± 13%
  41   4   41 ± 4%   64   8   64 ± 8% 
  83   9   83 ± 9%   19   2   19 ± 2% 
  74   7   74 ± 7%   29   3   29 ± 3% 
  52   7   52 ± 7%   54   9   54 ± 9% 
  28   3   28 ± 3%   81   12   81 ± 12%
  56   5   56 ± 5%   49   5   49 ± 5% 
  36   2   36 ± 2%   72   5   72 ± 5% 
  33   2   33 ± 2%   75   5   75 ± 5% 
  49   7   49 ± 7%   57   10   57 ± 10%
  90   9   90 ± 9%   11   1   11 ± 1% 
  45   6   45 ± 6%   61   9   61 ± 9% 
  45   5   45 ± 5%   62   8   62 ± 8% 
  47   6   47 ± 6%   59   9   59 ± 9% 
  31   4   31 ± 4%   77   11   77 ± 11%
  31   3   31 ± 3%   77   10   77 ± 10%
  43   7   43 ± 7%   64   12   64 ± 12%
  23   4   23 ± 4%   86   16   86 ± 16%
  22   4   22 ± 4%   87   16   87 ± 16%
  102   8   102 ± 8%    −2   0   −2 ± 0% 
  101   13   101 ± 13%   −1   0   −1 ± 0% 
  99   1   99 ± 1%   1   0   1 ± 0%
  91   7   91 ± 7%   10   1   10 ± 1% 
  81   8   81 ± 8%   21   2   21 ± 2% 
  11   2   11 ± 2%   93   19   93 ± 19%
  17   3   17 ± 3%   92   17   92 ± 17%
  15   2   15 ± 2%   89   17   89 ± 17%
  11   2   11 ± 2%   93   18   93 ± 18%
  15   3   15 ± 3%   91   22   91 ± 22%
  28   3   28 ± 3%   79   10   79 ± 10%
  8   1    8 ± 1%   95   19   95 ± 19%
  43   6   43 ± 6%   63   9   63 ± 9% 
  23   5   23 ± 5%   80   19   80 ± 19%
  23   5   23 ± 5%   80   20   80 ± 20%
  25   4   25 ± 4%   81   16   81 ± 16%
  17   2   17 ± 2%   91   15   91 ± 15%
  11   2   11 ± 2%   92   22   92 ± 22%
  12   1   12 ± 1%   92   11   92 ± 11%
  19   3   19 ± 3%   87   16   87 ± 16%
  87   12    87 ± 12%   14   2   14 ± 2% 
  41   4   41 ± 4%   66   8   66 ± 8% 
  35   1   35 ± 1%   72   1   72 ± 1% 
  68   5   68 ± 5%   36   3   36 ± 3% 
  58   5   58 ± 5%   47   5   47 ± 5% 
  73   8   73 ± 8%   30   4   30 ± 4% 
  62   8   62 ± 8%   42   7   42 ± 7% 
  18   1   18 ± 1%   91   4   91 ± 4% 
  11   3   11 ± 3%   93   33   93 ± 33%
  96   4   96 ± 4%   4   0   4 ± 0%
  45   7   45 ± 7%   58   10   58 ± 10%
  15   3   15 ± 3%   89   19   89 ± 19%
  51   3   51 ± 3%   52   4   52 ± 4% 
  93   6   93 ± 6%   8   1   8 ± 1%
  36   3   36 ± 3%   66   7   66 ± 7% 
  27   2   27 ± 2%   76   7   76 ± 7% 
  81   18    81 ± 18%   21   5   21 ± 5% 
 
(57)

Claims

1. A double stranded ribonucleic acid (dsRNA) for inhibiting expression of a human JC virus genome in a cell, wherein said dsRNA comprises a sense strand and an antisense strand that together form a region of complementarity less than 30 base pairs in length, wherein the portion of the antisense strand that is complementary to the sense strand comprises at least 15 contiguous nucleotides complementary to 5′-UGUUGAAUGUUGGGUUCCU-3′ (SEQ ID NO: 935), and wherein said dsRNA, upon contact with a cell expressing said JC virus, inhibits expression of said JC virus genome.
2. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.
3. The dsRNA of claim 2, wherein said 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.
4. The dsRNA of claim 2, wherein said 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.
5. A cell comprising the dsRNA of claim 1.
6. 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.
7. A method for inhibiting the expression of a gene from the 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.
8. A method of inhibiting the replication of a JC virus in a patient comprising administering to the patient an amount of the dsRNA of claim 1 effective to inhibit replication of the virus.
9. A vector for inhibiting the expression of a gene from JC virus in a cell, said vector being capable of:
a) expressing the dsRNA of claim 1 as a single RNA molecule with two complementary regions; or alternatively
b) expressing each strand of the dsRNA of claim 1 under the control of a separate promoter.
10. A cell comprising the vector of claim 9.
11. The dsRNA of claim 1, wherein said antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 132.
12. The dsRNA of claim 1 wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 131 and said antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 132.
13. The dsRNA of claim 1 wherein the sense strand consists of SEQ ID NO: 131 and said antisense strand consists of SEQ ID NO: 132.
14. A composition comprising a plurality of vectors wherein one vector comprises a regulatory sequence operably linked to a nucleotide sequence encoding the sense strand of claim 1, and another vector comprises a regulatory sequence operably linked to a nucleotide sequence encoding the antisense strand of claim 1.
15. A cell comprising the composition of claim 14.
*****