# Patent

## Title
Compositions and methods for inhibiting expression of a gene from the JC virus

## 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.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/252,414, filed on Oct. 4, 2011, which is a continuation of U.S. application Ser. No. 12/720,465, filed Mar. 9, 2010 (U.S. Pat. No. 8,058,257), which is a continuation of U.S. application Ser. No. 11/741,205, filed Apr. 27, 2007 (U.S. Pat. No. 7,691,824) and claims the benefit of U.S. Provisional Application No. 60/795,765, filed Apr. 28, 2006. All of the prior applications are incorporated herein by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 27, 2013, is named 22811US_CRF_sequencelisting.txt and is 310,917 bytes in size.


FIELD OF THE INVENTION
This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of one of the genes of the JC virus and the use of the dsRNA to treat pathological processes mediated by JC virus infection, such as PML.
BACKGROUND OF THE INVENTION
Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system which results from reactivation of the latent polyomavirus JC virus (JCV) and its productive replication in glial cells of the human brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18). Once a rare disease primarily seen in patients with impaired immune systems due to lymphoproliferative and myeloproliferative disorders, PML has become one of the major neurologic problems among patients with AIDS (Cinque, P., (2003). J. Neurovirol. 9(Suppl. 1):88-92).
It has been reported that between 4 and 8% of AIDS patients exhibit signs of PML, and JCV has been detected in the cerebrospinal fluid of affected patients, suggesting that there is active replication of the virus in the brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18, Clifford, D. B., (2001) J. Neurovirol. 4:279). In addition, PML has recently been seen in patients undergoing experimental treatment with Tsybari, an anti VLA4 antibody, in combination with interferon. The histological hallmarks of PML include multifocal demyelinated lesions with enlarged eosinophilic nuclei in oligodendrocytes and enlarged bizarre astrocytes with lobulated hyperchromatic nuclei within white matter tracts of the brain (Cinque, P., (2003). J. Neurovirol. 9(Suppl. 1):88-92), although in some instances atypical features that include a unifocal pattern of demyelination and involvement of the gray matter have been reported (Sweeney, B. J., (1994). J. Neurol. Neurosurg. Psychiatry 57:994-997). Earlier observations from in vitro cell culture studies and an in vivo evaluation of JCV in clinical samples led to early assumptions that oligodendrocytes and astrocytes are the only cells which support productive viral infections (Gordon, J. (1998) Int. J. Mol. Med. 1:647-655). Accordingly, molecular studies have provided evidence for cell-type-specific transcription of the viral early genome in cells derived from the central nervous system (Raj, G. V., (1995) Virology 10:283-291). However, subsequent studies have shown low, but detectable, levels of JCV gene expression in normeural cells, including B cells, and noticeably high levels of production of the viral early protein in several neural and normeural tumor cells in humans (Gordon, J. (1998) Int. J. Mol. Med. 1:647-655, Khalili, K., 2003. Oncogene 22:5181-5191).
Like the other polyomaviruses, JCV is a small DNA virus whose genome can be divided into three regions that encompass the transcription control region; the genes responsible for the expression of the viral early protein, T antigen; and the genes encoding the viral late proteins, VP1, VP2, and VP3. In addition, the late genome is also responsible for production of an auxiliary viral protein, agnoprotein. T-antigen expression is pivotal for initiation of the viral lytic cycle, as this protein stimulates transcription of the late genes and induces the process of viral DNA replication. Recent studies have ascribed an important role for agnoprotein in the transcription and replication of JCV, as inhibition of its production significantly reduced viral gene expression and replication (M. Safak et al., unpublished observations). Furthermore, the agnoprotein dysregulates the cell cycle by altering the expression of several cyclins and their associated kinases (Darbinyan, A., (2002) Oncogene 21:5574-5581).
Thus far, there are no effective therapies for the suppression of JCV replication and the treatment of PML. Cytosine arabinoside (AraC) has been tested for the treatment of PML patients, and the outcome in some instances revealed a remission of JCV-associated demyelination (Aksamit, A. (2001) J. Neurovirol. 7:386-390). Reports from the AIDS Clinical Trial Group Organized Trial 243, however, have suggested that there is no difference in the survival of human immunodeficiency virus type 1 (HIV-1)-infected patients with PML and that of the control population, although in other reports it has been suggested that the failure of AraC in the AIDS Clinical Trial Group trial may have been due to insufficient delivery of the AraC via the intravenous and intrathecal routes (Levy, R. M., (2001) J. Neurovirol. 7:382-385). Based on in vitro studies showing the ability of inhibitors of topoisomerase to suppress JCV DNA replication, the topoisomerase inhibitor topotecan was used for the treatment of AIDS-PML patients, and the results suggested that topotecan treatment may be associated with a decreased lesion size and prolonged survival (Royal, W., III, (2003) J. Neurovirol. 9:411-419).
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Recent reports have indicated that in vitro, RNAi may show some promise in reducing JC virus replication (Radhakrishnan, S. (2004) J. Vir. 78:7264-7269, Orba, Y. (2004) J. Vir. 78:7270-7273). However, the RNAi agents examined were not designed against all know JC Virus strains and were not selected for stability and other properties need for in vivo therapeutic RNAi agents. Accordingly, despite significant advances in the field of RNAi, there remains a need for an agent that can selectively and efficiently silence a gene in the JC virus using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit replication of the JC virus for use in treating pathological processes mediated by JC virus infection.
SUMMARY OF THE INVENTION
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the JC virus in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by JC viral infection, such as PML. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a gene from the JC Virus.
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression one of the genes of the JC virus and viral replication. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoded by a gene from the JC Virus, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing infected with the JC virus, inhibits the expression of a gene from the JC Virus by at least 40%.
For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1a and b and the second sequence is selected from the group consisting of the antisense sequences of Tables 1a and b. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such modified sequence will be based on a first sequence of said dsRNA selected from the group consisting of the sense sequences of Tables 1a and b and a second sequence selected from the group consisting of the antisense sequences of Tables 1a and 1b.
In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is generally a mammalian cell, such as a human cell.
In another embodiment, the invention provides a pharmaceutical composition for inhibiting the replication of the JC virus in an organism, generally a human subject, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier or delivery vehicle.
In another embodiment, the invention provides a method for inhibiting the expression of a gene in the JC Virus in a cell, comprising the following steps:

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


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



BRIEF DESCRIPTION OF THE FIGURES
No Figures are presented.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of a gene from the JC Virus in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by JC virus infection using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a gene from the JC Virus. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and or maintenance of JC virus infection and the occurrence of PML in a subject infected with the JC virus. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a gene from the JC Virus. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by JC viral infection, e.g. cancer, by targeting a gene involved in JC virus replication and/or maintenance in a cell.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a gene from the JC virus, as well as compositions and methods for treating diseases and disorders caused by the infection with the JC virus, such as PML. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of a gene from the JC Virus, together with a pharmaceutically acceptable carrier.
Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a gene in a gene from the JC Virus, and methods of using the pharmaceutical compositions to treat diseases caused by infection with the JC virus.
I. Definitions
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “JC virus” refers to the latent polyomavirus JC Virus that has a reference sequence NC—001699. In addition, further accession numbers of various JCVirus sequences are AB038249.1-AB038255.1, AB048545.1-AB048582.1, AB074575.1-AB074591.1, AB077855.1-AB077879.1, AB081005.1-AB081030.1, AB081600.1-AB081618.1, AB081654.1, AB092578.1-AB092587.1, AB103387.1, AB103402.1-AB103423.1, AB104487.1, AB113118.1-AB113145.1, AB118651.1-AB118659.1, AB126981.1-AB127027.1, AB127342.1, AB127344.1, AB127346.1-AB127349.1, AB127352.1-AB127353.1, AB198940.1-AB198954.1, AB220939.1-AB220943.1, AF004349.1-AF004350.1, AF015526.1-AF015537.1, AF015684.1, AF030085.1, AF281599.1-AF281626.1, AF295731.1-AF295739.1, AF300945.1-AF300967.1, AF363830.1-AF363834.1, AF396422.1-AF396435.1, AY121907.1-AY121915.1, NC—001699.1, U61771.1, U73500.1-U73502.1.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene from the JC Virus, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding JC virus). For example, a polynucleotide is complementary to at least a part of a JC virus mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding JC virus.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence” and “inhibit the expression of”, in as far as they refer to a gene from the JC Virus, herein refer to the at least partial suppression of the expression of a gene from the JC Virus, as manifested by a reduction of the amount of mRNA transcribed from a gene from the JC Virus which may be isolated from a first cell or group of cells in which a gene from the JC Virus is transcribed and which has or have been treated such that the expression of a gene from the JC Virus is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to JC virus genome transcription, e.g. the amount of protein encoded by a gene from the JC Virus, or the number of cells displaying a certain phenotype, e.g. infection with the JC Virus. In principle, JC virus genome silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a gene from the JC Virus by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of a gene from the JC Virus is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, a gene from the JC Virus is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, a gene from the JC Virus is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention.
As used herein in the context of JC virus expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by JC virus infection. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by JC virus expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by JC virus infection or an overt symptom of pathological processes mediated by JC virus expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by JC virus infection, the patient's history and age, the stage of pathological processes mediated by JC virus infection, and the administration of other anti-pathological processes mediated by JC virus infection.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a gene from the JC Virus in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a gene from the JC Virus, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing the gene from the JC virus, inhibits the expression of the JC virus gene by at least 40%.
The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a gene from the JC Virus, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s).
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, a gene from the JC Virus is the human JC virus genome. In specific embodiments, the antisense strand of the dsRNA comprises the sense sequences of Tables 1a and b and the second sequence is selected from the group consisting of the antisense sequences of Tables 1a and b. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1a and b can readily be determined using the target sequence and the flanking JC virus sequence.
In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Tables 1a and b. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of a gene from the JC Virus. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1a and b and the second oligonucleotide is described as the antisense strand in Tables 1a and b
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1a and b, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1a and b minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1a and b, and differing in their ability to inhibit the expression of a gene from the JC Virus in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1a and b can readily be made using the JC virus sequence and the target sequence provided.
In addition, the RNAi agents provided in Tables 1a and b identify a site in the JC virus mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1a and b coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a gene from the JC Virus. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target JC virus genome produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1a and b.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a gene from the JC Virus, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a gene from the JC Virus. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a gene from the JC Virus is important, especially if the particular region of complementarity in a gene from the JC Virus is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular—CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]n, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other preferred modifications include 2′-methoxy(2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. dsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-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.
Vector Encoded RNAi Agents
The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.
dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
III. Pharmaceutical Compositions Comprising dsRNA
In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a gene from the JC Virus and/or viral infection, such as PML. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.
The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of a gene from the JC Virus. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of a gene from the JC Virus.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by JC virus expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Liposomes
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, 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 GM1 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).
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, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an dsRNA RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNA dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by JC virus expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Caused by Expression of a Gene from the JC Virus
The invention relates in particular to the use of a dsRNA or a pharmaceutical composition prepared therefrom for the treatment or prevention of pathological conditions associated with JC Virus infection, e.g., PML. Owing to the inhibitory effect on JC virus expression, an dsRNA according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life, particularly in a patient being treated with an anti-VLA4 antibody as part of treatment for MS.
The invention furthermore relates to the use of an dsRNA or a pharmaceutical composition thereof for treating PML in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.
Methods for Inhibiting Expression of a Gene from the JC Virus
In yet another aspect, the invention provides a method for inhibiting the expression of a gene from the JC Virus in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target JC virus genome is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target JC virus gene. Compositions and methods for inhibiting the expression of these JC virus genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of a gene from the JC Virus, to the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Design of JCV siRNAs
Full-length genome sequences to JC virus available on Apr. 10, 2006, were obtained, resulting in a target pool of 388 sequences (accession numbers: AB038249.1-AB038255.1; AB048545.1-AB048582.1; AB074575.1-AB074591.1; AB077855.1-AB077879.1; AB081005.1-AB081030.1; AB081600.1-AB081618.1; AB081654.1; AB092578.1-AB092587.1; AB103387.1; AB103402.1-AB103423.1; AB104487.1; AB113118.1-AB113145.1; AB118651.1-AB118659.1; AB126981.1-AB127027.1; AB127342.1; AB127344.1; AB127346.1-AB127349.1; AB127352.1-AB127353.1; AB198940.1-AB198954.1; AB220939.1-AB220943.1; AF004349.1-AF004350.1; AF015526.1-AF015537.1; AF015684.1; AF030085.1; AF281599.1-AF281626.1; AF295731.1-AF295739.1; AF300945.1-AF300967.1; AF363830.1-AF363834.1; AF396422.1-AF396435.1; AY121907.1-AY121915.1; NC—001699.1; U61771.1; U73500.1-U73502.1). NC—001699 was defined as reference sequence.
The siRNA selection process was run as follows: ClustalW multiple alignment was used to generate a global alignment of all sequences from the target pool. An IUPAC consensus sequence was then generated.
All conserved 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.
In order to rank candidate target sequences and their respective siRNAs and select appropriate ones, their predicted potential for interacting with irrelevant targets (off-target potential) was taken as a ranking parameter. siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.
For predicting siRNA-specific off-target potential, the following assumptions were made:

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


To identify potential off-target genes, 19mer input sequences were subjected to a homology search against publically available human mRNA sequences.
To this purpose, fastA (version 3.4) searches were performed with all 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.
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:

    
    
        Number of mismatches in non-seed region
        Number of mismatches in seed region
        Number of mismatches in cleavage site region
    
    


The off-target score was calculated for considering assumption 1 to 3 as follows:




  
    Off
    ⁢
    
      -
    
    ⁢
    target
    ⁢
    
      
    
    ⁢
    score
  
  =
  
    
      number
      ⁢
      
        
      
      ⁢
      of
      ⁢
      
        
      
      ⁢
      seed
      ⁢
      
        
      
      ⁢
      mismatches
      *
      10
    
    +
    
      number
      ⁢
      
        
      
      ⁢
      of
      ⁢
      
        
      
      ⁢
      cleavage
      ⁢
      
        
      
      ⁢
      site
      ⁢
      
        
      
      ⁢
      mismatches
      *
      1.2
    
    +
    
      number
      ⁢
      
        
      
      ⁢
      of
      ⁢
      
        
      
      ⁢
      non
      ⁢
      
        -
      
      ⁢
      seed
      ⁢
      
        
      
      ⁢
      mismatches
      *
      1
    
  




The most relevant off-target gene for input each 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.
In order to generate a ranking for siRNAs, calculated relevant off-target scores were transferred into a result table. All siRNAs were sorted according to the off-target score (descending).
An off-target score of 2.2 was defined as cut-off for siRNA selection (specificity criterion). In addition, all sequences with only one mismatch in the seed region were eliminated from the screening set. The selection procedure resulted in a set of 93 JCV specific siRNAs (Table 1a).
An expanded screening was generated by re-calculating the predicted specificity based on the newly available human RefSeq database (Human mRNA sequences in RefSeq release version 21 (downloaded Jan. 12, 2007)) and selecting only 208 siRNAs that did not contain more than 3 G's in a row and had an off-target score of at least 2 for the antisense strand (Table 1b).
Synthesis of JCV siRNAs
All siRNAs were synthesized in 0.2 μmole synthesis scale on an ABI3900 DNA synthesizer according to standard procedures.
For the initial screening set (93 different siRNA sequences), 4 different strategies of chemical modification were used:

    a) exo/endo light (EEL): —sense strand: 2′-O-methyl @ all pyrimidines, PTO between nucleotides 20 and 21 (counting from 5′-end), dTdT at 3′-end (nucleotides 20 and 21)
    —antisense strand: 2′-O-methyl at pyrimidines in 5′-UA-3′ and 5′-CA-3′ motives, PTO between nucleotides 20 and 21 (counting from 5′-end), dTdT at 3′-end (nucleotides 20 and 21)
    b) EEL plus 2′-O-methyl in position 2 of antisense strand (only if no 5′-UA-3′ and 5′-CA-3′ at 5′-end, otherwise already covered by EEL)
    c) EEL plus 2′-O-methyl in position 2 of sense strand (only if no pyrimidine in position 2, otherwise already covered by EEL)
    d) EEL plus 2′-O-methyl in position 2 of sense and antisense strand (only if not already covered by a, b, and c) (Table 1a)


For the expanded screening set (208 different siRNA sequences), siRNAs were composed of unmodified RNA oligonucleotides with dT/dT overhangs (dTdT at 3′-end (nucleotides 20 and 21) of antisense and sense strands) (Table 1b).
Screening of JCV siRNAs
Construction of Reporter-Systems Encoding JCV Transcripts
The sequence of the early JCV transcript (E) was synthesized at GENEART (Regensburg, Germany) and cloned into GENEART standard vectors. The sequence of the late JCV transcript was subdivided in a first approach into two fragments: L1, including the transcript sequence of the VP1 protein, and LA23, including the sequences of VP2, VP3 and the Agnoprotein. Due to cloning problems with fragment LA23, this sequence was subdivided in a second approach into two fragments (LA23 1-700 and LA23 701-1438). All sequences were synthesized at GENEART and cloned into GENEART standard vectors. All fragments (E, L1, LA23 1-700 and LA23 701-1438) were subcloned into psiCheck-2 (Promega, Mannheim, Germany) via XhoI and NotI (both NEB, Frankfurt, Germany), resulting in constructs with the JCV sequences between the stop-codon and the polyA-signal of Renilla luciferase.








L1



(SEQ ID NO: 931)



CTCGAGACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCATATGAAGATGGCCCCA



 


ACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGTTCCAAAACTTCTTATAAGAGGAGGAGT


 


AGAAGTTCTAGAAGTTAAAACTGGGGTTGACTCAATTACAGAGGTAGAATGCTTTTTAACTCCAG


 


AAATGGGTGACCCAGATGAGCATCTTAGGGGTTTTAGTAAGTCAATTTCTATATCAGATACATTT


 


GAAAGTGACTCCCCAAATAAGGACATGCTTCCTTGTTACAGTGTGGCCAGAATTCCACTACCCAA


 


TCTAAATGAGGATCTAACCTGTGGAAATATACTAATGTGGGAGGCTGTGACCTTAAAAACTGAGG


 


TTCTAGGGGTGACAACTTTGATGAATGTGCACTCTAATGGTCAAGCAACTCATGACAATGGTGCA


 


GGAAAGCCAGTGCAGGGCACCAGCTTTCATTTTTTTTCTGTTGGCGGGGAGGCTTTAGAATTACA


 


GGGGGTGGTTTTTAATTACAGAACAAAGTACCCAGATGGAACAATTTTTCCAAAGAATGCAACAG


 


TGCAATCTCAAGTAATGAACACAGAGCACAAGGCGTACCTAGATAAGAACAAAGCATATCCTGTT


 


GAATGTTGGGTTCCTGATCCCACCAGAAATGAAAACACAAGATATTTTGGGACACTAACAGGAGG


 


AGAAAATGTTCCTCCAGTTCTTCATATAACAAACACTGCCACAACAGTGCTGCTTGATGAATTTG


 


GTGTTGGGCCACTTTGCAAAGGTGACAACTTGTATTTGTCAGCTGTTGATGTTTGTGGAATGTTT


 


ACTAACAGATCTGGTTCCCAGCAGTGGAGAGGACTGTCCAGATATTTTAAGGTTCAGCTCAGAAA


 


AAGGAGGGTTAAAAACCCCTACCCAATTTCTTTCCTTCTTACTGATTTGATTAACAGAAGGACCC


 


CTAGAGTTGATGGGCAACCTATGTATGGTATGGATGCTCAGGTAGAGGAGGTTAGAGTTTTTGAG


 


GGGACAGAGGAACTTCCAGGGGACCCAGACATGATGAGATATGTTGACAGATATGGACAGTTGCA


 


AACAAAGATGCTGTAATCAAAATCCTTTATTGTAATATGCAGTACATTTTAATAAAGTATAACCA


 


GCTTTACTTTACAGTTGCAGTCATGCGGCCGC


 


E


(SEQ ID NO: 932)



CTCGAGCCGCCTCCAAGCTTACTCAGAAGTAGTAAGGGCGTGGAGGCTTTTTAGGAGGCC



 


AGGGAAATTCCCTTGTTTTTCCCTTTTTTGCAGTAATTTTTTGCTGCAAAAAGCTAAAATGGACA


 


AAGTGCTGAATAGGGAGGAATCCATGGAGCTTATGGATTTATTAGGCCTTGATAGGTCTGCATGG


 


GGGAACATTCCTGTCATGAGAAAAGCTTATCTGAAAAAATGCAAAGAACTCCACCCTGATAAAGG


 


TGGGGACGAAGACAAGATGAAGAGAATGAATTTTTTATATAAAAAAATGGAACAAGGTGTAAAAG


 


TTGCTCATCAGCCTGATTTTGGTACATGGAATAGTTCAGAGGTTGGTTGTGATTTTCCTCCTAAT


 


TCTGATACCCTTTATTGCAAGGAATGGCCTAACTGTGCCACTAATCCTTCAGTGCATTGCCCCTG


 


TTTAATGTGCATGCTAAAATTAAGGCATAGAAACAGAAAATTTTTAAGAAGCAGCCCACTTGTGT


 


GGATAGATTGCTATTGCTTTGATTGCTTCAGACAATGGTTTGGGTGTGACTTAACCCAAGAAGCT


 


CTTCATTGCTGGGAGAAAGTTCTTGGAGACACCCCCTACAGGGATCTAAAGCTTTAAGTGCCAAC


 


CTATGGAACAGATGAATGGGAATCCTGGTGGAATACATTTAATGAGAAGTGGGATGAAGACCTGT


 


TTTGCCATGAAGAAATGTTTGCCAGTGATGATGAAAACACAGGATCCCAACACTCTACCCCACCT


 


AAAAAGAAAAAAAAGGTAGAAGACCCTAAAGACTTTCCTGTAGATCTGCATGCATTCCTCAGTCA


 


AGCTGTGTTTAGTAATAGAACTGTTGCTTCTTTTGCTGTGTATACCACTAAAGAAAAAGCTCAAA


 


TTTTATATAAGAAACTTATGGAAAAATATTCTGTAACTTTTATAAGTAGACATGGTTTTGGGGGT


 


CATAATATTTTGTTTTTCTTAACACCACATAGACATAGAGTGTCAGCAATTAATAACTACTGTCA


 


AAAACTATGTACCTTTAGTTTTTTAATTTGTAAAGGTGTGAATAAGGAATACTTGTTTTATAGTG


 


CCCTGTGTAGACAGCCATATGCAGTAGTGGAAGAAAGTATTCAGGGGGGCCTTAAGGAGCATGAC


 


TTTAACCCAGAAGAACCAGAAGAAACTAAGCAGGTTTCATGGAAATTAGTTACACAGTATGCCTT


 


GGAAACCAAGTGTGAGGATGTTTTTTTGCTTATGGGCATGTACTTAGACTTTCAGGAAAACCCAC


 


AGCAATGCAAAAAATGTGAAAAAAAGGATCAGCCAAATCACTTTAACCATCATGAAAAACACTAT


 


TATAATGCCCAAATTTTTGCAGATAGCAAAAATCAAAAAAGCATTTGCCAGCAGGCTGTTGATAC


 


TGTAGCAGCCAAACAAAGGGTTGACAGCATCCACATGACCAGAGAAGAAATGTTAGTTGAAAGGT


 


TTAATTTCTTGCTTGATAAAATGGACTTAATTTTTGGGGCACATGGCAATGCTGTTTTAGAGCAA


 


TATATGGCTGGGGTGGCCTGGATTCATTGCTTGCTGCCTCAAATGGACACTGTTATTTATGACTT


 


TCTAAAATGCATTGTATTAAACATTCCAAAAAAAAGGTACTGGCTATTCAAGGGGCCAATAGACA


 


GTGGCAAAACTACTTTAGCTGCAGCTTTACTTGATCTCTGTGGGGGAAAGTCATTAAATGTTAAT


 


ATGCCATTAGAAAGATTAAACTTTGAATTAGGAGTGGGTATAGATCAGTTTATGGTTGTATTTGA


 


GGATGTAAAAGGCACTGGTGCAGAGTCAAGGGATTTACCTTCAGGGCATGGCATAAGCAACCTTG


 


ATTGCTTAAGAGATTACTTAGATGGAAGTGTAAAAGTTAATTTAGAGAGAAAACACCAAAACAAA


 


AGAACACAGGTGTTTCCACCTGGAATTGTAACCATGAATGAATATTCAGTGCCTAGAACTTTACA


 


GGCCAGATTTGTAAGGCAGATAGATTTTAGACCAAAGGCCTACCTGAGAAAATCACTAAGCTGCT


 


CTGAGTATTTGCTAGAAAAAAGGATTTTGCAAAGTGGTATGACTTTGCTTTTGCTTTTAATCTGG


 


TTTAGGCCAGTTGCTGACTTTGCAGCTGCCATTCATGAGAGGATTGTGCAGTGGAAAGAAAGGCT


 


GGATTTAGAAATAAGCATGTATACATTTTCTACTATGAAAGCTAATGTTGGTATGGGGAGACCCA


 


TTCTTGACTTTCCTAGAGAGGAAGATTCTGAAGCAGAAGACTCTGGACATGGATCAAGCACTGAA


 


TCACAATCACAATGCTTTTCCCAGGTCTCAGAAGCCTCTGGTGCAGACACACAGGAAAACTGCAC


 


TTTTCACATCTGTAAAGGCTTTCAATGTTTCAAAAAACCAAAGACCCCTCCCCCAAAATAACTGC


 


AACTGTGCGGCCGC


 


LA23 1-700


(SEQ ID NO: 933)



CTCGAGCAGCTAACAGCCAGTAAACAAAGCACAAGGGGAAGTGGAAAGCAGCCAAGGGAACATGT



 


TTTGCGAGCCAGAGCTGTTTTGGCTTGTCACCAGCTGGCCATGGTTCTTCGCCAGCTGTCACGTA


 


AGGCTTCTGTGAAAGTTAGTAAAACCTGGAGTGGAACTAAAAAAAGAGCTCAAAGGATTTTAATT


 


TTTTTGTTAGAATTTTTGCTGGACTTTTGCACAGGTGAAGACAGTGTAGACGGGAAAAAAAGACA


 


GAGACACAGTGGTTTGACTGAGCAGACATACAGTGCTTTGCCTGAACCAAAAGCTACATAGGTAA


 


GTAATGTTTTTTTTTGTGTTTTCAGGTTCATGGGTGCCGCACTTGCACTTTTGGGGGACCTAGTT


 


GCTACTGTTTCTGAGGCTGCTGCTGCCACAGGATTTTCAGTAGCTGAAATTGCTGCTGGAGAGGC


 


TGCTGCTACTATAGAAGTTGAAATTGCATCCCTTGCTACTGTAGAGGGGATTACAAGTACCTCTG


 


AGGCTATAGCTGCTATAGGCCTTACTCCTGAAACATATGCTGTAATAACTGGAGCTCCGGGGGCT


 


GTAGCTGGGTTTGCTGCATTGGTTCAAACTGTAACTGGTGGTAGTGCTATTGCTCAGTTGGGATA


 


TAGATTTTTTGCTGACTGGGATCATAAAGTTTCAACAGTTGGGCTTTTTCGCGGCCGC


 


LA23 701-1438


(SEQ ID NO: 934)



CTCGAGAGCAGCCAGCTATGGCTTTACAATTATTTAATCCAGAAGACTACTATGATATTTTATTT



 


CCTGGAGTGAATGCCTTTGTTAACAATATTCACTATTTAGATCCTAGACATTGGGGCCCGTCCTT


 


GTTCTCCACAATCTCCCAGGCTTTTTGGAATCTTGTTAGAGATGATTTGCCAGCCTTAACCTCTC


 


AGGAAATTCAGAGAAGAACCCAAAAACTATTTGTTGAAAGTTTAGCAAGGTTTTTGGAAGAAACT


 


ACTTGGGCAATAGTTAATTCACCAGCTAACTTATATAATTATATTTCAGACTATTATTCTAGATT


 


GTCTCCAGTTAGGCCCTCTATGGTAAGGCAAGTTGCCCAAAGGGAGGGAACCTATATTTCTTTTG


 


GCCACTCATACACCCAAAGTATAGATGATGCAGACAGCATTCAAGAAGTTACCCAAAGGCTAGAT


 


TTAAAAACCCCAAATGTGCAATCTGGTGAATTTATAGAAAGAAGTATTGCACCAGGAGGTGCAAA


 


TCAAAGATCTGCTCCTCAATGGATGTTGCCTTTACTTTTAGGGTTGTACGGGACTGTAACACCTG


 


CTCTTGAAGCATATGAAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGT


 


TCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAACTGGGGTTGACTCAATTA


 


CAGAGGTAGAATGCTGCGGCCGC






Screen of JCV siRNAs in Transfected Cells
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 LipofectamineTM 2000 (Invitrogen GmbH, Karlsruhe, Germany), with the plasmid diluted in Opti-MEM to a final volume of 12.5 μl per well, prepared as a mastermix for the whole plate.
4 h after plasmid transfection, growth medium was removed from cells and replaced by 100 μl/well of fresh medium. siRNA transfections were performed using LipofectamineTM 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 μM. Each siRNA was tested in quadruplicate for each concentration.
Cells were lysed by removing growth medium and application of 150 μl of a 1:1 mixture consisting of medium and substrate from the Dual-Glo Luciferase Assay System (Promega, Mannheim, Germany). The luciferase assay was performed according to the manufacturer's protocol for Dual-Glo Luciferase assay and luminescence was measured in a Victor-Light 1420 Luminescence Counter (Perkin Elmer, Rodgau-Jügesheim, Germany). Values obtained with Renilla luciferase were normalized to the respective values obtained with Firefly luciferase in order to correct for transfection efficacy. Renilla/Firefly luciferase activities obtained after transfection with siRNAs directed against a JCV gene were normalized to Renilla/Firefly luciferase activities obtained after transfection of an unrelated control siRNA set to 100%. Tables 1a and b provides the results where the siRNAs, the sequences of which are given in Tables 1a and b, were tested at a single dose of 30 nM. The percentage inhibition±standard deviation, compared to the unrelated control siRNA, is indicated in the column ‘Remaining luciferase activity (% of control)’. A number of JCV siRNAs at 30 nM were effective at reducing levels of the targeted mRNA by more than 70% in Cos-7 cells (i.e. remaining luciferase activity was less than 30%).
Selected JCV siRNAs from the single dose screen were further characterized by dose response curves. Transfections of JCV siRNAs for generation of dose response curves were performed with the following siRNA concentrations according to the above protocol:

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


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







TABLE 2







IC50s










Duplex
Mean IC50



name
[nM]














AD-12599
2.37



AD-12622
0.57



AD-12666
3.7



AD-12677
0.49



AD-12709
0.19



AD-12710
0.47



AD-12712
2.33



AD-12722
0.12



AD-12724
0.26



AD-12728
0.8



AD-12761
1.2



AD-12763
0.95



AD-12767
0.09



AD-12768
0.19



AD-12769
0.35



AD-12771
0.35



AD-12774
0.13



AD-12775
0.18



AD-12777
0.17



AD-12778
12.65



AD-12781
0.18



AD-12784
0.44



AD-12795
0.65



AD-12813
0.2



AD-12818
1.88



AD-12821
0.07



AD-12823
0.46



AD-12824
0.25



AD-12825
0.52



AD-12827
0.15



AD-12829
0.14



AD-12842
0.44










Screen of JCV siRNAs Against Live JC Virus in SVG-A Cells
Cells and Virus
SVG-A cells (human fetal glial cells transformed by SV40 T antigen) obtained from Walter Atwood at Brown University were cultured in Eagle's Minimum Essential Media (ATCC, Manassas, Va.) supplemented to contain 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, Calif.), Penicillin 100 U/ml, Streptomycin 100 ug/ml (Invitrogen, Carlsbad Calif.) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Thermo Electron Corporation, Ashville, N.C.). The Mad-1-SVEA strain of JCV obtained from Walter Atwood at Brown University was used in all experiments; viral stocks were prepared using SVG-A cells according to standard published methods (Liu and Atwood, Propagation and assay of the JC Virus, Methods Mol. Biol. 2001; 165:9-17).
Prophylaxis Assay
SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hours prior to transfection in the media described above minus antibiotics. Cells were transfected with the indicated concentration of siRNA (10 nM, 50 nM, or 100 nM) using LipofectamineTM 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-SVEA 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.







TABLE 3







Prophylaxis Assay









Duplex
Targeted JCV
Remaining Virus (% of Luciferase Control)











Number
Transcript
50 nM
10 nM
100 nM














AD-12599
VP1
79.9
ND
ND


AD-12709
VP1
46.0
ND
ND


AD-12710
VP1
25.9
ND
ND


AD-12784
VP1
30.9
ND
ND


AD-12712
VP1
29.7
ND
ND


AD-12724
VP1
30.5
38.9
25.8


AD-12622
VP1
22.9
28.2
 9.1


AD-12728
VP1
21.1
22.2
ND


AD-12795
VP1
13.6
16.9
 8.5


AD-12842
VP1
16.0
23.4
12.7


AD-12761
VP1
26.4
52.3
ND


AD-12818
VP1
24.0
50.2
28.0


AD-12666
VP1
54.1
ND
ND


AD-12763
VP1
39.5
ND
ND


AD-12722
T Antigen
43.6
82.1
ND


AD-12813
T Antigen
21.5
48.8
19.4


AD-12767
T Antigen
37.6
52.2
30.9


AD-12821
T Antigen
33.0
51.2
30.8


AD-12774
T Antigen
74.0
89.2
ND


AD-12827
T Antigen
77.0
92.0
ND


AD-12775
T Antigen
81.6
95.4
ND


AD-12777
T Antigen
73.3
93.9
ND


AD-12829
T Antigen
78.6
93.6
ND


AD-12781
T Antigen
38.8
62.6
34.4


AD-12768
VP2/3
73.9
92.4
ND


AD-12771
VP2/3
51.6
83.6
ND


AD-12824
VP2/3
42.1
79.0
43.7


AD-12769
VP2/3
35.2
78.0
39.7


AD-12823
VP2/3
38.1
78.1
42.0


AD-12677
VP2/3
99.1
102.1 
ND


AD-12825
VP2/3
100.8
99.1
ND





ND indicates no data.






Post-Infection Treatment Assay
SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hours prior to infection in 10% FBS media. Cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock diluted in 2% FBS media. Cells were rocked by hand approximately 8-10 times to get equal virus binding across the entire coverslip every 15 minutes for one hour and then additional 10% FBS media was added. Twenty-four and forty-eight hours postinfection, cells were washed with 10% FBS media containing no antibiotics and then transfected with 50 nM of the indicated siRNA using LipofectamineTM 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.Z1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for control coverslips transfected with Luciferase siRNA. Table 4 shows the results of the post-infection treatment experiments. All of the siRNAs tested in the treatment assay showed significant antiviral activity against JCV, such that the remaining virus was significantly less than that in the luciferase siRNA control.







TABLE 4







Treatment Assay











Duplex
Targeted JCV
Remaining Virus (% of



Number
Transcript
Luciferase Control)















AD-12724
VP1
38.9



AD-12622
VP1
28.2



AD-12795
VP1
16.9



AD-12842
VP1
23.4



AD-12818
VP1
ND



AD-12813
T Antigen
48



AD-12767
T Antigen
56.9



AD-12821
T Antigen
75.8



AD-12781
T Antigen
75.8



AD-12824
VP2/3
60.4



AD-12769
VP2/3
70.7



AD-12823
VP2/3
72.4







ND indicates no data.






Prophylaxis Administration of JCV siRNAs Inhibits the Production of Active Progeny JC Virus
SVG-A cells were seeded in 6-well dishes 24 hours prior to transfection in the media described above minus antibiotics. Cells were transfected with 10 nM of the indicated siRNA using LipofectamineTM 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.







TABLE 5







Prophylaxis administration of JCV siRNAs inhibits the production


of active progeny JC virus capable of secondary infection









Remaining Virus (% of Luciferase Control)










Duplex
Targeted

Virus


Name
Transcript
Media
Preparation













AD-12622
VP1
30.8
24.9


AD-12842
VP1
33.3
26.9


AD-12813
T Antigen
57.8
38.7


AD-12824
VP2/3
83.6
57.6


AD-12769
VP2/3
79.1
52.2









Stability in Cerebrospinal Fluid (CSF) of Selected siRNAs Targeting JCV
Eleven Selected JCV siRNAs were Tested for Stability at 5 uM Over 48 h at 37° C. in Human CSF, as Well as in PBS for Comparison.
30 μl of human cerebrospinal fluid (CSF) was mixed with 3 μl of 50 μM duplex (siRNA) solution (150 pmole/well) in a 96-well plate, sealed to avoid evaporation and incubated for the indicated time at 37° C. Incubation of the siRNA in 30 μl PBS for 48 h served as a control for non-specific degradation. Reactions were stopped by the addition of 4 ul proteinase K (20 mg/ml) and 25 ul of proteinase K buffer, and an incubation for 20′ at 42° C. Samples were then spin filtered through a 0.2 μm 96 well filter plate at 3000 rpm for 20′. Incubation wells were washed with 50 ul Millipore water twice and the combined washing solutions were spin filtered also.
Samples were analyzed by ion exchange HPLC under denaturing conditions. Samples were transferred to single autosampler vials. IEX-HPLC analysis was performed under the following conditions: Dionex DNAPac PA200 (4×250 mm analytical column), temperature of 45° C. (denaturing conditions by pH=11), flow rate of 1 ml/min, injection volume of 50 ul, and detection wavelength of 260 nm with 1 nm bandwidth (reference wavelength 600 nm). In addition, the gradient conditions were as follows with HPLC Eluent A: 20 mM Na3PO4 in 10% ACN; pH=11 and HPLC Eluent B: 1 M NaBr in HPLC Eluent A:















Time
% A
% B




















0.00 min
75
25



1.00 min
75
25



19.0 min
38
62



19.5 min
0
100



21.5 min
0
100



22.0 min
75
25



24.0 min
75
25










Under the above denaturing IEX-HPLC conditions, the duplexes eluted as two separated single strands. All chromatograms were integrated automatically by the Dionex Chromeleon 6.60 HPLC software, and were adjusted manually as necessary. The area under the peak for each strand was calculated and the %-values for each intact full length product (FLP) for each time points were calculated by the following equation:

%-FLP(s/as;t=x)=(PeakArea(s/as);t=x/PeakArea(s/as);t=0min)*100%

All values were normalized to FLP at t=0 min. Table 6 provides the results after 48 hours of incubation in human CSF at 37° C. At least 75% of both antisense and sense strands of ten JCV siRNAs (AD-12622, AD-12724, AD-12767, AD-12769, AD-12795, AD-12813, AD-12818, AD-12823, AD-12824, AD-12842) were recovered, demonstrating that these siRNAs are highly stable in human CSF at 37° C. For AD-12821, 59% of the antisense and 97% of the sense strand was recovered after 48 h of incubation in human CSF at 37° C., showing that this siRNA has a half-life of greather than 48 h in human CSF at 37° C.








TABLE 6







Stability in human CSF











Duplex
% full length material after 48 hours












name
antisense
sense















AD-12622
93
105



AD-12724
90
106



AD-12767
85
104



AD-12769
100
104



AD-12795
86
109



AD-12813
94
98



AD-12818
75
99



AD-12821
59
97



AD-12823
98
98



AD-12824
84
98



AD-12842
87
102










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







TABLE 1a 







JCV Gene Walk; siRNAs targeting >95% of all stransins (>=out of 388);


Human specific pan-JCV: 208 siRNAs; allsiRNAs double overhang design;


dTdT, no modifications. 1a-1: sequences; 1a-2: assay results












Table 1a-1
position
SEQ

SEQ



duplex
in
ID
sense strand
ID
antisense strand


name
consensus
NO:
sequence (5′-3′)
NO:
sequence (5′-3′)





AD-14742
1533-1551
515
CUUAUAAGAGGAGGAGUAGTT
516
CUACUCCUCCUCUUAUAAGTT


 


AD-14743
1703-1721
517
CAUGCUUCCUUGUUACAGUTT
518
ACUGUAACAAGGAAGCAUGTT


 


AD-14744
1439-1457
519
UACGGGACUGUAACACCUGTT
520
CAGGUGUUACAGUCCCGUATT


 


AD-14745
1705-1723
521
UGCUUCCUUGUUACAGUGUTT
522
ACACUGUAACAAGGAAGCATT


 


AD-14746
2064-2082
523
CCUGUUGAAUGUUGGGUUCTT
524
GAACCCAACAUUCAACAGGTT


 


AD-14747
2067-2085
525
GUUGAAUGUUGGGUUCCUGTT
526
CAGGAACCCAACAUUCAACTT


 


AD-14748
2071-2089
527
AAUGUUGGGUUCCUGAUCCTT
528
GGAUCAGGAACCCAACAUUTT


 


AD-14749
2121-2139
529
ACACUAACAGGAGGAGAAATT
530
UUUCUCCUCCUGUUAGUGUTT


 


AD-14750
1535-1553
531
UAUAAGAGGAGGAGUAGAATT
532
UUCUACUCCUCCUCUUAUATT


 


AD-14751
1536-1554
533
AUAAGAGGAGGAGUAGAAGTT
534
CUUCUACUCCUCCUCUUAUTT


 


AD-14752
1445-1463
535
ACUGUAACACCUGCUCUUGTT
536
CAAGAGCAGGUGUUACAGUTT


 


AD-14753
1700-1718
537
GGACAUGCUUCCUUGUUACTT
538
GUAACAAGGAAGCAUGUCCTT


 


AD-14754
1702-1720
539
ACAUGCUUCCUUGUUACAGTT
540
CUGUAACAAGGAAGCAUGUTT


 


AD-14755
1704-1722
541
AUGCUUCCUUGUUACAGUGTT
542
CACUGUAACAAGGAAGCAUTT


 


AD-14756
2065-2083
543
CUGUUGAAUGUUGGGUUCCTT
544
GGAACCCAACAUUCAACAGTT


 


AD-14757
2070-2088
545
GAAUGUUGGGUUCCUGAUCTT
546
GAUCAGGAACCCAACAUUCTT


 


AD-14758
1441-1459
547
CGGGACUGUAACACCUGCUTT
548
AGCAGGUGUUACAGUCCCGTT


 


AD-14759
1443-1461
549
GGACUGUAACACCUGCUCUTT
550
AGAGCAGGUGUUACAGUCCTT


 


AD-14760
1444-1462
551
GACUGUAACACCUGCUCUUTT
552
AAGAGCAGGUGUUACAGUCTT


 


AD-14761
1609-1627
553
CUCCAGAAAUGGGUGACCCTT
554
GGGUCACCCAUUUCUGGAGTT


 


AD-14762
1537-1555
555
UAAGAGGAGGAGUAGAAGUTT
556
ACUUCUACUCCUCCUCUUATT


 


AD-14763
629-647
557
GAGGCUGCUGCUACUAUAGTT
558
CUAUAGUAGCAGCAGCCUCTT


 


AD-14764
656-674
559
AUUGCAUCCCUUGCUACUGTT
560
CAGUAGCAAGGGAUGCAAUTT


 


AD-14765
658-676
561
UGCAUCCCUUGCUACUGUATT
562
UACAGUAGCAAGGGAUGCATT


 


AD-14766
517-535
563
UUGUGUUUUCAGGUUCAUGTT
564
CAUGAACCUGAAAACACAATT


 


AD-14767
559-577
565
GGACCUAGUUGCUACUGUUTT
566
AACAGUAGCAACUAGGUCCTT


 


AD-14768
591-609
567
CUGCCACAGGAUUUUCAGUTT
568
ACUGAAAAUCCUGUGGCAGTT


 


AD-14769
638-656
569
GCUACUAUAGAAGUUGAAATT
570
UUUCAACUUCUAUAGUAGCTT


 


AD-14770
655-673
571
AAUUGCAUCCCUUGCUACUTT
572
AGUAGCAAGGGAUGCAAUUTT


 


AD-14771
561-579
573
ACCUAGUUGCUACUGUUUCTT
574
GAAACAGUAGCAACUAGGUTT


 


AD-14772
639-657
575
CUACUAUAGAAGUUGAAAUTT
576
AUUUCAACUUCUAUAGUAGTT


 


AD-14773
715-733
577
AGGCCUUACUCCUGAAACATT
578
UGUUUCAGGAGUAAGGCCUTT


 


AD-14774
716-734
579
GGCCUUACUCCUGAAACAUTT
580
AUGUUUCAGGAGUAAGGCCTT


 


AD-14775
326-344
581
GUAAAACCUGGAGUGGAACTT
582
GUUCCACUCCAGGUUUUACTT


 


AD-14776
518-536
583
UGUGUUUUCAGGUUCAUGGTT
584
CCAUGAACCUGAAAACACATT


 


AD-14777
520-538
585
UGUUUUCAGGUUCAUGGGUTT
586
ACCCAUGAACCUGAAAACATT


 


AD-14778
661-679
587
AUCCCUUGCUACUGUAGAGTT
588
CUCUACAGUAGCAAGGGAUTT


 


AD-14779
560-578
589
GACCUAGUUGCUACUGUUUTT
590
AAACAGUAGCAACUAGGUCTT


 


AD-14780
681-699
591
GGAUUACAAGUACCUCUGATT
592
UCAGAGGUACUUGUAAUCCTT


 


AD-14781
714-732
593
UAGGCCUUACUCCUGAAACTT
594
GUUUCAGGAGUAAGGCCUATT


 


AD-14782
377-395
595
UGUUAGAAUUUUUGCUGGATT
596
UCCAGCAAAAAUUCUAACATT


 


AD-14783
589-607
597
UGCUGCCACAGGAUUUUCATT
598
UGAAAAUCCUGUGGCAGCATT


 


AD-14784
594-612
599
CCACAGGAUUUUCAGUAGCTT
600
GCUACUGAAAAUCCUGUGGTT


 


AD-14785
648-666
601
AAGUUGAAAUUGCAUCCCUTT
602
AGGGAUGCAAUUUCAACUUTT


 


AD-14786
649-667
603
AGUUGAAAUUGCAUCCCUUTT
604
AAGGGAUGCAAUUUCAACUTT


 


AD-14787
587-605
605
GCUGCUGCCACAGGAUUUUTT
606
AAAAUCCUGUGGCAGCAGCTT


 


AD-14788
325-343
607
AGUAAAACCUGGAGUGGAATT
608
UUCCACUCCAGGUUUUACUTT


 


AD-14789
515-533
609
UUUUGUGUUUUCAGGUUCATT
610
UGAACCUGAAAACACAAAATT


 


AD-14790
516-534
611
UUUGUGUUUUCAGGUUCAUTT
612
AUGAACCUGAAAACACAAATT


 


AD-14791
519-537
613
GUGUUUUCAGGUUCAUGGGTT
614
CCCAUGAACCUGAAAACACTT


 


AD-14792
521-539
615
GUUUUCAGGUUCAUGGGUGTT
616
CACCCAUGAACCUGAAAACTT


 


AD-14793
522-540
617
UUUUCAGGUUCAUGGGUGCTT
618
GCACCCAUGAACCUGAAAATT


 


AD-14794
523-541
619
UUUCAGGUUCAUGGGUGCCTT
620
GGCACCCAUGAACCUGAAATT


 


AD-14795
616-634
621
AAUUGCUGCUGGAGAGGCUTT
622
AGCCUCUCCAGCAGCAAUUTT


 


AD-14796
657-675
623
UUGCAUCCCUUGCUACUGUTT
624
ACAGUAGCAAGGGAUGCAATT


 


AD-14797
761-779
625
GCUGUAGCUGGGUUUGCUGTT
626
CAGCAAACCCAGCUACAGCTT


 


AD-14798
645-663
627
UAGAAGUUGAAAUUGCAUCTT
628
GAUGCAAUUUCAACUUCUATT


 


AD-14799
647-665
629
GAAGUUGAAAUUGCAUCCCTT
630
GGGAUGCAAUUUCAACUUCTT


 


AD-14800
660-678
631
CAUCCCUUGCUACUGUAGATT
632
UCUACAGUAGCAAGGGAUGTT


 


AD-14801
324-342
633
UAGUAAAACCUGGAGUGGATT
634
UCCACUCCAGGUUUUACUATT


 


AD-14802
372-390
635
UUUUUUGUUAGAAUUUUUGTT
636
CAAAAAUUCUAACAAAAAATT


 


AD-14803
640-658
637
UACUAUAGAAGUUGAAAUUTT
638
AAUUUCAACUUCUAUAGUATT


 


AD-14804
562-580
639
CCUAGUUGCUACUGUUUCUTT
640
AGAAACAGUAGCAACUAGGTT


 


AD-14805
563-581
641
CUAGUUGCUACUGUUUCUGTT
642
CAGAAACAGUAGCAACUAGTT


 


AD-14806
566-584
643
GUUGCUACUGUUUCUGAGGTT
644
CCUCAGAAACAGUAGCAACTT


 


AD-14807
625-643
645
UGGAGAGGCUGCUGCUACUTT
646
AGUAGCAGCAGCCUCUCCATT


 


AD-14808
627-645
647
GAGAGGCUGCUGCUACUAUTT
648
AUAGUAGCAGCAGCCUCUCTT


 


AD-14809
628-646
649
AGAGGCUGCUGCUACUAUATT
650
UAUAGUAGCAGCAGCCUCUTT


 


AD-14810
632-650
651
GCUGCUGCUACUAUAGAAGTT
652
CUUCUAUAGUAGCAGCAGCTT


 


AD-14811
513-531
653
UUUUUUGUGUUUUCAGGUUTT
654
AACCUGAAAACACAAAAAATT


 


AD-14812
641-659
655
ACUAUAGAAGUUGAAAUUGTT
656
CAAUUUCAACUUCUAUAGUTT


 


AD-14813
323-341
657
UUAGUAAAACCUGGAGUGGTT
658
CCACUCCAGGUUUUACUAATT


 


AD-14814
717-735
659
GCCUUACUCCUGAAACAUATT
660
UAUGUUUCAGGAGUAAGGCTT


 


AD-14815
646-664
661
AGAAGUUGAAAUUGCAUCCTT
662
GGAUGCAAUUUCAACUUCUTT


 


AD-14816
592-610
663
UGCCACAGGAUUUUCAGUATT
664
UACUGAAAAUCCUGUGGCATT


 


AD-14817
590-608
665
GCUGCCACAGGAUUUUCAGTT
666
CUGAAAAUCCUGUGGCAGCTT


 


AD-14818
526-544
667
CAGGUUCAUGGGUGCCGCATT
668
UGCGGCACCCAUGAACCUGTT


 


AD-14819
615-633
669
AAAUUGCUGCUGGAGAGGCTT
670
GCCUCUCCAGCAGCAAUUUTT


 


AD-14820
617-635
671
AUUGCUGCUGGAGAGGCUGTT
672
CAGCCUCUCCAGCAGCAAUTT


 


AD-14821
652-670
673
UGAAAUUGCAUCCCUUGCUTT
674
AGCAAGGGAUGCAAUUUCATT


 


AD-14822
374-392
675
UUUUGUUAGAAUUUUUGCUTT
676
AGCAAAAAUUCUAACAAAATT


 


AD-14823
375-393
677
UUUGUUAGAAUUUUUGCUGTT
678
CAGCAAAAAUUCUAACAAATT


 


AD-14824
631-649
679
GGCUGCUGCUACUAUAGAATT
680
UUCUAUAGUAGCAGCAGCCTT


 


AD-14825
376-394
681
UUGUUAGAAUUUUUGCUGGTT
682
CCAGCAAAAAUUCUAACAATT


 


AD-14826
512-530
683
UUUUUUUGUGUUUUCAGGUTT
684
ACCUGAAAACACAAAAAAATT


 


AD-14827
1127-1145
685
GAAACUACUUGGGCAAUAGTT
686
CUAUUGCCCAAGUAGUUUCTT


 


AD-14828
1410-1428
687
AAUGGAUGUUGCCUUUACUTT
688
AGUAAAGGCAACAUCCAUUTT


 


AD-14829
1406-1424
689
CCUCAAUGGAUGUUGCCUUTT
690
AAGGCAACAUCCAUUGAGGTT


 


AD-14830
1418-1436
691
UUGCCUUUACUUUUAGGGUTT
692
ACCCUAAAAGUAAAGGCAATT


 


AD-14831
1126-1144
693
AGAAACUACUUGGGCAAUATT
694
UAUUGCCCAAGUAGUUUCUTT


 


AD-14832
1125-1143
695
AAGAAACUACUUGGGCAAUTT
696
AUUGCCCAAGUAGUUUCUUTT


 


AD-14833
1419-1437
697
UGCCUUUACUUUUAGGGUUTT
698
AACCCUAAAAGUAAAGGCATT


 


AD-14834
1420-1438
699
GCCUUUACUUUUAGGGUUGTT
700
CAACCCUAAAAGUAAAGGCTT


 


AD-14835
1422-1440
701
CUUUACUUUUAGGGUUGUATT
702
UACAACCCUAAAAGUAAAGTT


 


AD-14836
1423-1441
703
UUUACUUUUAGGGUUGUACTT
704
GUACAACCCUAAAAGUAAATT


 


AD-14837
1425-1443
705
UACUUUUAGGGUUGUACGGTT
706
CCGUACAACCCUAAAAGUATT


 


AD-14838
1123-1141
707
GGAAGAAACUACUUGGGCATT
708
UGCCCAAGUAGUUUCUUCCTT


 


AD-14839
1409-1427
709
CAAUGGAUGUUGCCUUUACTT
710
GUAAAGGCAACAUCCAUUGTT


 


AD-14840
1413-1431
711
GGAUGUUGCCUUUACUUUUTT
712
AAAAGUAAAGGCAACAUCCTT


 


AD-14841
1416-1434
713
UGUUGCCUUUACUUUUAGGTT
714
CCUAAAAGUAAAGGCAACATT


 


AD-14842
1414-1432
715
GAUGUUGCCUUUACUUUUATT
716
UAAAAGUAAAGGCAACAUCTT


 


AD-14843
911-929
717
CCAGAAGACUACUAUGAUATT
718
UAUCAUAGUAGUCUUCUGGTT


 


AD-14844
910-928
719
UCCAGAAGACUACUAUGAUTT
720
AUCAUAGUAGUCUUCUGGATT


 


AD-14845
1120-1138
721
UUUGGAAGAAACUACUUGGTT
722
CCAAGUAGUUUCUUCCAAATT


 


AD-14846
1404-1422
723
CUCCUCAAUGGAUGUUGCCTT
724
GGCAACAUCCAUUGAGGAGTT


 


AD-14847
1337-1355
725
CCAAAUGUGCAAUCUGGUGTT
726
CACCAGAUUGCACAUUUGGTT


 


AD-14848
1338-1356
727
CAAAUGUGCAAUCUGGUGATT
728
UCACCAGAUUGCACAUUUGTT


 


AD-14849
1397-1415
729
AGAUCUGCUCCUCAAUGGATT
730
UCCAUUGAGGAGCAGAUCUTT


 


AD-14850
1407-1425
731
CUCAAUGGAUGUUGCCUUUTT
732
AAAGGCAACAUCCAUUGAGTT


 


AD-14851
4157-4175
733
GCUCAAAUUUUAUAUAAGATT
734
UCUUAUAUAAAAUUUGAGCTT


 


AD-14852
4795-4813
735
AGCCUGAUUUUGGUACAUGTT
736
CAUGUACCAAAAUCAGGCUTT


 


AD-14853
4156-4174
737
CUCAAAUUUUAUAUAAGAATT
738
UUCUUAUAUAAAAUUUGAGTT


 


AD-14854
5002-5020
739
ACAAAGUGCUGAAUAGGGATT
740
UCCCUAUUCAGCACUUUGUTT


 


AD-14855
4792-4810
741
CUGAUUUUGGUACAUGGAATT
742
UUCCAUGUACCAAAAUCAGTT


 


AD-14856
4790-4808
743
GAUUUUGGUACAUGGAAUATT
744
UAUUCCAUGUACCAAAAUCTT


 


AD-14857
4801-4819
745
CUCAUCAGCCUGAUUUUGGTT
746
CCAAAAUCAGGCUGAUGAGTT


 


AD-14858
4622-4640
747
AGCCCACUUGUGUGGAUAGTT
748
CUAUCCACACAAGUGGGCUTT


 


AD-14859
4997-5015
749
GUGCUGAAUAGGGAGGAAUTT
750
AUUCCUCCCUAUUCAGCACTT


 


AD-14860
5094-5112
751
AGUAAGGGCGUGGAGGCUUTT
752
AAGCCUCCACGCCCUUACUTT


 


AD-14861
4564-4582
753
GUGACUUAACCCAAGAAGCTT
754
GCUUCUUGGGUUAAGUCACTT


 


AD-14862
5095-5113
755
UAGUAAGGGCGUGGAGGCUTT
756
AGCCUCCACGCCCUUACUATT


 


AD-14863
4800-4818
757
UCAUCAGCCUGAUUUUGGUTT
758
ACCAAAAUCAGGCUGAUGATT


 


AD-14864
4265-4283
759
GUAGAAGACCCUAAAGACUTT
760
AGUCUUUAGGGUCUUCUACTT


 


AD-14865
4267-4285
761
AGGUAGAAGACCCUAAAGATT
762
UCUUUAGGGUCUUCUACCUTT


 


AD-14866
4270-4288
763
AAAAGGUAGAAGACCCUAATT
764
UUAGGGUCUUCUACCUUUUTT


 


AD-14867
4269-4287
765
AAAGGUAGAAGACCCUAAATT
766
UUUAGGGUCUUCUACCUUUTT


 


AD-14868
2874-2892
767
GAUUGUGCAGUGGAAAGAATT
768
UUCUUUCCACUGCACAAUCTT


 


AD-14869
2875-2893
769
GGAUUGUGCAGUGGAAAGATT
770
UCUUUCCACUGCACAAUCCTT


 


AD-14870
3950-3968
771
UGUAGACAGCCAUAUGCAGTT
772
CUGCAUAUGGCUGUCUACATT


 


AD-14871
3896-3914
773
CAUGACUUUAACCCAGAAGTT
774
CUUCUGGGUUAAAGUCAUGTT


 


AD-14872
4990-5008
775
AUAGGGAGGAAUCCAUGGATT
776
UCCAUGGAUUCCUCCCUAUTT


 


AD-14873
4994-5012
777
CUGAAUAGGGAGGAAUCCATT
778
UGGAUUCCUCCCUAUUCAGTT


 


AD-14874
5000-5018
779
AAAGUGCUGAAUAGGGAGGTT
780
CCUCCCUAUUCAGCACUUUTT


 


AD-14875
4563-4581
781
UGACUUAACCCAAGAAGCUTT
782
AGCUUCUUGGGUUAAGUCATT


 


AD-14876
3895-3913
783
AUGACUUUAACCCAGAAGATT
784
UCUUCUGGGUUAAAGUCAUTT


 


AD-14877
4262-4280
785
GAAGACCCUAAAGACUUUCTT
786
GAAAGUCUUUAGGGUCUUCTT


 


AD-14878
4162-4180
787
AAAAAGCUCAAAUUUUAUATT
788
UAUAAAAUUUGAGCUUUUUTT


 


AD-14879
4798-4816
789
AUCAGCCUGAUUUUGGUACTT
790
GUACCAAAAUCAGGCUGAUTT


 


AD-14880
4799-4817
791
CAUCAGCCUGAUUUUGGUATT
792
UACCAAAAUCAGGCUGAUGTT


 


AD-14881
5006-5024
793
AUGGACAAAGUGCUGAAUATT
794
UAUUCAGCACUUUGUCCAUTT


 


AD-14882
4264-4282
795
UAGAAGACCCUAAAGACUUTT
796
AAGUCUUUAGGGUCUUCUATT


 


AD-14883
4268-4286
797
AAGGUAGAAGACCCUAAAGTT
798
CUUUAGGGUCUUCUACCUUTT


 


AD-14884
4623-4641
799
CAGCCCACUUGUGUGGAUATT
800
UAUCCACACAAGUGGGCUGTT


 


AD-14885
4788-4806
801
UUUUGGUACAUGGAAUAGUTT
802
ACUAUUCCAUGUACCAAAATT


 


AD-14886
4993-5011
803
UGAAUAGGGAGGAAUCCAUTT
804
AUGGAUUCCUCCCUAUUCATT


 


AD-14887
4995-5013
805
GCUGAAUAGGGAGGAAUCCTT
806
GGAUUCCUCCCUAUUCAGCTT


 


AD-14888
4996-5014
807
UGCUGAAUAGGGAGGAAUCTT
808
GAUUCCUCCCUAUUCAGCATT


 


AD-14889
3952-3970
809
UGUGUAGACAGCCAUAUGCTT
810
GCAUAUGGCUGUCUACACATT


 


AD-14890
4595-4613
811
UGCUUUGAUUGCUUCAGACTT
812
GUCUGAAGCAAUCAAAGCATT


 


AD-14891
4596-4614
813
UUGCUUUGAUUGCUUCAGATT
814
UCUGAAGCAAUCAAAGCAATT


 


AD-14892
4597-4615
815
AUUGCUUUGAUUGCUUCAGTT
816
CUGAAGCAAUCAAAGCAAUTT


 


AD-14893
4599-4617
817
CUAUUGCUUUGAUUGCUUCTT
818
GAAGCAAUCAAAGCAAUAGTT


 


AD-14894
4726-4744
819
AUUGCAAGGAAUGGCCUAATT
820
UUAGGCCAUUCCUUGCAAUTT


 


AD-14895
4753-4771
821
AUUUUCCUCCUAAUUCUGATT
822
UCAGAAUUAGGAGGAAAAUTT


 


AD-14896
4802-4820
823
GCUCAUCAGCCUGAUUUUGTT
824
CAAAAUCAGGCUGAUGAGCTT


 


AD-14897
4803-4821
825
UGCUCAUCAGCCUGAUUUUTT
826
AAAAUCAGGCUGAUGAGCATT


 


AD-14898
4806-4824
827
AGUUGCUCAUCAGCCUGAUTT
828
AUCAGGCUGAUGAGCAACUTT


 


AD-14899
5091-5109
829
AAGGGCGUGGAGGCUUUUUTT
830
AAAAAGCCUCCACGCCCUUTT


 


AD-14900
5093-5111
831
GUAAGGGCGUGGAGGCUUUTT
832
AAAGCCUCCACGCCCUUACTT


 


AD-14901
4259-4277
833
GACCCUAAAGACUUUCCUGTT
834
CAGGAAAGUCUUUAGGGUCTT


 


AD-14902
3901-3919
835
AGGAGCAUGACUUUAACCCTT
836
GGGUUAAAGUCAUGCUCCUTT


 


AD-14903
4757-4775
837
UGUGAUUUUCCUCCUAAUUTT
838
AAUUAGGAGGAAAAUCACATT


 


AD-14904
4758-4776
839
UUGUGAUUUUCCUCCUAAUTT
840
AUUAGGAGGAAAAUCACAATT


 


AD-14905
4562-4580
841
GACUUAACCCAAGAAGCUCTT
842
GAGCUUCUUGGGUUAAGUCTT


 


AD-14906
4585-4603
843
GCUUCAGACAAUGGUUUGGTT
844
CCAAACCAUUGUCUGAAGCTT


 


AD-14907
4587-4605
845
UUGCUUCAGACAAUGGUUUTT
846
AAACCAUUGUCUGAAGCAATT


 


AD-14908
4588-4606
847
AUUGCUUCAGACAAUGGUUTT
848
AACCAUUGUCUGAAGCAAUTT


 


AD-14909
4591-4609
849
UUGAUUGCUUCAGACAAUGTT
850
CAUUGUCUGAAGCAAUCAATT


 


AD-14910
5003-5021
851
GACAAAGUGCUGAAUAGGGTT
852
CCCUAUUCAGCACUUUGUCTT


 


AD-14911
4165-4183
853
AAGAAAAAGCUCAAAUUUUTT
854
AAAAUUUGAGCUUUUUCUUTT


 


AD-14912
4166-4184
855
AAAGAAAAAGCUCAAAUUUTT
856
AAAUUUGAGCUUUUUCUUUTT


 


AD-14913
4263-4281
857
AGAAGACCCUAAAGACUUUTT
858
AAAGUCUUUAGGGUCUUCUTT


 


AD-14914
4274-4292
859
AAAAAAAAGGUAGAAGACCTT
860
GGUCUUCUACCUUUUUUUUTT


 


AD-14915
4266-4284
861
GGUAGAAGACCCUAAAGACTT
862
GUCUUUAGGGUCUUCUACCTT


 


AD-14916
4272-4290
863
AAAAAAGGUAGAAGACCCUTT
864
AGGGUCUUCUACCUUUUUUTT


 


AD-14917
4271-4289
865
AAAAAGGUAGAAGACCCUATT
866
UAGGGUCUUCUACCUUUUUTT


 


AD-14918
4559-4577
867
UUAACCCAAGAAGCUCUUCTT
868
GAAGAGCUUCUUGGGUUAATT


 


AD-14919
4789-4807
869
AUUUUGGUACAUGGAAUAGTT
870
CUAUUCCAUGUACCAAAAUTT


 


AD-14920
4998-5016
871
AGUGCUGAAUAGGGAGGAATT
872
UUCCUCCCUAUUCAGCACUTT


 


AD-14921
5070-5088
873
GAGGCCAGGGAAAUUCCCUTT
874
AGGGAAUUUCCCUGGCCUCTT


 


AD-14922
4158-4176
875
AGCUCAAAUUUUAUAUAAGTT
876
CUUAUAUAAAAUUUGAGCUTT


 


AD-14923
5065-5083
877
CAGGGAAAUUCCCUUGUUUTT
878
AAACAAGGGAAUUUCCCUGTT


 


AD-14924
2872-2890
879
UUGUGCAGUGGAAAGAAAGTT
880
CUUUCUUUCCACUGCACAATT


 


AD-14925
4782-4800
881
UACAUGGAAUAGUUCAGAGTT
882
CUCUGAACUAUUCCAUGUATT


 


AD-14926
4783-4801
883
GUACAUGGAAUAGUUCAGATT
884
UCUGAACUAUUCCAUGUACTT


 


AD-14927
5064-5082
885
AGGGAAAUUCCCUUGUUUUTT
886
AAAACAAGGGAAUUUCCCUTT


 


AD-14928
5071-5089
887
GGAGGCCAGGGAAAUUCCCTT
888
GGGAAUUUCCCUGGCCUCCTT


 


AD-14929
3951-3969
889
GUGUAGACAGCCAUAUGCATT
890
UGCAUAUGGCUGUCUACACTT


 


AD-14930
3949-3967
891
GUAGACAGCCAUAUGCAGUTT
892
ACUGCAUAUGGCUGUCUACTT


 


AD-14931
4355-4373
893
GAAGACCUGUUUUGCCAUGTT
894
CAUGGCAAAACAGGUCUUCTT


 


AD-14932
4363-4381
895
AGUGGGAUGAAGACCUGUUTT
896
AACAGGUCUUCAUCCCACUTT


 


AD-14933
4356-4374
897
UGAAGACCUGUUUUGCCAUTT
898
AUGGCAAAACAGGUCUUCATT


 


AD-14934
4361-4379
899
UGGGAUGAAGACCUGUUUUTT
900
AAAACAGGUCUUCAUCCCATT


 


AD-14935
4560-4578
901
CUUAACCCAAGAAGCUCUUTT
902
AAGAGCUUCUUGGGUUAAGTT


 


AD-14936
2873-2891
903
AUUGUGCAGUGGAAAGAAATT
904
UUUCUUUCCACUGCACAAUTT


 


AD-14937
4730-4748
905
CUUUAUUGCAAGGAAUGGCTT
906
GCCAUUCCUUGCAAUAAAGTT


 


AD-14938
3899-3917
907
GAGCAUGACUUUAACCCAGTT
908
CUGGGUUAAAGUCAUGCUCTT


 


AD-14939
4756-4774
909
GUGAUUUUCCUCCUAAUUCTT
910
GAAUUAGGAGGAAAAUCACTT


 


AD-14940
4590-4608
911
UGAUUGCUUCAGACAAUGGTT
912
CCAUUGUCUGAAGCAAUCATT


 


AD-14941
4159-4177
913
AAGCUCAAAUUUUAUAUAATT
914
UUAUAUAAAAUUUGAGCUUTT


 


AD-14942
2743-2761
915
CUGGACAUGGAUCAAGCACTT
916
GUGCUUGAUCCAUGUCCAGTT


 


AD-14943
4155-4173
917
UCAAAUUUUAUAUAAGAAATT
918
UUUCUUAUAUAAAAUUUGATT


 


AD-14944
2871-2889
919
UGUGCAGUGGAAAGAAAGGTT
920
CCUUUCUUUCCACUGCACATT


 


AD-14945
4786-4804
921
UUGGUACAUGGAAUAGUUCTT
922
GAACUAUUCCAUGUACCAATT


 


AD-14946
4364-4382
923
AAGUGGGAUGAAGACCUGUTT
924
ACAGGUCUUCAUCCCACUUTT


 


AD-14947
4359-4377
925
GGAUGAAGACCUGUUUUGCTT
926
GCAAAACAGGUCUUCAUCCTT


 


AD-14948
2744-2762
927
UCUGGACAUGGAUCAAGCATT
928
UGCUUGAUCCAUGUCCAGATT


 


AD-14949
4787-4805
929
UUUGGUACAUGGAAUAGUUTT
930
AACUAUUCCAUGUACCAAATT


























Residual


Relative





luciferase


siRNA activity





activity
SD of
Residual
(normalized to
SD of
Relative


Table 1a-2
(relative to
residual
luciferase
positive
relative
siRNA


duplex
control siRNA
luciferase
activity +/−
control luc-
siRNA
activity +/− 


name
treated cells)
activity
SD
siRNA)
activity
SD





















AD-14742
40.85
4.38
41 ± 4%
82.24
8.83
82 ± 9%


 


AD-14743
20.92
4.10
21 ± 4%
109.97
21.56
110 ± 22%


 


AD-14744
62.20
4.47
62 ± 4%
52.56
3.78
53 ± 4%


 


AD-14745
43.97
2.60
44 ± 3%
77.91
4.60
78 ± 5%


 


AD-14746
24.52
1.96
25 ± 2%
104.96
8.38
105 ± 8% 


 


AD-14747
32.67
4.51
33 ± 5%
93.62
12.94
 94 ± 13%


 


AD-14748
93.99
3.23
94 ± 3%
8.36
0.29
 8 ± 0%


 


AD-14749
55.16
2.81
55 ± 3%
62.35
3.18
62 ± 3%


 


AD-14750
30.86
3.11
31 ± 3%
96.14
9.70
 96 ± 10%


 


AD-14751
54.44
4.03
54 ± 4%
63.35
4.69
63 ± 5%


 


AD-14752
53.88
7.58
54 ± 8%
64.13
9.02
64 ± 9%


 


AD-14753
35.24
7.45
35 ± 7%
90.05
19.03
 90 ± 19%


 


AD-14754
70.39
2.80
70 ± 3%
41.17
1.64
41 ± 2%


 


AD-14755
41.80
1.60
42 ± 2%
80.93
3.10
81 ± 3%


 


AD-14756
56.69
3.05
57 ± 3%
60.22
3.24
60 ± 3%


 


AD-14757
39.16
2.16
39 ± 2%
84.60
4.67
85 ± 5%


 


AD-14758
39.79
2.95
40 ± 3%
83.72
6.22
84 ± 6%


 


AD-14759
30.62
1.01
31 ± 1%
96.48
3.20
96 ± 3%


 


AD-14760
28.14
2.74
28 ± 3%
99.93
9.72
100 ± 10%


 


AD-14761
67.42
2.83
67 ± 3%
45.30
1.90
45 ± 2%


 


AD-14762
36.10
1.30
36 ± 1%
88.85
3.21
89 ± 3%


 


AD-14763
49.39
6.77
49 ± 7%
78.14
10.71
 78 ± 11%


 


AD-14764
74.04
5.32
74 ± 5%
40.09
2.88
40 ± 3%


 


AD-14765
50.84
10.47
 51 ± 10%
75.91
15.63
 76 ± 16%


 


AD-14766
72.59
3.55
73 ± 4%
42.32
2.07
42 ± 2%


 


AD-14767
34.82
7.41
35 ± 7%
100.63
21.40
101 ± 21%


 


AD-14768
48.68
6.31
49 ± 6%
79.24
10.27
 79 ± 10%


 


AD-14769
39.07
5.53
39 ± 6%
94.08
13.31
 94 ± 13%


 


AD-14770
45.59
5.89
46 ± 6%
84.01
10.85
 84 ± 11%


 


AD-14771
45.57
4.10
46 ± 4%
84.04
7.56
84 ± 8%


 


AD-14772
33.12
3.64
33 ± 4%
103.26
11.36
103 ± 11%


 


AD-14773
37.38
5.72
37 ± 6%
96.69
14.78
 97 ± 15%


 


AD-14774
42.38
4.41
42 ± 4%
88.96
9.26
89 ± 9%


 


AD-14775
46.59
3.00
47 ± 3%
82.47
5.31
82 ± 5%


 


AD-14776
71.28
8.67
71 ± 9%
44.35
5.40
44 ± 5%


 


AD-14777
64.55
6.21
65 ± 6%
54.74
5.26
55 ± 5%


 


AD-14778
60.45
8.91
60 ± 9%
61.07
9.00
61 ± 9%


 


AD-14779
32.46
0.82
32 ± 1%
104.27
2.63
104 ± 3% 


 


AD-14780
22.96
2.86
23 ± 3%
118.94
14.81
119 ± 15%


 


AD-14781
56.99
9.43
57 ± 9%
66.41
10.99
 66 ± 11%


 


AD-14782
29.90
8.74
30 ± 9%
108.24
31.65
108 ± 32%


 


AD-14783
42.63
6.57
43 ± 7%
88.58
13.66
 89 ± 14%


 


AD-14784
67.06
1.35
67 ± 1%
50.86
1.03
51 ± 1%


 


AD-14785
48.90
3.32
49 ± 3%
78.89
5.35
79 ± 5%


 


AD-14786
27.74
2.06
28 ± 2%
111.57
8.29
112 ± 8% 


 


AD-14787
38.77
6.24
39 ± 6%
94.53
15.22
 95 ± 15%


 


AD-14788
32.84
8.60
33 ± 9%
103.70
27.17
104 ± 27%


 


AD-14789
46.96
1.70
47 ± 2%
81.89
2.96
82 ± 3%


 


AD-14790
43.61
4.90
44 ± 5%
87.06
9.79
 87 ± 10%


 


AD-14791
35.55
4.34
36 ± 4%
99.51
12.15
100 ± 12%


 


AD-14792
38.22
3.51
38 ± 4%
95.38
8.75
95 ± 9%


 


AD-14793
90.85
5.92
91 ± 6%
14.13
0.92
14 ± 1%


 


AD-14794
83.37
3.27
83 ± 3%
25.68
1.01
26 ± 1%


 


AD-14795
55.06
3.61
55 ± 4%
69.38
4.55
69 ± 5%


 


AD-14796
30.98
5.78
31 ± 6%
106.56
19.89
107 ± 20%


 


AD-14797
28.95
3.15
29 ± 3%
109.70
11.95
110 ± 12%


 


AD-14798
67.39
3.70
67 ± 4%
50.35
2.76
50 ± 3%


 


AD-14799
66.83
4.72
67 ± 5%
51.21
3.61
51 ± 4%


 


AD-14800
33.26
5.72
33 ± 6%
103.04
17.71
103 ± 18%


 


AD-14801
39.15
4.57
39 ± 5%
93.96
10.97
 94 ± 11%


 


AD-14802
91.20
5.35
91 ± 5%
13.58
0.80
14 ± 1%


 


AD-14803
34.15
7.94
34 ± 8%
101.67
23.64
102 ± 24%


 


AD-14804
30.08
6.54
30 ± 7%
107.96
23.48
108 ± 23%


 


AD-14805
32.44
4.27
32 ± 4%
104.31
13.73
104 ± 14%


 


AD-14806
35.62
3.11
36 ± 3%
99.41
8.67
99 ± 9%


 


AD-14807
28.27
7.28
28 ± 7%
110.76
28.52
111 ± 29%


 


AD-14808
30.29
3.96
30 ± 4%
107.63
14.08
108 ± 14%


 


AD-14809
31.59
4.46
32 ± 4%
105.63
14.91
106 ± 15%


 


AD-14810
30.11
5.71
30 ± 6%
107.91
20.46
108 ± 20%


 


AD-14811
55.27
6.82
55 ± 7%
69.06
8.52
69 ± 9%


 


AD-14812
45.27
5.99
45 ± 6%
84.51
11.19
 85 ± 11%


 


AD-14813
77.97
7.01
78 ± 7%
34.01
3.06
34 ± 3%


 


AD-14814
29.54
3.56
30 ± 4%
108.78
13.09
109 ± 13%


 


AD-14815
65.04
3.18
65 ± 3%
53.97
2.64
54 ± 3%


 


AD-14816
64.03
4.63
64 ± 5%
55.53
4.02
56 ± 4%


 


AD-14817
37.83
2.89
38 ± 3%
95.99
7.33
96 ± 7%


 


AD-14818
28.88
5.60
29 ± 6%
109.82
21.30
110 ± 21%


 


AD-14819
92.90
4.87
93 ± 5%
10.97
0.58
11 ± 1%


 


AD-14820
75.41
3.69
75 ± 4%
37.97
1.86
38 ± 2%


 


AD-14821
73.08
6.22
73 ± 6%
41.57
3.54
42 ± 4%


 


AD-14822
86.39
9.34
86 ± 9%
21.02
2.27
21 ± 2%


 


AD-14823
96.50
10.46
 97 ± 10%
5.40
0.59
 5 ± 1%


 


AD-14824
32.62
3.41
33 ± 3%
104.03
10.89
104 ± 11%


 


AD-14825
102.71
7.66
103 ± 8% 
−4.18
0.31
−4 ± 0%


 


AD-14826
92.45
5.66
92 ± 6%
11.66
0.71
12 ± 1%


 


AD-14827
63.46
16.38
 63 ± 16%
46.00
11.88
 46 ± 12%


 


AD-14828
45.99
15.21
 46 ± 15%
67.99
22.49
 68 ± 22%


 


AD-14829
40.54
16.03
 41 ± 16%
74.86
29.60
 75 ± 30%


 


AD-14830
117.10
3.66
117 ± 4% 
−21.52
0.67
−22 ± 1% 


 


AD-14831
54.78
21.12
 55 ± 21%
56.93
21.95
 57 ± 22%


 


AD-14832
67.07
10.81
 67 ± 11%
41.46
6.68
41 ± 7%


 


AD-14833
71.52
11.90
 72 ± 12%
35.85
5.97
36 ± 6%


 


AD-14834
58.05
16.37
 58 ± 16%
52.81
14.89
 53 ± 15%


 


AD-14835
93.36
5.43
93 ± 5%
8.36
0.49
 8 ± 0%


 


AD-14836
108.84
4.85
109 ± 5% 
−11.13
0.50
−11 ± 0% 


 


AD-14837
106.68
10.06
107 ± 10%
−8.41
0.79
−8 ± 1%


 


AD-14838
37.06
6.68
37 ± 7%
79.23
14.28
 79 ± 14%


 


AD-14839
36.03
7.54
36 ± 8%
80.53
16.84
 81 ± 17%


 


AD-14840
38.51
5.90
39 ± 6%
77.40
11.86
 77 ± 12%


 


AD-14841
110.86
8.91
111 ± 9% 
−13.67
1.10
−14 ± 1% 


 


AD-14842
34.83
5.51
35 ± 6%
82.04
12.98
 82 ± 13%


 


AD-14843
23.75
6.04
24 ± 6%
95.99
24.41
 96 ± 24%


 


AD-14844
27.47
5.29
27 ± 5%
91.30
17.57
 91 ± 18%


 


AD-14845
93.12
4.70
93 ± 5%
8.67
0.44
 9 ± 0%


 


AD-14846
81.72
8.26
82 ± 8%
23.01
2.33
23 ± 2%


 


AD-14847
77.89
5.29
78 ± 5%
27.83
1.89
28 ± 2%


 


AD-14848
44.40
4.95
44 ± 5%
69.99
7.81
70 ± 8%


 


AD-14849
46.41
5.08
46 ± 5%
67.46
7.38
67 ± 7%


 


AD-14850
35.52
6.70
36 ± 7%
81.17
15.31
 81 ± 15%


 


AD-14851
36.07
1.13
36 ± 1%
102.63
3.22
103 ± 3% 


 


AD-14852
67.98
6.75
68 ± 7%
51.41
5.11
51 ± 5%


 


AD-14853
69.44
3.07
69 ± 3%
49.05
2.17
49 ± 2%


 


AD-14854
29.12
6.88
29 ± 7%
113.79
26.89
114 ± 27%


 


AD-14855
36.04
7.07
36 ± 7%
102.68
20.14
103 ± 20%


 


AD-14856
33.61
7.93
34 ± 8%
106.57
25.15
107 ± 25%


 


AD-14857
50.76
8.76
51 ± 9%
79.04
13.64
 79 ± 14%


 


AD-14858
53.60
7.26
54 ± 7%
74.49
10.09
 74 ± 10%


 


AD-14859
39.07
9.34
39 ± 9%
97.82
23.38
 98 ± 23%


 


AD-14860
62.78
6.85
63 ± 7%
59.75
6.52
60 ± 7%


 


AD-14861
87.47
1.86
87 ± 2%
20.12
0.43
20 ± 0%


 


AD-14862
79.95
4.02
80 ± 4%
32.19
1.62
32 ± 2%


 


AD-14863
30.46
4.49
30 ± 4%
111.64
16.46
112 ± 16%


 


AD-14864
33.18
5.07
33 ± 5%
107.26
16.38
107 ± 16%


 


AD-14865
26.25
3.98
26 ± 4%
118.39
17.96
118 ± 18%


 


AD-14866
36.73
1.24
37 ± 1%
101.57
3.44
102 ± 3% 


 


AD-14867
33.16
3.13
33 ± 3%
107.30
10.12
107 ± 10%


 


AD-14868
29.91
4.56
30 ± 5%
112.52
17.16
113 ± 17%


 


AD-14869
28.24
3.66
28 ± 4%
115.20
14.91
115 ± 15%


 


AD-14870
50.37
3.04
50 ± 3%
79.67
4.81
80 ± 5%


 


AD-14871
39.37
5.11
39 ± 5%
97.32
12.63
 97 ± 13%


 


AD-14872
34.71
4.12
35 ± 4%
104.82
12.43
105 ± 12%


 


AD-14873
32.14
1.79
32 ± 2%
108.93
6.07
109 ± 6% 


 


AD-14874
101.77
4.87
102 ± 5% 
−2.85
0.14
−3 ± 0%


 


AD-14875
80.81
4.39
81 ± 4%
30.80
1.67
31 ± 2%


 


AD-14876
30.74
1.88
31 ± 2%
111.18
6.81
111 ± 7% 


 


AD-14877
57.38
2.84
57 ± 3%
68.42
3.39
68 ± 3%


 


AD-14878
70.23
3.35
70 ± 3%
47.79
2.28
48 ± 2%


 


AD-14879
79.03
7.72
79 ± 8%
33.66
3.29
34 ± 3%


 


AD-14880
21.65
2.46
22 ± 2%
125.78
14.28
126 ± 14%


 


AD-14881
27.66
1.71
28 ± 2%
116.13
7.17
116 ± 7% 


 


AD-14882
34.01
2.94
34 ± 3%
105.93
9.16
106 ± 9% 


 


AD-14883
40.62
3.22
41 ± 3%
95.33
7.56
95 ± 8%


 


AD-14884
35.73
5.94
36 ± 6%
103.18
17.14
103 ± 17%


 


AD-14885
47.40
7.65
47 ± 8%
84.45
13.63
 84 ± 14%


 


AD-14886
37.23
3.94
37 ± 4%
100.76
10.67
101 ± 11%


 


AD-14887
42.94
7.26
43 ± 7%
91.61
15.50
 92 ± 15%


 


AD-14888
32.58
4.06
33 ± 4%
108.24
13.50
108 ± 14%


 


AD-14889
83.09
2.98
83 ± 3%
27.15
0.97
27 ± 1%


 


AD-14890
59.49
2.94
59 ± 3%
65.04
3.22
65 ± 3%


 


AD-14891
21.93
5.52
22 ± 6%
125.32
31.52
125 ± 32%


 


AD-14892
72.69
2.19
73 ± 2%
43.84
1.32
44 ± 1%


 


AD-14893
24.43
7.07
24 ± 7%
121.32
35.11
121 ± 35%


 


AD-14894
33.84
5.08
34 ± 5%
106.20
15.95
106 ± 16%


 


AD-14895
21.68
4.46
22 ± 4%
125.73
25.84
126 ± 26%


 


AD-14896
26.99
5.01
27 ± 5%
117.20
21.73
117 ± 22%


 


AD-14897
29.04
2.72
29 ± 3%
113.92
10.67
114 ± 11%


 


AD-14898
32.64
4.87
33 ± 5%
108.14
16.13
108 ± 16%


 


AD-14899
61.71
4.59
62 ± 5%
61.47
4.57
61 ± 5%


 


AD-14900
31.01
2.84
31 ± 3%
110.75
10.14
111 ± 10%


 


AD-14901
31.47
1.57
31 ± 2%
110.01
5.49
110 ± 5% 


 


AD-14902
76.99
0.55
77 ± 1%
36.95
0.26
37 ± 0%


 


AD-14903
20.55
3.55
21 ± 4%
127.55
22.05
128 ± 22%


 


AD-14904
22.65
6.87
23 ± 7%
124.18
37.68
124 ± 38%


 


AD-14905
56.98
4.94
57 ± 5%
69.07
5.99
69 ± 6%


 


AD-14906
34.20
3.66
34 ± 4%
105.63
11.29
106 ± 11%


 


AD-14907
28.59
8.12
29 ± 8%
114.64
32.56
115 ± 33%


 


AD-14908
34.08
3.36
34 ± 3%
105.82
10.44
106 ± 10%


 


AD-14909
76.57
2.33
77 ± 2%
37.61
1.15
38 ± 1%


 


AD-14910
46.50
4.14
46 ± 4%
85.89
7.64
86 ± 8%


 


AD-14911
29.62
2.02
30 ± 2%
112.99
7.69
113 ± 8% 


 


AD-14912
22.27
0.48
22 ± 0%
124.78
2.69
125 ± 3% 


 


AD-14913
59.80
2.85
60 ± 3%
64.53
3.08
65 ± 3%


 


AD-14914
93.21
5.10
93 ± 5%
10.90
0.60
11 ± 1%


 


AD-14915
25.99
4.45
26 ± 4%
118.82
20.34
119 ± 20%


 


AD-14916
48.20
1.46
48 ± 1%
83.16
2.51
83 ± 3%


 


AD-14917
41.03
3.07
41 ± 3%
94.67
7.08
95 ± 7%


 


AD-14918
110.62
6.34
111 ± 6% 
−17.04
0.98
−17 ± 1% 


 


AD-14919
73.66
3.68
74 ± 4%
42.29
2.11
42 ± 2%


 


AD-14920
19.80
1.72
20 ± 2%
128.75
11.20
129 ± 11%


 


AD-14921
33.13
1.14
33 ± 1%
107.34
3.71
107 ± 4% 


 


AD-14922
52.94
6.99
53 ± 7%
63.41
8.37
63 ± 8%


 


AD-14923
33.77
8.92
34 ± 9%
89.23
23.56
 89 ± 24%


 


AD-14924
64.47
10.96
 64 ± 11%
47.86
8.13
48 ± 8%


 


AD-14925
97.16
7.57
97 ± 8%
3.83
0.30
 4 ± 0%


 


AD-14926
27.29
8.79
27 ± 9%
97.96
31.56
 98 ± 32%


 


AD-14927
27.02
10.01
 27 ± 10%
98.33
36.42
 98 ± 36%


 


AD-14928
76.75
4.78
77 ± 5%
31.32
1.95
31 ± 2%


 


AD-14929
32.92
9.44
33 ± 9%
90.38
25.93
 90 ± 26%


 


AD-14930
31.00
9.40
31 ± 9%
92.97
28.21
 93 ± 28%


 


AD-14931
31.36
8.73
31 ± 9%
92.48
25.74
 92 ± 26%


 


AD-14932
32.42
9.01
32 ± 9%
91.05
25.29
 91 ± 25%


 


AD-14933
39.94
6.96
40 ± 7%
80.92
14.10
 81 ± 14%


 


AD-14934
42.94
7.66
43 ± 8%
76.88
13.71
 77 ± 14%


 


AD-14935
47.74
8.48
48 ± 8%
70.41
12.51
 70 ± 13%


 


AD-14936
35.21
4.02
35 ± 4%
87.29
9.97
 87 ± 10%


 


AD-14937
89.25
3.53
89 ± 4%
14.48
0.57
14 ± 1%


 


AD-14938
29.38
6.46
29 ± 6%
95.15
20.91
 95 ± 21%


 


AD-14939
26.45
8.33
26 ± 8%
99.09
31.21
 99 ± 31%


 


AD-14940
77.50
6.51
78 ± 7%
30.31
2.55
30 ± 3%


 


AD-14941
36.40
10.76
 36 ± 11%
85.68
25.32
 86 ± 25%


 


AD-14942
65.11
5.84
65 ± 6%
47.01
4.22
47 ± 4%


 


AD-14943
89.96
4.69
90 ± 5%
13.52
0.71
14 ± 1%


 


AD-14944
48.98
5.85
49 ± 6%
68.74
8.21
69 ± 8%


 


AD-14945
43.45
3.29
43 ± 3%
76.18
5.77
76 ± 6%


 


AD-14946
41.25
1.26
41 ± 1%
79.15
2.43
79 ± 2%


 


AD-14947
42.10
7.34
42 ± 7%
78.01
13.60
 78 ± 14%


 


AD-14948
42.39
5.75
42 ± 6%
77.62
10.54
 78 ± 11%


 


AD-14949
27.68
5.79
28 ± 6%
97.44
20.39
 97 ± 20%









Tables 1b-1 and 1b-2








TABLE 1b 







siRNAs targeting JCV transcripts for primary screen; 1b-1: sequences;


1b-2: assay results. C* column describes chemistries as follows:













Table 1b-1


SEQ

SEQ



duplex

position in
ID
sense strand
ID
antisense strand


name
C*
consensus
NO:
sequence (5′-3′)
NO:
sequence (5′-3′)
















AD-12598
a
1426-1444
1
AcuuuuAGGGuuGuAcGGGTsT
2
CcCGuAcAACCCuAAAAGUTsT


 


AD-12708
b
1426-1444
3
AcuuuuAGGGuuGuAcGGGTsT
4
CCCGuAcAACCCuAAAAGUTsT


 


AD-12599
a
1427-1445
5
cuuuuAGGGuuGuAcGGGATsT
6
UcCCGuAcAACCCuAAAAGTsT


 


AD-12709
b
1427-1445
7
cuuuuAGGGuuGuAcGGGATsT
8
UCCCGuAcAACCCuAAAAGTsT


 


AD-12600
a
2026-2044
9
cAGAGcAcAAGGcGuAccuTsT
10
AgGuACGCCUUGUGCUCUGTsT


 


AD-12710
b
2026-2044
11
cAGAGcAcAAGGcGuAccuTsT
12
AGGuACGCCUUGUGCUCUGTsT


 


AD-12784
c
2026-2044
13
caGAGcAcAAGGcGuAccuTsT
14
AGGuACGCCUUGUGCUCUGTsT


 


AD-12832
d
2026-2044
15
caGAGcAcAAGGcGuAccuTsT
16
AgGuACGCCUUGUGCUCUGTsT


 


AD-12601
a
1431-1449
17
uAGGGuuGuAcGGGAcuGuTsT
18
AcAGUCCCGuAcAACCCuATsT


 


AD-12785
c
1431-1449
19
uaGGGuuGuAcGGGAcuGuTsT
20
AcAGUCCCGuAcAACCCuATsT


 


AD-12602
a
1432-1450
21
AGGGuuGuAcGGGAcuGuATsT
22
uacAGUCCCGuAcAACCCUTsT


 


AD-12711
b
1432-1450
23
AGGGuuGuAcGGGAcuGuATsT
24
uAcAGUCCCGuAcAACCCUTsT


 


AD-12786
c
1432-1450
25
AgGGuuGuAcGGGAcuGuATsT
26
uAcAGUCCCGuAcAACCCUTsT


 


AD-12833
d
1432-1450
27
AgGGuuGuAcGGGAcuGuATsT
28
uacAGUCCCGuAcAACCCUTsT


 


AD-12603
a
1436-1454
29
uuGuAcGGGAcuGuAAcAcTsT
30
GuGUuAcAGUCCCGuAcAATsT


 


AD-12712
b
1436-1454
31
uuGuAcGGGAcuGuAAcAcTsT
32
GUGUuAcAGUCCCGuAcAATsT


 


AD-12604
a
4794-4812
33
GccuGAuuuuGGuAcAuGGTsT
34
CcAUGuACcAAAAUcAGGCTsT


 


AD-12605
a
5099-5117
35
GAAGuAGuAAGGGcGuGGATsT
36
UccACGCCCUuACuACUUCTsT


 


AD-12713
b
5099-5117
37
GAAGuAGuAAGGGcGuGGATsT
38
UCcACGCCCUuACuACUUCTsT


 


AD-12787
c
5099-5117
39
GaAGuAGuAAGGGcGuGGATsT
40
UCcACGCCCUuACuACUUCTsT


 


AD-12834
d
5099-5117
41
GaAGuAGuAAGGGcGuGGATsT
42
UccACGCCCUuACuACUUCTsT


 


AD-12606
a
713-731
43
AuAGGccuuAcuccuGAAATsT
44
UuUcAGGAGuAAGGCCuAUTsT


 


AD-12714
b
713-731
45
AuAGGccuuAcuccuGAAATsT
46
UUUcAGGAGuAAGGCCuAUTsT


 


AD-12607
a
3946-3964
47
GAcAGccAuAuGcAGuAGuTsT
48
AcuACUGcAuAUGGCUGUCTsT


 


AD-12715
b
3946-3964
49
GAcAGccAuAuGcAGuAGuTsT
50
ACuACUGcAuAUGGCUGUCTsT


 


AD-12788
c
3946-3964
51
GacAGccAuAuGcAGuAGuTsT
52
ACuACUGcAuAUGGCUGUCTsT


 


AD-12835
d
3946-3964
53
GacAGccAuAuGcAGuAGuTsT
54
AcuACUGcAuAUGGCUGUCTsT


 


AD-12608
a
1128-1146
55
AAAcuAcuuGGGcAAuAGuTsT
56
AcuAUUGCCcAAGuAGUUUTsT


 


AD-12716
b
1128-1146
57
AAAcuAcuuGGGcAAuAGuTsT
58
ACuAUUGCCcAAGuAGUUUTsT


 


AD-12789
c
1128-1146
59
AaAcuAcuuGGGcAAuAGuTsT
60
ACuAUUGCCcAAGuAGUUUTsT


 


AD-12836
d
1128-1146
61
AaAcuAcuuGGGcAAuAGuTsT
62
AcuAUUGCCcAAGuAGUUUTsT


 


AD-12609
a
525-543
63
ucAGGuucAuGGGuGccGcTsT
64
GcGGcACCcAUGAACCUGATsT


 


AD-12717
b
525-543
65
ucAGGuucAuGGGuGccGcTsT
66
GCGGcACCcAUGAACCUGATsT


 


AD-12610
a
5096-5114
67
GuAGuAAGGGcGuGGAGGcTsT
68
GcCUCcACGCCCUuACuACTsT


 


AD-12718
b
5096-5114
69
GuAGuAAGGGcGuGGAGGcTsT
70
GCCUCcACGCCCUuACuACTsT


 


AD-12611
a
4727-4745
71
uAuuGcAAGGAAuGGccuATsT
72
uaGGCcAUUCCUUGcAAuATsT


 


AD-12719
b
4727-4745
73
uAuuGcAAGGAAuGGccuATsT
74
uAGGCcAUUCCUUGcAAuATsT


 


AD-12790
c
4727-4745
75
uauuGcAAGGAAuGGccuATsT
76
uAGGCcAUUCCUUGcAAuATsT


 


AD-12837
d
4727-4745
77
uauuGcAAGGAAuGGccuATsT
78
uaGGCcAUUCCUUGcAAuATsT


 


AD-12612
a
5097-5115
79
AGuAGuAAGGGcGuGGAGGTsT
80
CcUCcACGCCCUuACuACUTsT


 


AD-12720
b
5097-5115
81
AGuAGuAAGGGcGuGGAGGTsT
82
CCUCcACGCCCUuACuACUTsT


 


AD-12791
c
5097-5115
83
AguAGuAAGGGcGuGGAGGTsT
84
CCUCcACGCCCUuACuACUTsT


 


AD-12838
d
5097-5115
85
AguAGuAAGGGcGuGGAGGTsT
86
CcUCcACGCCCUuACuACUTsT


 


AD-12613
a
4601-4619
87
uGcuAuuGcuuuGAuuGcuTsT
88
AgcAAUcAAAGcAAuAGcATsT


 


AD-12721
b
4601-4619
89
uGcuAuuGcuuuGAuuGcuTsT
90
AGcAAUcAAAGcAAuAGcATsT


 


AD-12792
c
4601-4619
91
ugcuAuuGcuuuGAuuGcuTsT
92
AGcAAUcAAAGcAAuAGcATsT


 


AD-12839
d
4601-4619
93
ugcuAuuGcuuuGAuuGcuTsT
94
AgcAAUcAAAGcAAuAGcATsT


 


AD-12614
a
4600-4618
95
GcuAuuGcuuuGAuuGcuuTsT
96
AaGcAAUcAAAGcAAuAGCTsT


 


AD-12722
b
4600-4618
97
GcuAuuGcuuuGAuuGcuuTsT
98
AAGcAAUcAAAGcAAuAGCTsT


 


AD-12615
a
1421-1439
99
ccuuuAcuuuuAGGGuuGuTsT
100
AcAACCCuAAAAGuAAAGGTsT


 


AD-12616
a
1424-1442
101
uuAcuuuuAGGGuuGuAcGTsT
102
CguAcAACCCuAAAAGuAATsT


 


AD-12723
b
1424-1442
103
uuAcuuuuAGGGuuGuAcGTsT
104
CGuAcAACCCuAAAAGuAATsT


 


AD-12617
a
1403-1421
105
GcuccucAAuGGAuGuuGcTsT
106
GcAAcAUCcAUUGAGGAGCTsT


 


AD-12618
a
1534-1552
107
uuAuAAGAGGAGGAGuAGATsT
108
UcuACUCCUCCUCUuAuAATsT


 


AD-12724
b
1534-1552
109
uuAuAAGAGGAGGAGuAGATsT
110
UCuACUCCUCCUCUuAuAATsT


 


AD-12619
a
5098-5116
111
AAGuAGuAAGGGcGuGGAGTsT
112
CuCcACGCCCUuACuACUUTsT


 


AD-12725
b
5098-5116
113
AAGuAGuAAGGGcGuGGAGTsT
114
CUCcACGCCCUuACuACUUTsT


 


AD-12793
c
5098-5116
115
AaGuAGuAAGGGcGuGGAGTsT
116
CUCcACGCCCUuACuACUUTsT


 


AD-12840
d
5098-5116
117
AaGuAGuAAGGGcGuGGAGTsT
118
CuCcACGCCCUuACuACUUTsT


 


AD-12620
a
1430-1448
119
uuAGGGuuGuAcGGGAcuGTsT
120
caGUCCCGuAcAACCCuAATsT


 


AD-12726
b
1430-1448
121
uuAGGGuuGuAcGGGAcuGTsT
122
cAGUCCCGuAcAACCCuAATsT


 


AD-12621
a
1701-1719
123
GAcAuGcuuccuuGuuAcATsT
124
UguAAcAAGGAAGcAUGUCTsT


 


AD-12727
b
1701-1719
125
GAcAuGcuuccuuGuuAcATsT
126
UGuAAcAAGGAAGcAUGUCTsT


 


AD-12794
c
1701-1719
127
GacAuGcuuccuuGuuAcATsT
128
UGuAAcAAGGAAGcAUGUCTsT


 


AD-12841
d
1701-1719
129
GacAuGcuuccuuGuuAcATsT
130
UguAAcAAGGAAGcAUGUCTsT


 


AD-12622
a
2066-2084
131
uGuuGAAuGuuGGGuuccuTsT
132
AgGAACCcAAcAUUcAAcATsT


 


AD-12728
b
2066-2084
133
uGuuGAAuGuuGGGuuccuTsT
134
AGGAACCcAAcAUUcAAcATsT


 


AD-12795
c
2066-2084
135
uguuGAAuGuuGGGuuccuTsT
136
AGGAACCcAAcAUUcAAcATsT


 


AD-12842
d
2066-2084
137
uguuGAAuGuuGGGuuccuTsT
138
AgGAACCcAAcAUUcAAcATsT


 


AD-12623
a
4561-4579
139
AcuuAAcccAAGAAGcucuTsT
140
AgAGCUUCUUGGGUuAAGUTsT


 


AD-12729
b
4561-4579
141
AcuuAAcccAAGAAGcucuTsT
142
AGAGCUUCUUGGGUuAAGUTsT


 


AD-12624
a
4797-4815
143
ucAGccuGAuuuuGGuAcATsT
144
UguACcAAAAUcAGGCUGATsT


 


AD-12730
b
4797-4815
145
ucAGccuGAuuuuGGuAcATsT
146
UGuACcAAAAUcAGGCUGATsT


 


AD-12625
a
1428-1446
147
uuuuAGGGuuGuAcGGGAcTsT
148
GuCCCGuAcAACCCuAAAATsT


 


AD-12731
b
1428-1446
149
uuuuAGGGuuGuAcGGGAcTsT
150
GUCCCGuAcAACCCuAAAATsT


 


AD-12626
a
1429-1447
151
uuuAGGGuuGuAcGGGAcuTsT
152
AgUCCCGuAcAACCCuAAATsT


 


AD-12732
b
1429-1447
153
uuuAGGGuuGuAcGGGAcuTsT
154
AGUCCCGuAcAACCCuAAATsT


 


AD-12627
a
662-680
155
ucccuuGcuAcuGuAGAGGTsT
156
CcUCuAcAGuAGcAAGGGATsT


 


AD-12733
b
662-680
157
ucccuuGcuAcuGuAGAGGTsT
158
CCUCuAcAGuAGcAAGGGATsT


 


AD-12628
a
663-681
159
cccuuGcuAcuGuAGAGGGTsT
160
CcCUCuAcAGuAGcAAGGGTsT


 


AD-12734
b
663-681
161
cccuuGcuAcuGuAGAGGGTsT
162
CCCUCuAcAGuAGcAAGGGTsT


 


AD-12629
a
1402-1420
163
uGcuccucAAuGGAuGuuGTsT
164
caAcAUCcAUUGAGGAGcATsT


 


AD-12735
b
1402-1420
165
uGcuccucAAuGGAuGuuGTsT
166
cAAcAUCcAUUGAGGAGcATsT


 


AD-12796
c
1402-1420
167
ugcuccucAAuGGAuGuuGTsT
168
cAAcAUCcAUUGAGGAGcATsT


 


AD-12843
d
1402-1420
169
ugcuccucAAuGGAuGuuGTsT
170
caAcAUCcAUUGAGGAGcATsT


 


AD-12630
a
1398-1416
171
GAucuGcuccucAAuGGAuTsT
172
AuCcAUUGAGGAGcAGAUCTsT


 


AD-12736
b
1398-1416
173
GAucuGcuccucAAuGGAuTsT
174
AUCcAUUGAGGAGcAGAUCTsT


 


AD-12797
c
1398-1416
175
GaucuGcuccucAAuGGAuTsT
176
AUCcAUUGAGGAGcAGAUCTsT


 


AD-12844
d
1398-1416
177
GaucuGcuccucAAuGGAuTsT
178
AuCcAUUGAGGAGcAGAUCTsT


 


AD-12631
a
1399-1417
179
AucuGcuccucAAuGGAuGTsT
180
caUCcAUUGAGGAGcAGAUTsT


 


AD-12737
b
1399-1417
181
AucuGcuccucAAuGGAuGTsT
182
cAUCcAUUGAGGAGcAGAUTsT


 


AD-12632
a
1400-1418
183
ucuGcuccucAAuGGAuGuTsT
184
AcAUCcAUUGAGGAGcAGATsT


 


AD-12633
a
1401-1419
185
cuGcuccucAAuGGAuGuuTsT
186
AacAUCcAUUGAGGAGcAGTsT


 


AD-12738
b
1401-1419
187
cuGcuccucAAuGGAuGuuTsT
188
AAcAUCcAUUGAGGAGcAGTsT


 


AD-12634
a
1435-1453
189
GuuGuAcGGGAcuGuAAcATsT
190
UgUuAcAGUCCCGuAcAACTsT


 


AD-12739
b
1435-1453
191
GuuGuAcGGGAcuGuAAcATsT
192
UGUuAcAGUCCCGuAcAACTsT


 


AD-12635
a
1437-1455
193
uGuAcGGGAcuGuAAcAccTsT
194
GgUGUuAcAGUCCCGuAcATsT


 


AD-12740
b
1437-1455
195
uGuAcGGGAcuGuAAcAccTsT
196
GGUGUuAcAGUCCCGuAcATsT


 


AD-12798
c
1437-1455
197
uguAcGGGAcuGuAAcAccTsT
198
GGUGUuAcAGUCCCGuAcATsT


 


AD-12845
d
1437-1455
199
uguAcGGGAcuGuAAcAccTsT
200
GgUGUuAcAGUCCCGuAcATsT


 


AD-12636
a
1438-1456
201
GuAcGGGAcuGuAAcAccuTsT
202
AgGUGUuAcAGUCCCGuACTsT


 


AD-12741
b
1438-1456
203
GuAcGGGAcuGuAAcAccuTsT
204
AGGUGUuAcAGUCCCGuACTsT


 


AD-12637
a
4796-4814
205
cAGccuGAuuuuGGuAcAuTsT
206
AuGuACcAAAAUcAGGCUGTsT


 


AD-12742
b
4796-4814
207
cAGccuGAuuuuGGuAcAuTsT
208
AUGuACcAAAAUcAGGCUGTsT


 


AD-12799
c
4796-4814
209
caGccuGAuuuuGGuAcAuTsT
210
AUGuACcAAAAUcAGGCUGTsT


 


AD-12846
d
4796-4814
211
caGccuGAuuuuGGuAcAuTsT
212
AuGuACcAAAAUcAGGCUGTsT


 


AD-12638
a
4992-5010
213
GAAuAGGGAGGAAuccAuGTsT
214
caUGGAUUCCUCCCuAUUCTsT


 


AD-12743
b
4992-5010
215
GAAuAGGGAGGAAuccAuGTsT
216
cAUGGAUUCCUCCCuAUUCTsT


 


AD-12800
c
4992-5010
217
GaAuAGGGAGGAAuccAuGTsT
218
cAUGGAUUCCUCCCuAUUCTsT


 


AD-12847
d
4992-5010
219
GaAuAGGGAGGAAuccAuGTsT
220
caUGGAUUCCUCCCuAUUCTsT


 


AD-12639
a
4999-5017
221
AAGuGcuGAAuAGGGAGGATsT
222
UcCUCCCuAUUcAGcACUUTsT


 


AD-12744
b
4999-5017
223
AAGuGcuGAAuAGGGAGGATsT
224
UCCUCCCuAUUcAGcACUUTsT


 


AD-12801
c
4999-5017
225
AaGuGcuGAAuAGGGAGGATsT
226
UCCUCCCuAUUcAGcACUUTsT


 


AD-12848
d
4999-5017
227
AaGuGcuGAAuAGGGAGGATsT
228
UcCUCCCuAUUcAGcACUUTsT


 


AD-12640
a
630-648
229
AGGcuGcuGcuAcuAuAGATsT
230
UcuAuAGuAGcAGcAGCCUTsT


 


AD-12745
b
630-648
231
AGGcuGcuGcuAcuAuAGATsT
232
UCuAuAGuAGcAGcAGCCUTsT


 


AD-12802
c
630-648
233
AgGcuGcuGcuAcuAuAGATsT
234
UCuAuAGuAGcAGcAGCCUTsT


 


AD-12849
d
630-648
235
AgGcuGcuGcuAcuAuAGATsT
236
UcuAuAGuAGcAGcAGCCUTsT


 


AD-12641
a
3947-3965
237
AGAcAGccAuAuGcAGuAGTsT
238
CuACUGcAuAUGGCUGUCUTsT


 


AD-12803
c
3947-3965
239
AgAcAGccAuAuGcAGuAGTsT
240
CuACUGcAuAUGGCUGUCUTsT


 


AD-12642
a
524-542
241
uucAGGuucAuGGGuGccGTsT
242
CgGcACCcAUGAACCUGAATsT


 


AD-12746
b
524-542
243
uucAGGuucAuGGGuGccGTsT
244
CGGcACCcAUGAACCUGAATsT


 


AD-12643
a
3948-3966
245
uAGAcAGccAuAuGcAGuATsT
246
uaCUGcAuAUGGCUGUCuATsT


 


AD-12747
b
3948-3966
247
uAGAcAGccAuAuGcAGuATsT
248
uACUGcAuAUGGCUGUCuATsT


 


AD-12804
c
3948-3966
249
uaGAcAGccAuAuGcAGuATsT
250
uACUGcAuAUGGCUGUCuATsT


 


AD-12850
d
3948-3966
251
uaGAcAGccAuAuGcAGuATsT
252
uaCUGcAuAUGGCUGUCuATsT


 


AD-12644
a
3900-3918
253
GGAGcAuGAcuuuAAcccATsT
254
UgGGUuAAAGUcAUGCUCCTsT


 


AD-12748
b
3900-3918
255
GGAGcAuGAcuuuAAcccATsT
256
UGGGUuAAAGUcAUGCUCCTsT


 


AD-12805
c
3900-3918
257
GgAGcAuGAcuuuAAcccATsT
258
UGGGUuAAAGUcAUGCUCCTsT


 


AD-12851
d
3900-3918
259
GgAGcAuGAcuuuAAcccATsT
260
UgGGUuAAAGUcAUGCUCCTsT


 


AD-12645
a
1417-1435
261
GuuGccuuuAcuuuuAGGGTsT
262
CcCuAAAAGuAAAGGcAACTsT


 


AD-12749
b
1417-1435
263
GuuGccuuuAcuuuuAGGGTsT
264
CCCuAAAAGuAAAGGcAACTsT


 


AD-12646
a
4565-4583
265
uGuGAcuuAAcccAAGAAGTsT
266
CuUCUUGGGUuAAGUcAcATsT


 


AD-12750
b
4565-4583
267
uGuGAcuuAAcccAAGAAGTsT
268
CUUCUUGGGUuAAGUcAcATsT


 


AD-12806
c
4565-4583
269
uguGAcuuAAcccAAGAAGTsT
270
CUUCUUGGGUuAAGUcAcATsT


 


AD-12852
d
4565-4583
271
uguGAcuuAAcccAAGAAGTsT
272
CuUCUUGGGUuAAGUcAcATsT


 


AD-12647
a
4598-4616
273
uAuuGcuuuGAuuGcuucATsT
274
UgAAGcAAUcAAAGcAAuATsT


 


AD-12751
b
4598-4616
275
uAuuGcuuuGAuuGcuucATsT
276
UGAAGcAAUcAAAGcAAuATsT


 


AD-12807
c
4598-4616
277
uauuGcuuuGAuuGcuucATsT
278
UGAAGcAAUcAAAGcAAuATsT


 


AD-12853
d
4598-4616
279
uauuGcuuuGAuuGcuucATsT
280
UgAAGcAAUcAAAGcAAuATsT


 


AD-12648
a
2060-2078
281
AuAuccuGuuGAAuGuuGGTsT
282
CcAAcAUUcAAcAGGAuAUTsT


 


AD-12649
a
4729-4747
283
uuuAuuGcAAGGAAuGGccTsT
284
GgCcAUUCCUUGcAAuAAATsT


 


AD-12752
b
4729-4747
285
uuuAuuGcAAGGAAuGGccTsT
286
GGCcAUUCCUUGcAAuAAATsT


 


AD-12650
a
1122-1140
287
uGGAAGAAAcuAcuuGGGcTsT
288
GcCcAAGuAGUUUCUUCcATsT


 


AD-12753
b
1122-1140
289
uGGAAGAAAcuAcuuGGGcTsT
290
GCCcAAGuAGUUUCUUCcATsT


 


AD-12808
c
1122-1140
291
ugGAAGAAAcuAcuuGGGcTsT
292
GCCcAAGuAGUUUCUUCcATsT


 


AD-12854
d
1122-1140
293
ugGAAGAAAcuAcuuGGGcTsT
294
GcCcAAGuAGUUUCUUCcATsT


 


AD-12651
a
4261-4279
295
AAGAcccuAAAGAcuuuccTsT
296
GgAAAGUCUUuAGGGUCUUTsT


 


AD-12754
b
4261-4279
297
AAGAcccuAAAGAcuuuccTsT
298
GGAAAGUCUUuAGGGUCUUTsT


 


AD-12809
c
4261-4279
299
AaGAcccuAAAGAcuuuccTsT
300
GGAAAGUCUUuAGGGUCUUTsT


 


AD-12855
d
4261-4279
301
AaGAcccuAAAGAcuuuccTsT
302
GgAAAGUCUUuAGGGUCUUTsT


 


AD-12652
a
1412-1430
303
uGGAuGuuGccuuuAcuuuTsT
304
AaAGuAAAGGcAAcAUCcATsT


 


AD-12755
b
1412-1430
305
uGGAuGuuGccuuuAcuuuTsT
306
AAAGuAAAGGcAAcAUCcATsT


 


AD-12810
c
1412-1430
307
ugGAuGuuGccuuuAcuuuTsT
308
AAAGuAAAGGcAAcAUCcATsT


 


AD-12856
d
1412-1430
309
ugGAuGuuGccuuuAcuuuTsT
310
AaAGuAAAGGcAAcAUCcATsT


 


AD-12653
a
4592-4610
311
uuuGAuuGcuucAGAcAAuTsT
312
AuUGUCUGAAGcAAUcAAATsT


 


AD-12756
b
4592-4610
313
uuuGAuuGcuucAGAcAAuTsT
314
AUUGUCUGAAGcAAUcAAATsT


 


AD-12654
a
4991-5009
315
AAuAGGGAGGAAuccAuGGTsT
316
CcAUGGAUUCCUCCCuAUUTsT


 


AD-12811
c
4991-5009
317
AauAGGGAGGAAuccAuGGTsT
318
CcAUGGAUUCCUCCCuAUUTsT


 


AD-12655
a
5004-5022
319
GGAcAAAGuGcuGAAuAGGTsT
320
CcuAUUcAGcACUUUGUCCTsT


 


AD-12757
b
5004-5022
321
GGAcAAAGuGcuGAAuAGGTsT
322
CCuAUUcAGcACUUUGUCCTsT


 


AD-12812
c
5004-5022
323
GgAcAAAGuGcuGAAuAGGTsT
324
CCuAUUcAGcACUUUGUCCTsT


 


AD-12857
d
5004-5022
325
GgAcAAAGuGcuGAAuAGGTsT
326
CcuAUUcAGcACUUUGUCCTsT


 


AD-12656
a
5005-5023
327
uGGAcAAAGuGcuGAAuAGTsT
328
CuAUUcAGcACUUUGUCcATsT


 


AD-12813
c
5005-5023
329
ugGAcAAAGuGcuGAAuAGTsT
330
CuAUUcAGcACUUUGUCcATsT


 


AD-12657
a
654-672
331
AAAuuGcAucccuuGcuAcTsT
332
GuAGcAAGGGAUGcAAUUUTsT


 


AD-12814
c
654-672
333
AaAuuGcAucccuuGcuAcTsT
334
GuAGcAAGGGAUGcAAUUUTsT


 


AD-12658
a
659-677
335
GcAucccuuGcuAcuGuAGTsT
336
CuAcAGuAGcAAGGGAUGCTsT


 


AD-12659
a
4273-4291
337
AAAAAAAGGuAGAAGAcccTsT
338
GgGUCUUCuACCUUUUUUUTsT


 


AD-12758
b
4273-4291
339
AAAAAAAGGuAGAAGAcccTsT
340
GGGUCUUCuACCUUUUUUUTsT


 


AD-12815
c
4273-4291
341
AaAAAAAGGuAGAAGAcccTsT
342
GGGUCUUCuACCUUUUUUUTsT


 


AD-12858
d
4273-4291
343
AaAAAAAGGuAGAAGAcccTsT
344
GgGUCUUCuACCUUUUUUUTsT


 


AD-12660
a
2025-2043
345
AcAGAGcAcAAGGcGuAccTsT
346
GguACGCCUUGUGCUCUGUTsT


 


AD-12759
b
2025-2043
347
AcAGAGcAcAAGGcGuAccTsT
348
GGuACGCCUUGUGCUCUGUTsT


 


AD-12661
a
4791-4809
349
uGAuuuuGGuAcAuGGAAuTsT
350
AuUCcAUGuACcAAAAUcATsT


 


AD-12760
b
4791-4809
351
uGAuuuuGGuAcAuGGAAuTsT
352
AUUCcAUGuACcAAAAUcATsT


 


AD-12816
c
4791-4809
353
ugAuuuuGGuAcAuGGAAuTsT
354
AUUCcAUGuACcAAAAUcATsT


 


AD-12859
d
4791-4809
355
ugAuuuuGGuAcAuGGAAuTsT
356
AuUCcAUGuACcAAAAUcATsT


 


AD-12662
a
1433-1451
357
GGGuuGuAcGGGAcuGuAATsT
358
UuAcAGUCCCGuAcAACCCTsT


 


AD-12817
c
1433-1451
359
GgGuuGuAcGGGAcuGuAATsT
360
UuAcAGUCCCGuAcAACCCTsT


 


AD-12663
a
1434-1452
361
GGuuGuAcGGGAcuGuAAcTsT
362
GuuAcAGUCCCGuAcAACCTsT


 


AD-12761
b
1434-1452
363
GGuuGuAcGGGAcuGuAAcTsT
364
GUuAcAGUCCCGuAcAACCTsT


 


AD-12818
c
1434-1452
365
GguuGuAcGGGAcuGuAAcTsT
366
GUuAcAGUCCCGuAcAACCTsT


 


AD-12860
d
1434-1452
367
GguuGuAcGGGAcuGuAAcTsT
368
GuuAcAGUCCCGuAcAACCTsT


 


AD-12664
a
1440-1458
369
AcGGGAcuGuAAcAccuGcTsT
370
GcAGGUGUuAcAGUCCCGUTsT


 


AD-12665
a
1442-1460
371
GGGAcuGuAAcAccuGcucTsT
372
GaGcAGGUGUuAcAGUCCCTsT


 


AD-12762
b
1442-1460
373
GGGAcuGuAAcAccuGcucTsT
374
GAGcAGGUGUuAcAGUCCCTsT


 


AD-12819
c
1442-1460
375
GgGAcuGuAAcAccuGcucTsT
376
GAGcAGGUGUuAcAGUCCCTsT


 


AD-12861
d
1442-1460
377
GgGAcuGuAAcAccuGcucTsT
378
GaGcAGGUGUuAcAGUCCCTsT


 


AD-12666
a
1608-1626
379
AcuccAGAAAuGGGuGAccTsT
380
GgUcACCcAUUUCUGGAGUTsT


 


AD-12763
b
1608-1626
381
AcuccAGAAAuGGGuGAccTsT
382
GGUcACCcAUUUCUGGAGUTsT


 


AD-12667
a
4793-4811
383
ccuGAuuuuGGuAcAuGGATsT
384
UccAUGuACcAAAAUcAGGTsT


 


AD-12764
b
4793-4811
385
ccuGAuuuuGGuAcAuGGATsT
386
UCcAUGuACcAAAAUcAGGTsT


 


AD-12668
a
5001-5019
387
cAAAGuGcuGAAuAGGGAGTsT
388
CuCCCuAUUcAGcACUUUGTsT


 


AD-12765
b
5001-5019
389
cAAAGuGcuGAAuAGGGAGTsT
390
CUCCCuAUUcAGcACUUUGTsT


 


AD-12820
c
5001-5019
391
caAAGuGcuGAAuAGGGAGTsT
392
CUCCCuAUUcAGcACUUUGTsT


 


AD-12862
d
5001-5019
393
caAAGuGcuGAAuAGGGAGTsT
394
CuCCCuAUUcAGcACUUUGTsT


 


AD-12669
a
5066-5084
395
ccAGGGAAAuucccuuGuuTsT
396
AacAAGGGAAUUUCCCUGGTsT


 


AD-12766
b
5066-5084
397
ccAGGGAAAuucccuuGuuTsT
398
AAcAAGGGAAUUUCCCUGGTsT


 


AD-12670
a
5069-5087
399
AGGccAGGGAAAuucccuuTsT
400
AaGGGAAUUUCCCUGGCCUTsT


 


AD-12767
b
5069-5087
401
AGGccAGGGAAAuucccuuTsT
402
AAGGGAAUUUCCCUGGCCUTsT


 


AD-12821
c
5069-5087
403
AgGccAGGGAAAuucccuuTsT
404
AAGGGAAUUUCCCUGGCCUTsT


 


AD-12863
d
5069-5087
405
AgGccAGGGAAAuucccuuTsT
406
AaGGGAAUUUCCCUGGCCUTsT


 


AD-12671
a
564-582
407
uAGuuGcuAcuGuuucuGATsT
408
UcAGAAAcAGuAGcAACuATsT


 


AD-12822
c
564-582
409
uaGuuGcuAcuGuuucuGATsT
410
UcAGAAAcAGuAGcAACuATsT


 


AD-12672
a
633-651
411
cuGcuGcuAcuAuAGAAGuTsT
412
AcUUCuAuAGuAGcAGcAGTsT


 


AD-12768
b
633-651
413
cuGcuGcuAcuAuAGAAGuTsT
414
ACUUCuAuAGuAGcAGcAGTsT


 


AD-12673
a
634-652
415
uGcuGcuAcuAuAGAAGuuTsT
416
AaCUUCuAuAGuAGcAGcATsT


 


AD-12769
b
634-652
417
uGcuGcuAcuAuAGAAGuuTsT
418
AACUUCuAuAGuAGcAGcATsT


 


AD-12823
c
634-652
419
ugcuGcuAcuAuAGAAGuuTsT
420
AACUUCuAuAGuAGcAGcATsT


 


AD-12864
d
634-652
421
ugcuGcuAcuAuAGAAGuuTsT
422
AaCUUCuAuAGuAGcAGcATsT


 


AD-12674
a
635-653
423
GcuGcuAcuAuAGAAGuuGTsT
424
caACUUCuAuAGuAGcAGCTsT


 


AD-12770
b
635-653
425
GcuGcuAcuAuAGAAGuuGTsT
426
cAACUUCuAuAGuAGcAGCTsT


 


AD-12675
a
636-654
427
cuGcuAcuAuAGAAGuuGATsT
428
UcAACUUCuAuAGuAGcAGTsT


 


AD-12676
a
637-655
429
uGcuAcuAuAGAAGuuGAATsT
430
UucAACUUCuAuAGuAGcATsT


 


AD-12771
b
637-655
431
uGcuAcuAuAGAAGuuGAATsT
432
UUcAACUUCuAuAGuAGcATsT


 


AD-12824
c
637-655
433
ugcuAcuAuAGAAGuuGAATsT
434
UUcAACUUCuAuAGuAGcATsT


 


AD-12865
d
637-655
435
ugcuAcuAuAGAAGuuGAATsT
436
UucAACUUCuAuAGuAGcATsT


 


AD-12677
a
912-930
437
cAGAAGAcuAcuAuGAuAuTsT
438
AuAUcAuAGuAGUCUUCUGTsT


 


AD-12825
c
912-930
439
caGAAGAcuAcuAuGAuAuTsT
440
AuAUcAuAGuAGUCUUCUGTsT


 


AD-12678
a
4153-4171
441
AAAuuuuAuAuAAGAAAcuTsT
442
AgUUUCUuAuAuAAAAUUUTsT


 


AD-12772
b
4153-4171
443
AAAuuuuAuAuAAGAAAcuTsT
444
AGUUUCUuAuAuAAAAUUUTsT


 


AD-12826
c
4153-4171
445
AaAuuuuAuAuAAGAAAcuTsT
446
AGUUUCUuAuAuAAAAUUUTsT


 


AD-12866
d
4153-4171
447
AaAuuuuAuAuAAGAAAcuTsT
448
AgUUUCUuAuAuAAAAUUUTsT


 


AD-12679
a
4779-4797
449
AuGGAAuAGuucAGAGGuuTsT
450
AaCCUCUGAACuAUUCcAUTsT


 


AD-12773
b
4779-4797
451
AuGGAAuAGuucAGAGGuuTsT
452
AACCUCUGAACuAUUCcAUTsT


 


AD-12680
a
4780-4798
453
cAuGGAAuAGuucAGAGGuTsT
454
AcCUCUGAACuAUUCcAUGTsT


 


AD-12774
b
4780-4798
455
cAuGGAAuAGuucAGAGGuTsT
456
ACCUCUGAACuAUUCcAUGTsT


 


AD-12827
c
4780-4798
457
cauGGAAuAGuucAGAGGuTsT
458
ACCUCUGAACuAUUCcAUGTsT


 


AD-12867
d
4780-4798
459
cauGGAAuAGuucAGAGGuTsT
460
AcCUCUGAACuAUUCcAUGTsT


 


AD-12681
a
4781-4799
461
AcAuGGAAuAGuucAGAGGTsT
462
CcUCUGAACuAUUCcAUGUTsT


 


AD-12775
b
4781-4799
463
AcAuGGAAuAGuucAGAGGTsT
464
CCUCUGAACuAUUCcAUGUTsT


 


AD-12682
a
4784-4802
465
GGuAcAuGGAAuAGuucAGTsT
466
CuGAACuAUUCcAUGuACCTsT


 


AD-12776
b
4784-4802
467
GGuAcAuGGAAuAGuucAGTsT
468
CUGAACuAUUCcAUGuACCTsT


 


AD-12828
c
4784-4802
469
GguAcAuGGAAuAGuucAGTsT
470
CUGAACuAUUCcAUGuACCTsT


 


AD-12868
d
4784-4802
471
GguAcAuGGAAuAGuucAGTsT
472
CuGAACuAUUCcAUGuACCTsT


 


AD-12683
a
4785-4803
473
uGGuAcAuGGAAuAGuucATsT
474
UgAACuAUUCcAUGuACcATsT


 


AD-12777
b
4785-4803
475
uGGuAcAuGGAAuAGuucATsT
476
UGAACuAUUCcAUGuACcATsT


 


AD-12829
c
4785-4803
477
ugGuAcAuGGAAuAGuucATsT
478
UGAACuAUUCcAUGuACcATsT


 


AD-12869
d
4785-4803
479
ugGuAcAuGGAAuAGuucATsT
480
UgAACuAUUCcAUGuACcATsT


 


AD-12684
a
719-737
481
cuuAcuccuGAAAcAuAuGTsT
482
cauAUGUUUcAGGAGuAAGTsT


 


AD-12778
b
719-737
483
cuuAcuccuGAAAcAuAuGTsT
484
cAuAUGUUUcAGGAGuAAGTsT


 


AD-12685
a
909-927
485
AuccAGAAGAcuAcuAuGATsT
486
UcAuAGuAGUCUUCUGGAUTsT


 


AD-12686
a
1119-1137
487
uuuuGGAAGAAAcuAcuuGTsT
488
caAGuAGUUUCUUCcAAAATsT


 


AD-12779
b
1119-1137
489
uuuuGGAAGAAAcuAcuuGTsT
490
cAAGuAGUUUCUUCcAAAATsT


 


AD-12687
a
1121-1139
491
uuGGAAGAAAcuAcuuGGGTsT
492
CccAAGuAGUUUCUUCcAATsT


 


AD-12780
b
1121-1139
493
uuGGAAGAAAcuAcuuGGGTsT
494
CCcAAGuAGUUUCUUCcAATsT


 


AD-12688
a
4357-4375
495
AuGAAGAccuGuuuuGccATsT
496
UgGcAAAAcAGGUCUUcAUTsT


 


AD-12781
b
4357-4375
497
AuGAAGAccuGuuuuGccATsT
498
UGGcAAAAcAGGUCUUcAUTsT


 


AD-12689
a
4358-4376
499
GAuGAAGAccuGuuuuGccTsT
500
GgcAAAAcAGGUCUUcAUCTsT


 


AD-12782
b
4358-4376
501
GAuGAAGAccuGuuuuGccTsT
502
GGcAAAAcAGGUCUUcAUCTsT


 


AD-12830
c
4358-4376
503
GauGAAGAccuGuuuuGccTsT
504
GGcAAAAcAGGUCUUcAUCTsT


 


AD-12870
d
4358-4376
505
GauGAAGAccuGuuuuGccTsT
506
GgcAAAAcAGGUCUUcAUCTsT


 


AD-12690
a
4360-4378
507
GGGAuGAAGAccuGuuuuGTsT
508
caAAAcAGGUCUUcAUCCCTsT


 


AD-12783
b
4360-4378
509
GGGAuGAAGAccuGuuuuGTsT
510
cAAAAcAGGUCUUcAUCCCTsT


 


AD-12831
c
4360-4378
511
GgGAuGAAGAccuGuuuuGTsT
512
cAAAAcAGGUCUUcAUCCCTsT


 


AD-12871
d
4360-4378
513
GgGAuGAAGAccuGuuuuGTsT
514
caAAAcAGGUCUUcAUCCCTsT










Description of chemistries:








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


b
exo/endo-light: sense strand: dTsdT + 2′0Me@all Py; antisense



strand: dTsdT + 2′0Me@ Py in uA, cA 


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


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



















TABLE 1b-2






Residual


Relative





luciferase


siRNA activity



activity
SD of
Residual
(normalized
SD of
Relative



(relative to
residual
luciferase
to positive
relative
siRNA


duplex
control siRNA
luciferase
activity +/−
control
siRNA
activity +/−


name
treated cells)
activity
SD
luc-siRNA)
activity
SD





















AD-12598
91
11
 91 ± 11%
9
2
9 ± 2%


AD-12708
32
5
32 ± 5%
76
17
76 ± 17%


AD-12599
25
6
25 ± 6%
79
13
79 ± 13%


AD-12709
16
4
16 ± 4%
97
26
97 ± 26%


AD-12600
79
9
79 ± 9%
21
3
21 ± 3% 


AD-12710
25
4
25 ± 4%
85
24
85 ± 24%


AD-12784
23
2
23 ± 2%
87
14
87 ± 14%


AD-12832
84
11
 84 ± 11%
18
4
18 ± 4% 


AD-12601
102
8
102 ± 8% 
−6
1
−6 ± 1% 


AD-12785
95
10
 95 ± 10%
6
1
6 ± 1%


AD-12602
107
9
107 ± 9% 
−11
2
−11 ± 2% 


AD-12711
70
4
70 ± 4%
34
3
34 ± 3% 


AD-12786
69
8
69 ± 8%
35
7
35 ± 7% 


AD-12833
94
8
94 ± 8%
7
1
7 ± 1%


AD-12603
100
9
100 ± 9% 
−4
1
−4 ± 1% 


AD-12712
27
5
27 ± 5%
82
16
82 ± 16%


AD-12604
15
2
15 ± 2%
94
13
94 ± 13%


AD-12605
94
5
94 ± 5%
7
0
7 ± 0%


AD-12713
61
10
 61 ± 10%
41
8
41 ± 8% 


AD-12787
55
6
55 ± 6%
47
6
47 ± 6% 


AD-12834
92
16
 92 ± 16%
8
2
8 ± 2%


AD-12606
78
3
78 ± 3%
25
1
25 ± 1% 


AD-12714
63
6
63 ± 6%
42
5
42 ± 5% 


AD-12607
101
9
101 ± 9% 
−1
0
−1 ± 0% 


AD-12715
101
5
101 ± 5% 
−1
0
−1 ± 0% 


AD-12788
85
18
 85 ± 18%
15
4
15 ± 4% 


AD-12835
95
9
95 ± 9%
6
1
6 ± 1%


AD-12608
103
13
103 ± 13%
−3
0
−3 ± 0% 


AD-12716
81
9
81 ± 9%
22
3
22 ± 3% 


AD-12789
61
4
61 ± 4%
44
4
44 ± 4% 


AD-12836
103
11
103 ± 11%
−3
0
−3 ± 0% 


AD-12609
108
19
108 ± 19%
−9
2
−9 ± 2% 


AD-12717
94
17
 94 ± 17%
7
1
7 ± 1%


AD-12610
88
9
88 ± 9%
14
2
14 ± 2% 


AD-12718
39
4
39 ± 4%
64
8
64 ± 8% 


AD-12611
38
6
38 ± 6%
69
12
69 ± 12%


AD-12719
26
4
26 ± 4%
78
13
78 ± 13%


AD-12790
17
3
17 ± 3%
87
18
87 ± 18%


AD-12837
22
4
22 ± 4%
81
16
81 ± 16%


AD-12612
100
6
100 ± 6% 
0
0
0 ± 0%


AD-12720
73
6
73 ± 6%
28
3
28 ± 3% 


AD-12791
46
9
46 ± 9%
57
12
57 ± 12%


AD-12838
97
15
 97 ± 15%
3
1
3 ± 1%


AD-12613
26
4
26 ± 4%
82
15
82 ± 15%


AD-12721
10
1
10 ± 1%
94
12
94 ± 12%


AD-12792
10
3
10 ± 3%
94
40
94 ± 40%


AD-12839
22
3
22 ± 3%
81
12
81 ± 12%


AD-12614
15
5
15 ± 5%
94
38
94 ± 38%


AD-12722
6
1
 6 ± 1%
98
26
98 ± 26%


AD-12615
93
4
93 ± 4%
8
0
8 ± 0%


AD-12616
95
4
95 ± 4%
5
0
5 ± 0%


AD-12723
73
7
73 ± 7%
30
3
30 ± 3% 


AD-12617
88
10
 88 ± 10%
13
2
13 ± 2% 


AD-12618
42
7
42 ± 7%
60
7
60 ± 7% 


AD-12724
21
5
21 ± 5%
89
32
89 ± 32%


AD-12619
95
7
95 ± 7%
6
1
6 ± 1%


AD-12725
71
2
71 ± 2%
30
1
30 ± 1% 


AD-12793
54
7
54 ± 7%
48
7
48 ± 7% 


AD-12840
94
9
94 ± 9%
7
1
7 ± 1%


AD-12620
106
7
106 ± 7% 
−8
1
−8 ± 1% 


AD-12726
100
7
100 ± 7% 
0
0
0 ± 0%


AD-12621
107
9
107 ± 9% 
−7
1
−7 ± 1% 


AD-12727
47
4
47 ± 4%
60
8
60 ± 8% 


AD-12794
40
8
40 ± 8%
67
20
67 ± 20%


AD-12841
78
13
 78 ± 13%
25
8
25 ± 8% 


AD-12622
16
4
16 ± 4%
92
29
92 ± 29%


AD-12728
25
6
25 ± 6%
84
29
84 ± 29%


AD-12795
23
3
23 ± 3%
86
20
86 ± 20%


AD-12842
19
4
19 ± 4%
91
20
91 ± 20%


AD-12623
103
9
103 ± 9% 
−3
0
−3 ± 0% 


AD-12729
84
8
84 ± 8%
17
2
17 ± 2% 


AD-12624
31
4
31 ± 4%
77
12
77 ± 12%


AD-12730
18
1
18 ± 1%
85
3
85 ± 3% 


AD-12625
94
10
 94 ± 10%
5
1
5 ± 1%


AD-12731
57
4
57 ± 4%
48
4
48 ± 4% 


AD-12626
99
7
99 ± 7%
0
0
0 ± 0%


AD-12732
82
5
82 ± 5%
20
1
20 ± 1% 


AD-12627
80
6
80 ± 6%
22
2
22 ± 2% 


AD-12733
65
6
65 ± 6%
39
4
39 ± 4% 


AD-12628
81
6
81 ± 6%
21
2
21 ± 2% 


AD-12734
82
7
82 ± 7%
21
2
21 ± 2% 


AD-12629
113
11
113 ± 11%
−14
2
−14 ± 2% 


AD-12735
90
9
90 ± 9%
11
1
11 ± 1% 


AD-12796
92
8
92 ± 8%
9
1
9 ± 1%


AD-12843
117
7
117 ± 7% 
−19
1
−19 ± 1% 


AD-12630
124
3
124 ± 3% 
−27
1
−27 ± 1% 


AD-12736
85
4
85 ± 4%
16
1
16 ± 1% 


AD-12797
52
1
52 ± 1%
53
1
53 ± 1% 


AD-12844
96
4
96 ± 4%
5
0
5 ± 0%


AD-12631
110
11
110 ± 11%
−12
1
−12 ± 1% 


AD-12737
115
13
115 ± 13%
−17
2
−17 ± 2% 


AD-12632
106
2
106 ± 2% 
−7
0
−7 ± 0% 


AD-12633
107
12
107 ± 12%
−8
1
−8 ± 1% 


AD-12738
88
5
88 ± 5%
14
1
14 ± 1% 


AD-12634
79
5
79 ± 5%
24
1
24 ± 1% 


AD-12739
69
8
69 ± 8%
35
6
35 ± 6% 


AD-12635
75
8
75 ± 8%
25
6
25 ± 6% 


AD-12740
65
8
65 ± 8%
40
8
40 ± 8% 


AD-12798
56
4
56 ± 4%
50
6
50 ± 6% 


AD-12845
74
6
74 ± 6%
30
3
30 ± 3% 


AD-12636
89
8
89 ± 8%
9
1
9 ± 1%


AD-12741
31
4
31 ± 4%
78
14
78 ± 14%


AD-12637
16
2
16 ± 2%
93
14
93 ± 14%


AD-12742
18
3
18 ± 3%
85
14
85 ± 14%


AD-12799
18
4
18 ± 4%
86
22
86 ± 22%


AD-12846
15
2
15 ± 2%
89
14
89 ± 14%


AD-12638
95
6
95 ± 6%
5
0
5 ± 0%


AD-12743
23
4
23 ± 4%
81
15
81 ± 15%


AD-12800
14
1
14 ± 1%
90
10
90 ± 10%


AD-12847
90
12
 90 ± 12%
10
2
10 ± 2% 


AD-12639
113
11
113 ± 11%
−15
2
−15 ± 2% 


AD-12744
42
4
42 ± 4%
60
7
60 ± 7% 


AD-12801
34
3
34 ± 3%
68
8
68 ± 8% 


AD-12848
114
3
114 ± 3% 
−14
0
−14 ± 0% 


AD-12640
96
11
 96 ± 11%
4
1
4 ± 1%


AD-12745
52
7
52 ± 7%
53
8
53 ± 8% 


AD-12802
74
9
74 ± 9%
29
4
29 ± 4% 


AD-12849
111
5
111 ± 5% 
−12
1
−12 ± 1% 


AD-12641
103
8
103 ± 8% 
−3
0
−3 ± 0% 


AD-12803
94
13
 94 ± 13%
6
1
6 ± 1%


AD-12642
105
3
105 ± 3% 
−6
0
−6 ± 0% 


AD-12746
100
9
100 ± 9% 
0
0
0 ± 0%


AD-12643
33
4
33 ± 4%
74
10
74 ± 10%


AD-12747
21
3
21 ± 3%
83
13
83 ± 13%


AD-12804
25
4
25 ± 4%
78
14
78 ± 14%


AD-12850
28
4
28 ± 4%
75
11
75 ± 11%


AD-12644
82
7
82 ± 7%
20
2
20 ± 2% 


AD-12748
25
4
25 ± 4%
78
14
78 ± 14%


AD-12805
23
7
23 ± 7%
80
30
80 ± 30%


AD-12851
61
7
61 ± 7%
41
5
41 ± 5% 


AD-12645
112
6
112 ± 6% 
−14
1
−14 ± 1% 


AD-12749
86
10
 86 ± 10%
16
2
16 ± 2% 


AD-12646
94
10
 94 ± 10%
6
1
6 ± 1%


AD-12750
93
11
 93 ± 11%
7
1
7 ± 1%


AD-12806
77
8
77 ± 8%
24
3
24 ± 3% 


AD-12852
96
4
96 ± 4%
5
0
5 ± 0%


AD-12647
27
3
27 ± 3%
81
11
81 ± 11%


AD-12751
29
6
29 ± 6%
74
19
74 ± 19%


AD-12807
31
2
31 ± 2%
72
6
72 ± 6% 


AD-12853
26
3
26 ± 3%
78
11
78 ± 11%


AD-12648
81
9
81 ± 9%
17
3
17 ± 3% 


AD-12649
92
9
92 ± 9%
8
1
8 ± 1%


AD-12752
71
9
71 ± 9%
30
5
30 ± 5% 


AD-12650
81
2
81 ± 2%
21
1
21 ± 1% 


AD-12753
57
1
57 ± 1%
48
1
48 ± 1% 


AD-12808
52
4
52 ± 4%
54
5
54 ± 5% 


AD-12854
77
5
77 ± 5%
26
2
26 ± 2% 


AD-12651
89
6
89 ± 6%
13
1
13 ± 1% 


AD-12754
88
7
88 ± 7%
12
1
12 ± 1% 


AD-12809
67
6
67 ± 6%
35
4
35 ± 4% 


AD-12855
88
10
 88 ± 10%
12
2
12 ± 2% 


AD-12652
91
2
91 ± 2%
10
0
10 ± 0% 


AD-12755
40
3
40 ± 3%
67
6
67 ± 6% 


AD-12810
35
1
35 ± 1%
72
3
72 ± 3% 


AD-12856
75
8
75 ± 8%
28
4
28 ± 4% 


AD-12653
79
8
79 ± 8%
23
3
23 ± 3% 


AD-12756
17
5
17 ± 5%
86
27
86 ± 27%


AD-12654
97
6
97 ± 6%
3
0
3 ± 0%


AD-12811
74
5
74 ± 5%
27
2
27 ± 2% 


AD-12655
46
6
46 ± 6%
59
9
59 ± 9% 


AD-12757
14
0
14 ± 0%
89
2
89 ± 2% 


AD-12812
12
3
12 ± 3%
92
28
92 ± 28%


AD-12857
35
7
35 ± 7%
70
17
70 ± 17%


AD-12656
10
3
10 ± 3%
99
33
99 ± 33%


AD-12813
9
1
 9 ± 1%
95
18
95 ± 18%


AD-12657
108
1
108 ± 1% 
−9
0
−9 ± 0% 


AD-12814
101
4
101 ± 4% 
−1
0
−1 ± 0% 


AD-12658
98
9
98 ± 9%
2
0
2 ± 0%


AD-12659
83
4
83 ± 4%
18
1
18 ± 1% 


AD-12758
80
14
 80 ± 14%
21
4
21 ± 4% 


AD-12815
25
3
25 ± 3%
79
11
79 ± 11%


AD-12858
67
4
67 ± 4%
35
2
35 ± 2% 


AD-12660
95
11
 95 ± 11%
8
3
8 ± 3%


AD-12759
66
7
66 ± 7%
39
6
39 ± 6% 


AD-12661
34
2
34 ± 2%
73
5
73 ± 5% 


AD-12760
10
3
10 ± 3%
94
30
94 ± 30%


AD-12816
12
4
12 ± 4%
92
37
92 ± 37%


AD-12859
33
1
33 ± 1%
72
2
72 ± 2% 


AD-12662
92
7
92 ± 7%
7
1
7 ± 1%


AD-12817
91
11
 91 ± 11%
10
2
10 ± 2% 


AD-12663
99
10
 99 ± 10%
3
1
3 ± 1%


AD-12761
20
5
20 ± 5%
89
22
89 ± 22%


AD-12818
20
4
20 ± 4%
90
20
90 ± 20%


AD-12860
93
11
 93 ± 11%
8
1
8 ± 1%


AD-12664
93
9
93 ± 9%
6
2
6 ± 2%


AD-12665
94
8
94 ± 8%
10
1
10 ± 1% 


AD-12762
58
8
58 ± 8%
47
10
47 ± 10%


AD-12819
49
6
49 ± 6%
58
9
58 ± 9% 


AD-12861
93
8
93 ± 8%
8
1
8 ± 1%


AD-12666
30
5
30 ± 5%
76
18
76 ± 18%


AD-12763
25
2
25 ± 2%
84
9
84 ± 9% 


AD-12667
65
10
 65 ± 10%
38
7
38 ± 7% 


AD-12764
34
7
34 ± 7%
69
17
69 ± 17%


AD-12668
34
4
34 ± 4%
73
10
73 ± 10%


AD-12765
13
3
13 ± 3%
91
22
91 ± 22%


AD-12820
11
2
11 ± 2%
93
17
93 ± 17%


AD-12862
19
4
19 ± 4%
87
22
87 ± 22%


AD-12669
22
3
22 ± 3%
87
12
87 ± 12%


AD-12766
11
4
11 ± 4%
93
39
93 ± 39%


AD-12670
45
3
45 ± 3%
61
5
61 ± 5% 


AD-12767
10
3
10 ± 3%
94
31
94 ± 31%


AD-12821
12
1
12 ± 1%
92
13
92 ± 13%


AD-12863
41
4
41 ± 4%
64
8
64 ± 8% 


AD-12671
83
9
83 ± 9%
19
2
19 ± 2% 


AD-12822
74
7
74 ± 7%
29
3
29 ± 3% 


AD-12672
52
7
52 ± 7%
54
9
54 ± 9% 


AD-12768
28
3
28 ± 3%
81
12
81 ± 12%


AD-12673
56
5
56 ± 5%
49
5
49 ± 5% 


AD-12769
36
2
36 ± 2%
72
5
72 ± 5% 


AD-12823
33
2
33 ± 2%
75
5
75 ± 5% 


AD-12864
49
7
49 ± 7%
57
10
57 ± 10%


AD-12674
90
9
90 ± 9%
11
1
11 ± 1% 


AD-12770
45
6
45 ± 6%
61
9
61 ± 9% 


AD-12675
45
5
45 ± 5%
62
8
62 ± 8% 


AD-12676
47
6
47 ± 6%
59
9
59 ± 9% 


AD-12771
31
4
31 ± 4%
77
11
77 ± 11%


AD-12824
31
3
31 ± 3%
77
10
77 ± 10%


AD-12865
43
7
43 ± 7%
64
12
64 ± 12%


AD-12677
23
4
23 ± 4%
86
16
86 ± 16%


AD-12825
22
4
22 ± 4%
87
16
87 ± 16%


AD-12678
102
8
102 ± 8% 
−2
0
−2 ± 0% 


AD-12772
101
13
101 ± 13%
−1
0
−1 ± 0% 


AD-12826
99
1
99 ± 1%
1
0
1 ± 0%


AD-12866
91
7
91 ± 7%
10
1
10 ± 1% 


AD-12679
81
8
81 ± 8%
21
2
21 ± 2% 


AD-12773
11
2
11 ± 2%
93
19
93 ± 19%


AD-12680
17
3
17 ± 3%
92
17
92 ± 17%


AD-12774
15
2
15 ± 2%
89
17
89 ± 17%


AD-12827
11
2
11 ± 2%
93
18
93 ± 18%


AD-12867
15
3
15 ± 3%
91
22
91 ± 22%


AD-12681
28
3
28 ± 3%
79
10
79 ± 10%


AD-12775
8
1
 8 ± 1%
95
19
95 ± 19%


AD-12682
43
6
43 ± 6%
63
9
63 ± 9% 


AD-12776
23
5
23 ± 5%
80
19
80 ± 19%


AD-12828
23
5
23 ± 5%
80
20
80 ± 20%


AD-12868
25
4
25 ± 4%
81
16
81 ± 16%


AD-12683
17
2
17 ± 2%
91
15
91 ± 15%


AD-12777
11
2
11 ± 2%
92
22
92 ± 22%


AD-12829
12
1
12 ± 1%
92
11
92 ± 11%


AD-12869
19
3
19 ± 3%
87
16
87 ± 16%


AD-12684
87
12
 87 ± 12%
14
2
14 ± 2% 


AD-12778
41
4
41 ± 4%
66
8
66 ± 8% 


AD-12685
35
1
35 ± 1%
72
1
72 ± 1% 


AD-12686
68
5
68 ± 5%
36
3
36 ± 3% 


AD-12779
58
5
58 ± 5%
47
5
47 ± 5% 


AD-12687
73
8
73 ± 8%
30
4
30 ± 4% 


AD-12780
62
8
62 ± 8%
42
7
42 ± 7% 


AD-12688
18
1
18 ± 1%
91
4
91 ± 4% 


AD-12781
11
3
11 ± 3%
93
33
93 ± 33%


AD-12689
96
4
96 ± 4%
4
0
4 ± 0%


AD-12782
45
7
45 ± 7%
58
10
58 ± 10%


AD-12830
15
3
15 ± 3%
89
19
89 ± 19%


AD-12870
51
3
51 ± 3%
52
4
52 ± 4% 


AD-12690
93
6
93 ± 6%
8
1
8 ± 1%


AD-12783
36
3
36 ± 3%
66
7
66 ± 7% 


AD-12831
27
2
27 ± 2%
76
7
76 ± 7% 


AD-12871
81
18
 81 ± 18%
21
5
21 ± 5%

## Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human JC virus genome in a cell, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising at least 16 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 330, wherein a region of complementarity between the antisense strand and a target sequence is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said JC virus, inhibits expression of said JC virus genome.

2. A cell comprising the dsRNA of claim 1.

3. 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.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier comprises a lipid.

5. A method for inhibiting the expression of a gene from a JC Virus in a cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a gene from a JC Virus, thereby inhibiting expression of a gene from the JC Virus in the cell.

6. A method of treating a pathological process mediated by JC virus expression comprising administering to a patient a therapeutically effective amount of the dsRNA of claim 1.

7. The dsRNA of claim 1, wherein said sense strand comprises at least 16 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:329.

8. A vector for inhibiting the expression of a gene from a JC Virus in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of claim 7.

9. A cell comprising the vector of claim 8.

10. The dsRNA of claim 1, wherein said antisense strand comprises the nucleotide sequence of SEQ ID NO:330.

11. The dsRNA of claim 1, wherein said sense strand comprises the nucleotide sequence of SEQ ID NO:329.

12. The dsRNA of claim 1, wherein said antisense strand consists of the nucleotide sequence of SEQ ID NO:330.

13. The dsRNA of claim 1, wherein said sense strand consists of the nucleotide sequence of SEQ ID NO:329.

14. The dsRNA of claim 1, wherein said antisense strand consists of the nucleotide sequence of SEQ ID NO:330 and said sense strand consists of the nucleotide sequence of SEQ ID NO:329.

15. The dsRNA of claim 1, wherein at least one nucleotide of said sense strand or said antisense strand is a modified nucleotide.

16. The dsRNA of claim 15, 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.

17. The dsRNA of claim 1, wherein said sense strand consists of the SEQ ID NO: 329 (ugGAcAAAGuGcuGAAuAGTsT) and the antisense strand consists of SEQ ID NO:330 (CuAUUcAGcACUUUGUCcATsT) and each strand includes a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”.

18. The dsRNA of claim 14, wherein at least one nucleotide of said sense strand or said antisense strand is a modified nucleotide, and 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.

19. A pharmaceutical composition for inhibiting the expression of a gene from a JC Virus in an organism, comprising the dsRNA of claim 17 and a pharmaceutically acceptable carrier.

20. A method of treating a pathological process mediated by JC virus expression comprising administering to a patient a therapeutically effective amount of the dsRNA of claim 17.