WO2007111998A2 - Dsrna compositions and methods for treating hpv infection - Google Patents

Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for treating human papilloma virus (HPV) infection. The dsRNA comprises 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 an HPV Target gene selected from among HPV E1, HPV E6 and the human E6AP gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by HPV infection and the expression of the E6AP gene using the pharmaceutical composition; and methods for inhibiting the expression of the HPV Target genes in a cell.

Description

dsRNA COMPOSITIONS AND METHODS FOR TREATING HPV INFECTION

Field of the Invention

This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to treat pathological processes mediated by human papillomavirus (HPV) infection, such as cervical cancer, anal cancer, HPV associated precancerous lesions, and genital warts.

Background of the Invention

Papillomaviruses (PV) are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been recognized in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-I) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human more than 100 types of papillomavirus (HPV) have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott— Raven Pub., Philadelphia, N.Y.; Bernard, H-U., 2005. J. Clin. Virol. 328: S1-S6).

Human papillomavirus (HPV) is one of the most prevalent sexually transmitted infections in the world. The majority of HPV infections are harmless. Some types of HPV cause genital warts, which appear as single or multiple bumps in the genital areas of men and women including the vagina, cervix, vulva (area outside of the vagina), penis, and rectum. Many people infected with HPV have no symptoms. While most HPV subtypes result in benign lesions, certain subtypes are considered high- risk and can lead to more serious lesions, such as cervical and anal dysplasia. Fifteen HPV types were recently classified as high-risk types (Munoz, N. et al. 2003. N. Engl. J. Med. 348(6):518-27.) These high-risk subtypes are genetically diverse, demonstrating >10% sequence divergence at the Ll gene, a major virus capsid protein. (Bernard, H-U., 2005. J. Clin. Virol. 328: S1-S6).

Women having HPV infection are often asymptomatic and may only discover their lesion after cervical screening. Cervical screening is widely performed using the Pap test. A Pap test is a histological evaluation of cervical tissue which is used to identify abnormal cervical cells. As part of a Pap test, the presence of HPV infection and the specific subtype may be determined with the use of nucleic acid based assays such as PCR or the commercial Hybrid Capture II technique (HCII) (Digene, Gaithersburg, Maryland, U.S. A).

Abnormal cervical cells, if identified, are graded as LSIL (low-grade squamous intraepithelial lesions) having a low risk of progressing to cancer (including CIN-I designated cells ("cervical intraepithelial neoplasia-1")); or HSIL (High-grade squamous intraepithelial lesions), including CIN-2 and CIN-3 designated cells, having a higher likelihood of progressing to cancer.

About 85% of low-grade lesions spontaneously regress, and the remainder either stay unchanged, or progress to high-grade lesions. About 10% of high-grade lesions, if left untreated, are expected to transform into cancerous tissues. HPV- 16 and HPV- 18 are most often associated with dysplasias, although several other transforming HPV subtypes are also associated with dysplasias.

Recent studies indicate that up to 89% of HIV positive homosexual males may be infected with these high-risk subtypes of HPV. HlV positive patients are also more likely to be infected with multiple subtypes of HPV at the same time, which is associated with a higher risk of dysplasia progression.

Evidence over the last two decades has led to a broad acceptance that HPV infection is necessary, though not sufficient, for the development of cervical cancer. The presence of HPV in cervical cancer is estimated at 99.7%. Anal cancer is thought to have a similar association between HPV infection and the development of anal dysplasia and anal cancer as is the case with cervical cancer. In one study of HIV negative patients with anal cancer, HPV infection was found in 88% of anal cancers. In the US in 2003, 12,200 new cases of cervical cancer and 4,100 cervical-cancer deaths were predicted along with 4,000 new cases of anal cancer and 500 anal- cancer deaths. While the incidence of cervical cancer has decreased in the last four decades due to widespread preventive screening, the incidence of anal cancer is increasing. The increase in anal cancer incidence may be attributed in part to HIV infection since HIV positive patients have a higher incidence of anal cancer than the general population. While anal cancer has an incidence of 0.9 cases per 100,000 in the general population, anal cancer has an incidence of 35 cases per 100,000 in the homosexual male population and 70-100 cases per 100,000 in the HIV positive homosexual male population. In fact, due to the high prevalence of anal dysplasia among HIV- infected patients and a growing trend of anal cancers, the 2003 USPHA/IDSA Guidelines for the Treatment of Opportunistic Infections in HIV Positive Patients will include treatment guidelines for patients diagnosed with anal dysplasia.

There is no known cure for HPV infection. There are treatments for genital warts, although they often disappear even without treatment. The method of treatment depends on factors such as the size and location of the genital warts. Among the treatments used are Imiquimod cream, 20 percent podophyllin antimitotic solution, 0.5 percent podofilox solution, 5 percent 5-fluorouracil cream, and Trichloroacetic acid. The use of podophyllin or podofilox is not recommended for pregnant women because they are absorbed by the skin and may cause birth defects. The use of 5-fluorouracil cream is also not recommended for pregnant women. Small genital warts can be physically removed by freezing (cryosurgery), burning (electrocautery) or laser treatment. Large warts that do not responded to other treatment may have to be removed by surgery. Genital warts have been known to return following physical removal; in these instances α-interferon has been directly injected into these warts. However, α -interferon is expensive, and its use does not reduce the rate of return of the genital warts.

As such there exists an unmet need for effective HPV treatment. Surprisingly, compounds have been discovered that meet this need, and provide other benefits as well. Recently, 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.

PCT Publication WO 03/008573 discloses a previous effort to develop a nucleic acid based medicament for the treatment of disease caused by HPV infection. This publication reports the use of two siRNAs directed to HPV mRNA to inhibit HPV replication in a cell based system; a related publication is found at Jiang, M. et al. 2005. N. A. R. 33(18): el 51.

Despite significant advances in the field of RNAi and advances in the treatment of pathological processes mediated by HPV infection, there remains a need for agents that can inhibit the progression of HPV infection and that can treat diseases associated with HPV infection. The challenge is exacerbated because such agents must be designed to inhibit all the high-risk HPV subtypes, which together display a wide degree of genotypic diversity.

Summary of the Invention

The invention provides a solution to the problem of treating diseases associated with HPV infection, by using double-stranded ribonucleic acid (dsRNA) to silence gene expression essential for HPV propagation. E6AP is a conserved gene of the human host species required by HPV for proliferation.

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the E6AP gene in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by the expression of the E6AP gene in connection with HPV infection, such as in cervical cancer and gential warts. 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 the E6AP gene.

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the E6AP gene. 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 encoding E6AP, 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 the E6AP, inhibits the expression of the E6AP gene 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 Table 1 and the second sequence is selected from the group consisting of the antisense sequences of Table 1. 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-modifϊed nucleotide, 2'-aIkyI-modifϊed 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 Table 1 and a second sequence selected from the group consisting of the antisense sequences of Table 1.

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 expression of the E6AP gene 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 the E6AP gene 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 encoding E6AP, 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 expressing the E6AP, inhibits expression of the E6AP gene 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 the E6AP gene, thereby inhibiting expression of the E6AP gene in the cell.

In another embodiment, the invention provides methods for treating, preventing or managing pathological processes mediated by HPV infection, e.g. cancer or gential warts, 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 the E6AP gene 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 the E6AP gene 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 a solution to the problem of treating diseases associated with HPV infection, by using double-stranded ribonucleic acid (dsRNA) to silence expression of genes essential for HPV proliferation. In particular, the dsRNA of the invention silence the HPV genes El or E6 or human E6AP, a conserved gene of the human host species required by HPV for proliferation. Herein, these genes are sometimes collectively called the HPV Target genes.

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the El, E6 or E6AP gene 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 the expression of the El, E6 or E6AP gene in association with HPV 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 the HPV Target mRNA transcript. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and/or maintenance of an HPV in mammals. Using cell-based and animal-based 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 the El, E6 or E6AP gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by HPV infection by targeting a host factor gene involved in the HPV life cycle.

Description of the HPV Targets: HPV El and E6 and human E6AP

The cellular ubiquitin ligase E6AP of the human host is implicated in the replication of HPV, particularly integrated (non-episomal) forms of HPV, through its complex with the E6 protein of the virus. E6 binds to many proteins regulating cell proliferation pathways and often provokes their degradation (Chakrabarti, O. and Krishna, S. 2003. J. Biosci. 28:337-348). E6 complexes with E6AP to target the tumor suppressor p53 for degradation (Scheffher, M. et al., 1990. Cell. 63:1129-1 136; and Scheffner, M. et al., 1993. Cell 75:495-505). By inactivating p53, the virus not only prevents p53-mediated apoptosis of the infected cells (Chakrabarti and Krishna, 2003) and facilitates the replication of its DNA that would otherwise be blocked by p53 (Lepik, D. et al. 1998. J. Virol. 72:6822-6831), but it also favors oncogenesis by decreasing p53- mediated control on genomic integrity (Thomas, M. et al. 1999. Oncogene. 18:7690-7700).

El and E6 are both described in considerable detail in "Papillomaviridae: The Viruses and Their Replication" by Peter M. Howley, pp. 947-978, in: Fundamental Virology, 3rd ed. Bernard N. Fields, David M. Knipe, and Peter M. Howley, eds. Lippincott-Raven Publishers, Philadelphia, 1996. The El ORF encodes a 68-76 kD protein essential for plasmid DNA replication. The full-length El product is a phosphorylated nuclear protein that binds to the origin of replication in the LCR of BPVl. El has also been shown to bind ATP and to bind in vitro to the full length E2 protein called the E2 transcription transactivator (E2TA), thereby enhancing viral transcription. Binding to E2 also strengthens the affinity of El for the origin of DNA replication. In HPV- 16, El has indirect effects on immortalization.

E6 is a small basic cell-transforming protein (e.g., the HPV 16 E6 comprises 151 amino acids), about 16—19 kD, which is localized to the nuclear matrix and non-nuclear membrane fraction. The E6 gene product contains four Cys— X-X-Cys motifs, indicating a potential for zinc binding; it may also act as a nucleic acid binding protein. In high-risk HPVs such as HPV- 16, E6 and E7 proteins are necessary and sufficient to immortalize their hosts — squamous epithelial cells. The E6 gene products of high-risk HPVs have been shown to complex with p53, and to promote its degradation.

The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of the HPV Target genes, as well as compositions and methods for treating diseases and disorders caused by HPV infection, e.g. cervical cancer and genital warts. 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 an HPV Target gene, together with a pharmaceutically acceptable carrier. An embodiment of the invention is the employment of more than one dsRNA, optionally targeting different HPV Target genes, in combination in a pharmaceutical formulation.

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 one or more HPV Target genes, and methods of using the pharmaceutical compositions to treat diseases caused by HPV infection.

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, "E6AP" refers to the ubiquitin protein ligase E3A (ube3A, also referred to as E6-associated protein or E6AP) gene or protein. Human mRNA sequences to E6AP representing different isoforms are provided as GenBank Accession numbers NM_130838.1, NM_130839.1, and NM_000462.2.

As used herein, "El" refers to the human papillomavirus type 16 (HPV16) El gene (GenBank accession number NC OO 1526, nucleotides 865 to 2813). As used herein, "E6" refers to the human papillomavirus type 16 (HPV 16) E6 gene (GenBank accession number NC_001526, nucleotides 65 to 559). Many variants of the El and E6 genes have also been publicly disclosed. These and future published El and E6 gene variants are intended to be covered herein by the use of "El" and "E6", unless specifically excluded by the context.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of one of the HPV Target genes, 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, 50⁰C or 70⁰C 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 E6AP). For example, a polynucleotide is complementary to at least a part of a E6AP mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding E6AP.

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 separate RNA molecules, such dsRNA are often referred to in the literature as siRNA ("short interfering RNA"). 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", "short hairpin RNA" or "shRNA". 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. In addition, as used in this specification, "dsRNA" may include chemical modifications to ribonucleotides, internucleoside linkages, end-groups, caps, and conjugated moieties, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by "dsRNA" for the purposes of this specification and claims.

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^f-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. For clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3' end or 5' end of an siRNA are not considered in determining whether an siRNA has an overhang or is blunt ended.

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 the HPV Target gene, herein refer to the at least partial suppression of the expression of the HPV Target, gene, as manifested by a reduction of the amount of mRNA transcribed from the HPV Target gene which may be isolated from a first cell or group of cells in which the HPV Target gene is transcribed and which has or have been treated such that the expression of the HPV Target gene 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 (mRNA in control cells) - (mRNA in treated cells) . ₀ (mRNA in control cells)

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to the HPV Target gene transcription, e.g. the amount of protein encoded by the HPV Target gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, HPV Target gene 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 the HPV Target gene 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 the E6AP gene is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the E6AP gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the E6AP gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. Table 2 provides a wide range of values for inhibition of transcription obtained in an in vitro assay using various E6AP dsRNA molecules at various concentrations. Likewise, Table 6 provides a wide range of values for the inhibition of transcription of El; and Table 8 provides a wide range of values for the inhibition of transcription of E6.

As used herein in the context of HPV infection, the terms "treat", "treatment", and the like, refer to relief from or alleviation of pathological processes mediated by HPV infection. Such description includes use of the therapeutic agents of the invention for prophylaxis or prevention of HPV infection, and relief from symptoms or pathologies caused by HPV 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 HPV infection), 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 HPV infection or an overt symptom of pathological processes mediated by HPV infection. 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 HPV infection, the patient's history and age, the stage of pathological processes mediated by HPV infection, and the administration of other anti-pathological agents.

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 a dsRNA 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 the HPV Target gene 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 the HPV Target gene, 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 said HPV Target gene, inhibits the expression of said HPV Target gene by at least 10%, 25%, or 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 the HPV Target gene, 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, the HPV Target gene is the human E6AP gene. In specific embodiments, the antisense strand of the dsRNA comprises a strand selected from the sense sequences of Table 1 and a second sequence selected from the group consisting of the antisense sequences of Table 1. Alternative antisense agents that target elsewhere in the target sequence provided in Table 1 can readily be determined using the target sequence and the flanking E6AP sequence.

In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Table 1. 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 the E6AP gene. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Table 1 and the second oligonucleotide is described as the antisense strand in Table 1. Table 1 provides a duplex name and sequence ID number for each preferred dsRNA.

In further embodiments, the dsRNA comprises at least one named duplex dsRNA selected from the groups of sequences provided in Table 5 (El dsRNA) or Table 7 (E6 dsRNA).

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 Table 1, Table 5 or Table 7, 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 Table 1, Table 5 or Table 7, 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 Table 1, Table 5 or Table 7, and differing in their ability to inhibit the expression of the HPV Target gene in a FACS assay or other 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 Table 1, Table 5 or Table 7 can readily be made using the reference sequence and the target sequence provided.

In addition, the RNAi agents provided in Table 1, Table 5 and Table 7 identify a site in the respective HPV Target 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 Table 1, Table 5 or Table 7 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the HPV Target gene. For example, the last 15 nucleotides of SEQ ID NO:1 (minus the added AA sequences) combined with the next 6 nucleotides from the target E6AP gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Table 1.

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 the HPV Target gene, 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 the HPV Target gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the HPV Target gene is important, especially if the particular region of complementarity in the HPV Target gene is known to have polymorphic sequence variation in the virus (if El or E6) or within the human population (for E6AP). 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, NY, USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2' modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non-natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.

Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Generally, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-chIoroethyl)amine; N-acetyl-N'-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D.J., and K.B. Hall, Biochem. (1996) 35:14665- 14670). In a particular embodiment, the 5'-end of the antisense strand and the 3'-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds. Table 1 provides examples of modified RNAi agents of the invention.

In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the degradation activities of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the degradation activity of cellular enzymes against nucleic acids are known in the art including, but not limited to, 2'- amino modifications, 2'-amino sugar modifications, 2'-F sugar modifications, 2'-F modifications, 2'-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2'-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1 116-8). Thus, at least one 2'-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, generally by a 2'-amino or a 2'-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a_. methylene bridge that connects the 2'-oxygen of ribose with the 4' -carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A.A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401- 5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D.A. and D.R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as vaginal epithelium. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor- mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides as well as dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1 ,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3'-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.

In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.

The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5' position of a nucleoside or oligonucleotide. In certain instances, an dsRNA bearing an aralkyl ligand attached to the 3'-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid- phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.

The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2'-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.

5.602.240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos.

6.262.241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2'-fluoro- oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand- bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

The incoφoration of a 2'-O-methyl, 2'-0-ethyI, 2'-O-propyI, 2'-O-allyl, 2'-O-aminoalkyl or 2'-deoxy-2'-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functional ized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2'-O- methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-aminoalkyl, 2'-O-allyl or 2'-deoxy-2'-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT Publication WO 200370918.

In some embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5'-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5'-position through a linking group. The amino group at the 5'- terminus can be prepared utilizing a 5'-Amino-Modifϊer C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the S'-position by the use of a ligand- nucleoside phosphoramidite wherein the ligand is linked to the 5'-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5'-terminus.

Examples of modified internucleoside linkages or 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'-S' 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 United States Patents relating to the preparation of the above phosphorus- atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 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; 5,541,306; 5,550,111 ; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.

Examples of modified intemucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar 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 United States patents relating to 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; 5,264,562; 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; 5,610,289; 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 certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4: 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l^-di-O-hexadecyl-rac-glycero-S-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., Biochem. Biophys. Acta, 1995, 1264:229), or an octadecyl amine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Then, 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides 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 oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate. The use of a cholesterol conjugate is particularly preferred since such a moiety can increase targeting vaginal epithelium cells, a site of HPV infection.

The instant disclosure describes a wide variety of embodiments of dsRNA that are useful to silence HPV Target genes and thus to treat HPV associated disorders. While the design of the specific therapeutic agent can take a variety of forms, certain functional characteristics will distinguish preferred dsRNA from other dsRNA. In particular, features such as good serum stability, high potency, lack of induced immune response, and good drug like behaviour, all measurable by those skilled in the art, will be tested to identify preferred dsRNA of the invention. In some situations, not all of these functional aspects will be present in the preferred dsRNA. But those skilled in the art are able to optimize these variables and others to select preferred compounds of the invention.

While many nucleotide modifications are possible, the inventors have identified patterns of chemical modifications which provide significantly improved pharmacological, immunological and ultimately therapeutic benefit. Table 9 sets out patterns of chemical modifications preferred for use with the duplex dsRNA set out in Table 1, Table 5 and Table 7 of the invention. Some of these modifications are also illustrated in Table 3.

Table 9

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 Hl 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 Hl 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 the HPV Target gene, such as pathological processes mediated by HPV infection. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for either topical administration in the cervix or systemic administration via parenteral delivery.

The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of the HPV Target gene. The present inventors have determined that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or suppress expression of the HPV Target gene, and in the case of warts or cervical or anal treatment, may be applied directly to the infected tissue.

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 of vaginal gel. 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. Sustained release formulations are well known in the art and are particularly useful for vaginal delivery of agents, such as could be used with the agents of the present invention. 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.

The inventors recognize that for a variety of reasons, including the variability of HPV genotypes, it may be desireable to treat HPV infection with more than one dsRNA of the invention at the same time. In an embodiment, a combination of dsRNA are selected to target the widest range of HPV genotypes, with the least complex mixture of dsRNA. A pharmaceutical composition of the invention comprising more than one type of dsRNA would be expected to contain dosages of individual dsRNA as described herein.

Combinations of dsRNA may be provided together in a single dosage form pharmaceutical composition. Alternatively, combination dsRNA may be provided in separate dosage forms, in which case they may be administered at the same time or at different times, and possibly by different means. The invention therefore contemplates pharmaceutical compositions comprising the desired combinations of dsRNA of the invention; and it also contemplates pharmaceutical compositions of single dsRNA which are intended to be provided as part of a combination regimen. In this latter case, the combination therapy invention is thereby a method of administering rather than a composition of matter.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by HPV infection. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.

Any method can be used to administer a dsRNA of the present invention to a mammal containing cells infected with HPV. For example, administration can be topical (e.g., vaginal, transdermal, etc); oral; or parenteral (e.g., by subcutaneous, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection), or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations).

Typically, when treating a mammal having cells infected with HPV, the dsRNA molecules are administered topically in a vaginal gel or cream. For example, dsRNAs formulated with or without liposomes can be topically applied directly to the cervix, anal tract or HPV lesions such as genital warts. For topical administration, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. Compositions for topical administration can be formulated in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Gels and creams may be formulated using polymers and permeabilizers known in the art. Gels or creams containing the dsRNA and associated excipients may be applied to the cervix using a cervical cap, vaginal diaphragm, coated condom, glove, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like can be added. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers).

In addition, dsRNA molecules can be administered to a mammal containing HPV- infected cells using non-viral methods, such as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge- associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, DOTAP (1,2- dioleoyl-3-trimethylammonium propane), DOTMA (N-[1, 2(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride), DOSPA (2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]- N,N-deimethyl-l-propanaminium), DOGS (dioctadecyl amido glycil spermine), and DC-chol (3,[N-N',N-dimethylethylenediamine)-carbamoyl]cholesterol).

Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including Lipofectin.RTM. (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene.TM. (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Moπϊssey, D. et al. 2005. Nat Biotechnol. 23(8): 1002-7.

Other non-viral methods of administering dsRNA molecules to a mammal containing HPV-infected cells include cationic lipid-based delivery systems (in addition to lipsomes) such as lipoplexes and nanoemulsions. Additionally, condensing polymeric delivery systems (i.e., DNA-polymer complexes, or "polyplexes") may be used, including but not limited to chitosans, poly(L-lysine)(PLL), polyethylenimine (PEI), dendrimers (e.g., polyamidoamine (PANAM) dendrimers), and poloxamines. Additionally, noncondensing polymeric delivery systems may be used, including but not limited to poloxamers, gelatin, PLGA (polylactic-co-glycolic acid), PVP (polyvinylpyrrolidone), and PVA (polyvinyl alcohol).

Procedures for the above-mentioned delivery or administration techniques are well known in the art. For instance, condensing polymeric delivery systems work by easily complexing with anionic DNA molecules; for example, poly(L-lysine)(PLL) works by forming a positively charged complex that interacts with negatively charged cell surface and subsequently undergoing rapid internalization.

Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to skin cells and cervical cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.

dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier" (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

In addition, dsRNA that target the HPV Target gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the E6AP gene can contain other therapeutic agents such as anti-inflammatory drugs (e.g., nonsteroidal anti-inflammatory drugs and corticosteroids) and antiviral drugs (e.g., ribivirin, vidarabine, acyclovir, and ganciclovir). In some embodiments, a composition can contain one or more dsRNAs having a sequence complementary to the HPV Target gene in combination with a keratolytic agent. Keratolytic agents are agents that separate or loosen the horny layer of the epidermis. An example of a keratolytic agent includes, without limitation, salicylic acid. Other examples are provided in U.S. Pat. No. 5,543,417. Keratolytic agents can be used in an amount effective to enhance the penetration of dsRNAs, for example, into tissues such as skin. For example, a keratolytic agent can be used in an amount that allows a dsRNA applied to a genital wart to penetrate throughout . the wart.

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 HPV infection. 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.

Combinations of dsRNA can be tested in vitro and in vivo using the same methods employed for identification of preferred single dsRNA. Such combinations may be selected based on a purely bioinformatics basis, wherein the minimum number of siRNA are selected which provide coverage over the widest range of genotypes. Alternatively, such combinations may be selected based on in vitro or in vivo evaluations along the lines of those described herein for single dsRNA agents. A preferred assay for testing combinations of dsRNA is to evaluate the phenotypic conseuqences of siRNA mediated HPV target knockdown in HPVl 6 positive cancer cell lines (e.g. SiHa or Caski, as described in, e.g., Hengstermann et al. (2005) Journal Vir. 79(14): 9296; and Butz et al. (2003) Oncogene 22: 5938), or in organotypic culture systems, as described in, e.g., Jeon et al. (1995) Journal Vir. 69(5):2989.

The inventors have identified certain preferred combinations of dsRNA which may be used to treat HPV infection. In the most general terms, the combination of dsRNA comprises more than one dsRNA selected from among Table 1 , Table 3, Table 5 and Table 7. Thus the invention contemplates the use of 2, 3, 4, 5 or more dsRNA duplexes selected from among Table 1, Table 3, Table 5 and Table 7 in a combination therapy. In principle, the smallest number of dsRNA is preferred for simplicity of the therapeutic product. This forces the selection of dsRNA which will cover the greatest number of deleterious or potentially deleterious HPV genotypes, and indeed may justify selection of a combination that does not necessarily cover all such HPV genotypes.

The following dsRNA are particularly amenable to combination:

From El: ND-9072; ND-9142; ND-9092; ND-9162; ND-9097; ND-9167; ND-9066; ND-9123; AL-DP-8082; AL-DP-8095;

From E6: ND-8903; ND-8991; ND-8914; ND-9002; ND-8906; ND-8994; ND-8943; ND-9031; ND-9032; ND-8920; ND-8952; ND-8951 ; ND-9008; ND-9040; ND-9039; AL-DP- 7783; AL-DP-7784;

From E6AP: AL-DP-7365; AL-DP-7371; AL-DP-7499; AL-DP-7545; AL-DP-7492; AL-DP-7473; AL-DP-7478; AL-DP-7554; AL-DP-7514; AL-DP-7397, ND-9300.

Methods for treating diseases caused by HPV infection

The methods and compositions described herein can be used to treat diseases and conditions caused by human papillomavirus, which can be the result of clinical or sub-clinical papillomavirus infections. Such diseases and conditions, herein sometimes called "HPV associated disorders" or "pathological processes mediated by HPV infection", include, e.g., epithelial malignancies, skin cancer (non-melanoma or melanoma), anogenital malignancies such as cervical cancer, HPV associated precancerous lesions (including LSIL or HSIL cervical tissue), anal carcinoma, malignant lesions, benign lesions, papillomacarcinomas, papilloadenocy stomas, papilloma neuropath icum, papillomatosis, cutaneous and mucosal papillomas, condylomas, fibroblastic tumors, and other pathological conditions associated with papillomavirus.

For example, the compositions described herein can be used to treat warts caused by HPV. Such warts include, e.g., common warts (verruca vulgaris), for example, palmar, plantar, and periungual warts; flat and filiform warts; anal, oral, pharyngeal, laryngeal, and tongue papillomas; and venereal warts (condyloma accuminata), also known as genital warts (for example, penile, vulvar, vaginal and cervical warts), which are one of the most serious manifestations of HPV infection. HPV DNA can be found in all grades of cervical intraepithelial neoplasia (CIN 1-111)_» and a specific subset of HPV types can be found in carcinoma in situ of the cervix. Consequently, women with genital warts, containing specific HPV types, are considered to be at high risk for the development of cervical cancer.

The most common disease associated with papillomavirus infection is benign skin warts, or common warts. Common warts generally contain HPV types 1, 2, 3, 4 or 10. Other conditions caused by papillomavirus include, e.g., laryngeal papillomas, which are benign epithelial tumors of the larynx. Two papillomavirus types, HPV-6 and HPV-1 1 , are most commonly associated with laryngeal papillomas. The compositions described herein can be used to treat these diseases and conditions.

The compositions described herein can also be used in the treatment of epidermodysplasia verruciformis (EV), a rare genetically transmitted disease characterized by disseminated flat warts that appear as small reddish macules.

In addition, the compositions described herein can be used to treat lesions resulting from cellular transformation for which HPV is an etiological agent, e.g., in the treatment of cervical cancer.

The compositions described herein can also be used in the treatment of HPV-induced dysplasias, e.g., penile, vulvar, cervical, vaginal oral, anal, and pharyngeal dysplasias, and in the treatment of HPV-induced cancers, e.g., penile, vulvar, cervical, vaginal, anal, oral, pharyngeal, and head and neck cancers.

The invention can also be practiced by including a specific dsRNA 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.

In a further alternative, the dsRNA targeting E6AP may be employed to treat neurological and behavioural disorders. E6AP has been implicated in neurological and behavioural disorders through the identification of E6AP mutations in patients having Angelman syndrome. Angelman syndrome (AS) is an imprinted neurobehavioral disorder characterized by mental retardation, absent speech, excessive laughter, seizures, ataxia, and a characteristic EEG pattern. (Hitchins, M.P. et al. 2004. Am J Med Genet A. 125(2): 167-72.) It would not, presumably, be the intent of treatment to induce such conditions; rather, as observed in many hereditary defects, this evidence that E6AP has a critical role in neurological and behavioural conditions also indicates that this target may have a variety of roles in human pathologies and is likely a suitable target for other diseases in this class where silencing of E6AP will compensate for other biochemical defects or diseases. As used herein "E6AP associated disorders" include the HPV associated disorders noted above and other neurological and behavioural disorders.

Methods for inhibiting expression of the E6AP gene

In yet another aspect, the invention provides a method for inhibiting the expression of the E6AP gene in a mammal. The method comprises administering a composition of Table 1 of the invention to the mammal such that expression of the target E6AP gene is silenced. Because of their high specificity, such dsRNAs of the invention specifically target RNAs (primary or processed) of the target E6AP gene. Compositions and methods for inhibiting the expression of these E6AP genes using such 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 the E6AP gene of 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, rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by topical/vaginal administration or 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

Gene Walking of the E6AP gene

siRNA design was carried out to identify si RNAs targeting human ubiquitin protein ligase E3A (ube3A, also referred to as E6AP). Human mRNA sequences to E6AP representing different isoforms (NM_130838.1, NMJ30839.1, NM 000462.2) were used.

The ClustalW multiple alignment function (Thompson J.D., et al., Nucleic Acids Res. 1994, 22:4673) of the BioEdit software was used with all human E6AP isoforms to identify mRNA sequence NM_130838.1 as shortest sequence as well as to confirm sequence conservation from position 5 to 4491 (end position) of the reference sequence, a requirement for efficient targeting of all E6AP isoforms.

All possible overlapping 19mers (representing siRNA sense strand sequences) spanning E6AP reference sequence NM l 30838.1 were identified, resulting in 4473 19mer candidate sequences. Combined, these candidate target sequences cover the 5'UTR, coding and 3'UTR domains of the E6AP mRNA, and the junction sites of these domains.

In order to rank and select siRNAs out of the pool of candidates, the 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) complementarity to a target gene in positions 2 to 9 (counting 5* to 3¹) of a strand (seed region) may be sufficient for interaction of that strand with the mRNA transcribed from the target gene and subsequent downregulation (Jackson AL, et al. Nat Biotechnol. 2003 Jun;21(6):635-7)

2) positions 1 and 19 of each strand are not relevant for off-target interactions

3) seed region may contribute more to off-target potential than rest of sequence 4) cleavage site region positions 10 and 11 (counting 5' to 3') of a strand may contribute more to off-target potential than the sequences 3' to the cleavage site (non-seed region), but not as much as the seed region

5) an off-target score can be calculated for each gene and each strand, based on complementarity of siRNA strand sequence to the gene's sequence and position of mismatches while considering assumptions 1 to 4

6) assuming potential abortion of sense strand activity by internal modifications introduced, only off-target potential of antisense strand will be relevant

7) the off-target potential of an siRNA can be inferred from the gene displaying the highest homology according to our criteria (best off-target gene), thus can be expressed by the off-target score of the respective gene

To identify potential off-target genes, 19mer antisense sequences were subjected to a homology search against publicly available human mRNA sequences. To this purpose, fastA (version 3.4) searches were performed with all 19mer candidate antisense sequences against the human RefSeq database. A Perl script was used to generate antisense sequences from the candidate I9mer sequences (perl script 2). fastA search was executed with parameter/value pairs -g 30 -f 30 -L -i -H in order to take into account the homology over the full length of the 19mer and to format the output suitable for the script analysis in the next step. The search resulted in a list of potential off-target genes for candidate siRNAs.

Further, fastA search parameters were applied with values -E 15000 in order to make database entries with more than 8 contiguous nucleobases identical to the 19mer sense strand sequences very likely to be transferred to a fastA output file while displaying the homology of the complete 19mer length (see assumption 1).

In order to identify the best off-target gene and its off-target score, the fastA output file was analyzed. The following off-target properties for each 19mer input sequence were extracted for each potential off-target gene: Number of mismatches in seed region

Number of mismatches in non-seed region

Number of mismatches in cleavage site region

The off-target score for each off-target gene was calculated as follows:

(number of seed mismatches multiplied by 10) + (number of cleavage site mismatches multiplied by 1.2) + number of non-seed mismatches

The lowest off-target score was extracted for each input 19mer sequence and successively written into an output file resulting in a list of off-target scores for all siRNAs corresponding to the input 19mer sequences.

In order to generate a ranking of siRNAs, off-target scores were entered into a result table. All siRNAs were finally sorted according descending to the off-target score and sequences containing stretches with more than 3 Gs in a row were excluded from selection.

The 156 siRNAs with an off-target score of >=3 were selected and synthesized (Table 1).

Table 1 dsRNA targeting E6AP

Target sequence of mRNA Sense strand antisense strand from human reference SEQ (target sequence) SEQ (guide sequence) SEQ sequence NM_130838 (human ID. having double ID. having ID. iso3) NO. overhang NO. double overhang NO. sequence of total 19mer duplex target site + AA at ends sequence (5 '-3') sequence (5 '-3'] name

AAAUACGAUGAAUCUACAAAAAA 1 AUACGAUGAAUCUACAAAATT ¹⁵⁷ UUUUGUAGAUUCAUCGUAUTT ³¹³ AL-DP- -7545 AAUGACUACAUUCUCAAUAAAAA 2 UGACUACAUUCUCAAUAAATT ¹⁵⁸ UUUAUUGAGAAUGUAGUCATT ³¹⁴ AL-DP'-7558 AAAGCCUGCACGAAUGAGUUUAA 3 AGCCUGCACGAAUGAGUUUTT ¹⁵⁹ AAACUCAUUCGUGCAGGCUTT ³¹⁵ AL-DP--7548 AAGGAUUGUCGAAAACCACUUAA 4 GGAUUGUCGAAAACCACUUTT ^λ60 AAGUGGUUUUCGACAAUCCTT ³¹⁶ AL-DP--7509 AACUCUCGAGAUCCUAAUUAUAA 5 CUCUCGAGAUCCUAAUUAUTT ¹⁶¹ AUAAUUAGGAUCUCGAGAGTT ³¹⁷ AL-DP--7492 AAAUGUGACUUACUUAACAGAAA 6 AUGUGACUUACUUAACAGATT ¹⁶² UCUGUUAAGUAAGUCACAUTT ³¹⁸ AL-DP--7554 AAGUAUACUCUCGAGAUCCUAAA 7 GUAUACUCUCGAGAUCCUATT ¹⁶³ UAGGAUCUCGAGAGUAUACTT ³¹⁹ AL-DP--7557 AAAGGUUACCUACAUCUCAUAAA 8 AGGUUACCUACAUCUCAUATT ¹⁶⁴ UAUGAGAUGUAGGUAACCUTT ³²° AL-DP -7476 AAAGUACUUAUUCAGACCAGAAA 9 AGUACUUAUUCAGACCAGATT ¹⁶⁵ UCUGGUCUGAAUAAGUACUTT ³²¹ AL-DP-^■7514 AAAUCCUAAUUAUCUGAAUUUAA 10 AUCCUAAUUAUCUGAAUUUTT ¹⁶⁶ AAAUUCAGAUAAUUAGGAUTT ³²² AL-DP-^■7540 AAAAGGAUAGGUGAUAGCUCAAA 11 AAGGAUAGGUGAUAGCUCATT ¹⁶⁷ UGAGCUAUCACCUAUCCUUTT ³²³ AL-DP--7397 AAGGAAGCCGGAAUCUAGAUUAA 12 GGAAGCCGGAAUCUAGAUUTT ¹⁶⁸ AAUCUAGAUUCCGGCUUCCTT ³²⁴ AL-DP-^■7526 AAUGCUUCGAAGUGCUUGAAAAA 13 UGCUUCGAAGUGCUUGAAATT ^l69 UUUCAAGCACUUCGAAGCATT ³²⁵ AL-DP--7473 AAUGGAUUGUCGAAAACCACUAA 14 UGGAUUGUCGAAAACCACUTT ¹⁷° AGUGGUUUUCGACAAUCCATT ³²⁶ AL-DP--7478 AACGGCUAGAGAUGAUCGCUAAA 15 CGGCUAGAGAUGAUCGCUATT ¹⁷¹ UAGCGAUCAUCUCUAGCCGTT ³²⁷ AL-DP--7553 AAACAGUCGAAAUCUAGUGAAAA 16 ACAGUCGAAAUCUAGUGAATT ¹⁷² UUCACUAGAUUUCGACUGUTT ³²⁸ AL-DP--7395 AAGAUCAGACUGUGGUCUAAAAA 17 GAUCAGACUGUGGUCUAAATT ¹⁷³ UUUAGACCACAGUCUGAUCTT ³²⁹ AL-DP -7522 AACUCGAGAUCCUAAUUAUCUAA 18 CUCGAGAUCCUAAUUAUCUTT ¹⁷⁴ AGAUAAUUAGGAUCUCGAGTT ³³⁰ AL-DP -7499 AAUAUCGUAAUGGAGAAUAGAAA 19 UAUCGUAAUGGAGAAUAGATT ¹⁷⁵ UCUAUUCUCCAUUACGAUATT ³³¹ AL-DP--7527 AACUCAAAGUUAGACGUGACCAA 20 CUCAAAGUUAGACGUGACCTT ¹⁷⁶ GGUCACGUCUAACUUUGAGTT ³³² AL-DP-^■7544 AAAGGAUAGGUGAUAGCUCACAA 21 AGGAUAGGUGAUAGCUCACTT ¹⁷⁷ GUGAGCUAUCACCUAUCCUTT ³³³ AL-DP--7489 AACACCUAACGUGGAAUGUGAAA 22 CACCUAACGUGGAAUGUGATT ¹⁷⁸ UCACAUUCCACGUUAGGUGTT ³³⁴ AL-DP--7365 AAAAUCGUUCAUUCAUUUACAAA 23 AAUCGUUCAUUCAUUUACATT ¹⁷⁹ UGUAAAUGAAUGAACGAUUTT ³³⁵ AL-DP--7390 AACUUGACGUAUCACAAUGUAAA 24 CUUGACGUAUCACAAUGUATT ¹⁸° UACAUUGUGAUACGUCAAGTT ³³⁶ AL-DP--7458 AAUGGUAUGUUCACAUACGAUAA 25 UGGUAUGUUCACAUACGAUTT ¹⁸¹ AUCGUAUGUGAACAUACCATT ³³⁷ AL-DP--7532 AAGAUAGGUGAUAGCUCACAGAA 26 GAUAGGUGAUAGCUCACAGTT ^λ82 CUGUGAGCUAUCACCUAUCTT ³³⁸ AL-DP -7546 AACCGGCUAGAGAUGAUCGCUAA 27 CCGGCUAGAGAUGAUCGCUTT ¹⁸³ AGCGAUCAUCUCUAGCCGGTT ³³⁹ AL-DP-^■7512

AACAUAGUACUGGGUCUGGCUAA 28 CAUAGUACUGGGUCUGGCUTT ¹⁸⁴ AGCCAGACCCAGUACUAUGTT 340 AL-DP-7470 AAAAUGUAUACUCUCGAGAUCAA 29 AAUGUAUACUCUCGAGAUCTT ¹⁸⁵ GAUCUCGAGAGUAUACAUUTT 341 AL-DP-7406 AAAACUUUUCGUGACUUGGGAAA 30 AACUUUUCGUGACUUGGGATT ¹⁸⁶ UCCCAAGUCACGAAAAGUUTT 342 AL-DP-7382 AAAAAGUUAGACGUGACCAUAAA 31 AAAGUUAGACGUGACCAUATT ¹⁸⁷ UAUGGUCACGUCUAACUUUTT 343 AL-DP-7547 AAUGAUUAGGGAGUUCUGGGAAA 32 UGAUUAGGGAGUUCUGGGATT ¹⁸⁸ UCCCAGAACUCCCUAAUCATT 344 AL-DP-7490 AAUACGAUGAAUCUACAAAAUAA 33 UACGAUGAAUCUACAAAAUTT ¹⁸⁹ AUUUUGUAGAUUCAUCGUATT 345 AL-DP-7493 AACUUGUCCGGCUAGAGAUGAAA 34 CUUGUCCGGCUAGAGAUGATT ¹⁹° UCAUCUCUAGCCGGACAAGTT 346 AL-DP-7529 AAUAUACUCUCGAGAUCCUAAAA 35 UAUACUCUCGAGAUCCUAATT ¹⁹¹ UUAGGAUCUCGAGAGUAUATT 347 AL-DP-7400 AAACUUGACGUAUCACMUGUAA 36 ACUUGACGUAUCACAAUGUTT ¹⁹² ACAUUGUGAUACGUCAAGUTT 348 AL-DP-7391 AAAACAGUCGAAAUCUAGUGAAA 37 AACAGUCGAAAUCUAGUGATT ^{X 93} UCACUAGAUUUCGACUGUUTT 349 AL-DP-7393 AAUCAUUAUCGUAAUGGAGAAAA 38 UCAUUAUCGUAAUGGAGAATT ¹⁹⁴ UUCUCCAUUACGAUAAUGATT 350 AL-DP-7511 AAAUAGUACUGGGUCUGGCUAAA 39 AUAGUACUGGGUCUGGCUATT ¹⁹⁵ UAGCCAGACCCAGUACUAUTT 351 AL-DP-7454 AACCUAACGUGGAAUGUGACUAA 40 CCUAACGUGGAAUGUGACUTT *⁹⁶ AGUCACAUUCCACGUUAGGTT 352 AL-DP-7450 AAUUGUCCGGCUAGAGAUGAUAA 41 UUGUCCGGCUAGAGAUGAUTT ¹⁹⁷ AUCAUCUCUAGCCGGACAATT 353 AL-DP-7533 AAACCUAACGUGGAAUGUGACAA 42 ACCUAACGUGGAAUGUGACTT ¹⁹⁸ GUCACAUUCCACGUUAGGUTT 354 AL-DP-7485 AAUUAACAGUCGAAAUCUAGUAA 43 UUAACAGUCGAAAUCUAGUTT ¹⁹⁹ ACUAGAUUUCGACUGUUAATT 355 AL-DP-7495 AAUUGGCAUAGUACUGGGUCUAA 44 UUGGCAUAGUACUGGGUCUTT ^2O° AGACCCAGUACUAUGCCAATT 356 AL-DP-7456 AAGAACUUUUCGUGACUUGGGAA 45 GAACUUUUCGUGACUUGGGTT ²⁰¹ CCCAAGUCACGAAAAGUUCTT 357 AL-DP-7538 on AAGUCCGGCUAGAGAUGAUCGAA 46 GUCCGGCUAGAGAUGAUCGTT ²⁰² CGAUCAUCUCUAGCCGGACTT 358 AL-DP-7377 AAGCCCUCGAGCUUUAUAAGAAA 47 GCCCUCGAGCUUUAUAAGATT ²⁰³ UCUUAUAAAGCUCGAGGGCTT 359 AL-DP-7405 AACUCGAGCUUUAUAAGAUUAAA 48 CUCGAGCUUUAUAAGAUUATT ²⁰⁴ UAAUCUUAUAAAGCUCGAGTT 360 AL-DP-7392 AAUGGCAUAGUACUGGGUCUGAA 49 UGGCAUAGUACUGGGUCUGTT ²⁰⁵ CAGACCCAGUACUAUGCCATT 361 AL-DP-7453 AAACGAAUGAGUUUUGUGCUUAA 50 ACGAAUGAGUUUUGUGCUUTT ²⁰⁶ AAGCACAAAACUCAUUCGUTT 362 AL-DP-7366 AAUUUCUUCGUAUGGAUAAUAAA 51 UUUCUUCGUAUGGAUAAUATT ²⁰⁷ UAUUAUCCAUACGAAGAAATT 363 AL-DP-7534 AAAGACGUGACCAUAUCAUAGAA 52 AGACGUGACCAUAUCAUAGTT ²⁰⁸ CUAUGAUAUGGUCACGUCUTT 364 AL-DP-7401 AAUAGUACUGGGUCUGGCUAUAA 53 UAGUACUGGGUCUGGCUAUTT ²⁰⁹ AUAGCCAGACCCAGUACUATT 365 AL-DP-7523 AACGUAUGGAUAAUAAUGCAGAA 54 CGUAUGGAUAAUAAUGCAGTT ²¹° CUGCAUUAUUAUCCAUACGTT 366 AL-DP-7555 AAUGGCUAUUUACAAUAACUGAA 55 UGGCUAUUUACAAUAACUGTT ²¹¹ CAGUUAUUGUAAAUAGCCATT 367 AL-DP-7536 AAAAUUCGCAUGUACAGUGAAAA 56 AAUUCGCAUGUACAGUGAATT ²¹² UUCACUGUACAUGCGAAUUTT 368 AL-DP-7371 AAAAUAGAAUUCGCAUGUACAAA 57 AAUAGAAUUCGCAUGUACATT ²¹³ UGUACAUGCGAAUUCUAUUTT 369 AL-DP-7372 AAUGGUAACCCAAUGAUGUAUAA 58 UGGUAACCCAAUGAUGUAUTT ²¹⁴ AUACAUCAUUGGGUUACCATT 370 AL-DP-7370 AAAGCCGGAAUCUAGAUUUCCAA 59 AGCCGGAAUCUAGAUUUCCTT ²¹⁵ GGAAAUCUAGAUUCCGGCUTT 371 AL-DP-7474 AAACUUUUCGUGACUUGGGAGAA 60 ACUUUUCGUGACUUGGGAGTT ²¹⁶ CUCCCAAGUCACGAAAAGUTT 372 AL-DP-7452 AAAGCCCUCGAGCUUUAUAAGAA 61 AGCCCUCGAGCUUUAUAAGTT ²¹⁷ CUUAUAAAGCUCGAGGGCUTT 373 AL-DP-7498

AAGAACGAAGAAUCACUGUUCAA 62 GAACGAAGAAUCACUGUUCTT 218 GAACAGUGAUUCUUCGUUCTT 374 AL-DP-7551 AAUAUUCUGACUACAUUCUCAAA 63 UAUUCUGACUACAUUCUCATT 219 UGAGAAUGUAGUCAGAAUATT 375 AL-DP-7552 AAGCAUCUAAUAGAACGCUACAA 64 GCAUCUAAUAGAACGCUACTT 220 GUAGCGUUCUAUUAGAUGCTT 376 AL-DP-7504 AAUCGAAAUCUAGUGAAUGAUAA 65 UCGAAAUCUAGUGAAUGAUTT 221 AUCAUUCACUAGAUUUCGATT 377 AL-DP-7467 AAAGUCGAAAUCUAGUGAAUGAA 66 AGUCGAAAUCUAGUGAAUGTT 222 CAUUCACUAGAUUUCGACUTT 378 AL-DP-7463 AAGAAAGGCGCUAGAAUUGAUAA 67 GAAAGGCGCUAGAAUUGAUTT 223 AUCAAUUCUAGCGCCUUUCTT 379 AL-DP-7399 AAAAAGGCGCUAGAAUUGAUUAA 68 AAAGGCGCUAGAAUUGAUUTT 224 AAUCAAUUCUAGCGCCUUUTT 380 AL-DP-7501 AAAGGCGCUAGAAUUGAUUUUAA 69 AGGCGCUAGAAUUGAUUUUTT 225 AAAAUCAAUUCUAGCGCCUTT 381 AL-DP-7385 AAAGCAUCUAAUAGAACGCUAAA 70 AGCAUCUAAUAGAACGCUATT 226 UAGCGUUCUAUUAGAUGCUTT 382 AL-DP-7480 AACAAAGCGAUGAGCAAGCUAAA 71 CAAAGCGAUGAGCAAGCUATT 227 UAGCUUGCUCAUCGCUUUGTT 383 AL-DP-7528 AACCAUGGUUGUCUACAGGAAAA 72 CCAUGGUUGUCUACAGGAATT 228 UUCCUGUAGACAACCAUGGTT 384 AL-DP-7535 AAAGAAAGGCGCUAGAAUUGAAA 73 AGAAAGGCGCUAGAAUUGATT 229 UCAAUUCUAGCGCCUUUCUTT 385 AL-DP-7403 AAAACGCUACUACCACCAGUUAA 74 AACGCUACUACCACCAGUUTT 230 AACUGGUGGUAGUAGCGUUTT 386 AL-DP-7380 AAGCGCUAGAAUUGAUUUUAAAA 75 GCGCUAGAAUUGAUUUUAATT 231 UUAAAAUCAAUUCUAGCGCTT 387 AL-DP-7364 AAGCACGUGAUCAGUGUUGCAAA 76 GCACGUGAUCAGUGUUGCATT 232 UGCAACACUGAUCACGUGCTT 388 AL-DP-7469 AAGAUAGUGUCCCAGUACAAAAA 77 GAUAGUGUCCCAGUACAAATT 233 UUUGUACUGGGACACUAUCTT 389 AL-DP-7518 AAUUUGCGUGAAAGUGUUACAAA 78 UUUGCGUGAAAGUGUUACATT 234 UGUAACACUUUCACGCAAATT 390 AL-DP-7464 AAAGUAUGUGCUACUUUUUUGAA 79 AGUAUGUGCUACUUUUUUGTT 235 CAAAAAAGUAGCACAUACUTT 391 AL-DP-7560 AAGUAUGUCGUCUUCAUGUGUAA 80 GUAUGUCGUCUUCAUGUGUTT 236 ACACAUGAAGACGACAUACTT 392 AL-DP-7461 AAGGUAGUCAAGCCUAUUGCAAA 81 GGUAGUCAAGCCUAUUGCATT 237 UGCAAUAGGCUUGACUACCTT 393 AL-DP-7472 AAACGUAACCUUCAAGUAUGUAA 82 ACGUAACCUUCAAGUAUGUTT 238 ACAUACUUGAAGGUUACGUTT 394 AL-DP-7459 AAACCACGUAACCUUCAAGUAAA 83 ACCACGUAACCUUCAAGUATT 239 UACUUGAAGGUUACGUGGUTT 395 AL-DP-7381 AACAGUAAGCUGACCUGGAAAAA 84 CAGUAAGCUGACCUGGAAATT 240 UUUCCAGGUCAGCUUACUGTT 396 AL-DP-7515 AAAGUAGGUUUACAUUACUGAAA 85 AGUAGGUUUACAUUACUGATT 241 UCAGUAAUGUAAACCUACUTT 397 AL-DP-7517 AAAAUGGUAGUCAAGCCUAUUAA 86 AAUGGUAGUCAAGCCUAUUTT 242 AAUAGGCUUGACUACCAUUTT 398 AL-DP-7521 AAGCCUAUUGCAACAAAGUUAAA 87 GCCUAUUGCAACAAAGUUATT 243 UAACUUUGUUGCAAUAGGCTT 399 AL-DP-7530 AAGACCACGUAACCUUCAAGUAA 88 GACCACGUAACCUUCAAGUTT 244 ACUUGAAGGUUACGUGGUCTT 400 AL-DP-7388 AAUGUUAAACGUUACUUUCAUAA 89 UGUUAAACGUUACUUUCAUTT 245 AUGAAAGUAACGUUUAACATT 401 AL-DP-7451 AAAUUGAAGCUAGCCGAAUGAAA 90 AUUGAAGCUAGCCGAAUGATT 246 UCAUUCGGCUAGCUUCAAUTT 402 AL-DP-7484 AAAUAUAACGAGGGAUAAAUUAA 91 AUAUAACGAGGGAUAAAUUTT 247 AAUUUAUCCCUCGUUAUAUTT 403 AL-DP-7376 AAACUUACUUAUUACCUAGAUAA 92 ACUUACUUAUUACCUAGAUTT 248 AUCUAGGUAAUAAGUAAGUTT 404 AL-DP-7500 AAUGUUCUCGUUGUUGUUUUAAA 93 UGUUCUCGUUGUUGUUUUATT 249 UAAAACAACAACGAGAACATT 405 AL-DP-7488 AAUUUUAAGGGUUAAAUCACUAA 94 UUUUAAGGGUUAAAUCACUTT 250 AGUGAUUUAACCCUUAAAATT 406 AL-DP-7541 AAAGUAACAGCACAACAAAϋUAA 95 AGUAACAGCACAACAAAUUTT 251 AAUUUGUUGUGCUGUUACUTT 407 AL-DP-7550

AACAACUCCUGCUCUGAGAUAAA 96 CAACUCCUGCUCUGAGAUATT 252 UAUCUCAGAGCAGGAGUUGTT 408 AL-DP-7776 AAGAUGUGACUUACUUAACAGAA 97 GAUGUGACUUACUUAACAGTT 253 CUGUUAAGUAAGUCACAUCTT 409 AL-DP-7777 AACAUUAUCGUAAUGGAGAAUAA 98 CAUUAUCGUAAUGGAGAAUTT 254 AUUCUCCAUUACGAUAAUGTT 410 AL-DP-7510 AACCAUUUUAUCGAGGCACGUAA 99 CCAUUUUAUCGAGGCACGUTT 255 ACGUGCCUCGAUAAAAUGGTT 411 AL-DP-7507 AAAGUAGCCAAUCCUCUUUCUAA 100 AGUAGCCAAUCCUCUUUCUTT 256 AGAAAGAGGAUUGGCUACUTT 412 AL-DP-7479 AAUAAUAGAACGCUACUACCAAA 101 UAAUAGAACGCUACUACCATT 257 UGGUAGUAGCGUUCUAUUATT 413 AL-DP-7542 AACUUCGUGCAACUGUAGUCAAA 102 CUUCGUGCAACUGUAGUCATT 258 UGACUACAGUUGCACGAAGTT 414 AL-DP-7494 AAUCAUAUGGUGACCAAUGAAAA 103 UCAUAUGGUGACCAAUGAATT 259 UUCAUUGGUCACCAUAUGATT 415 AL-DP-7531 AAUUAAUCCGUGUUAUUGGAAAA 104 UUAAUCCGUGUUAUUGGAATT 260 UUCCAAUAACACGGAUUAATT 416 AL-DP-7373 AAAUACGCUACCUUGAUGAAAAA 105 AUACGCUACCUUGAUGAAATT 261 UUUCAUCAAGGUAGCGUAUTT 417 AL-DP-7508 AAAGCUAGCCGAAUGAAGCGAAA 106 AGCUAGCCGAAUGAAGCGATT 262 UCGCUUCAUUCGGCUAGCUTT 418 AL-DP-7487 AAUACAUACGCUACCUUGAUGAA 107 UACAUACGCUACCUUGAUGTT 263 CAUCAAGGUAGCGUAUGUATT 419 AL-DP-7375 AAAAUAUAACGAGGGAUAAAUAA 108 AAUAUAACGAGGGAUAAAUTT 264 AUUUAUCCCUCGUUAUAUUTT 420 AL-DP-7462 AAGUUCUCGUUGUUGUUUUAAAA 109 GUUCUCGUUGUUGUUUUAATT 265 UUAAAACAACAACGAGAACTT 421 AL-DP-7513 AAUGAUUGACUGAUUGUUUUAAA 110 UGAUUGACUGAUUGUUUUATT 266 UAAAACAAUCAGUCAAUCATT 422 AL-DP-7455 AAUUUAAUCCGUGUUAUUGGAAA 111 UUUAAUCCGUGUUAUUGGATT 267 UCCAAUAACACGGAUUAAATT 423 AL-DP-7374 AAUGUCCGGCUAGAGAUGAUCAA 112 UGUCCGGCUAGAGAUGAUCTT 268 GAUCAUCUCUAGCCGGACATT 424 AL-DP-7475 AAGCUAGCCGAAUGAAGCGAGAA 113 GCUAGCCGAAUGAAGCGAGTT 269 CUCGCUUCAUUCGGCUAGCTT 425 AL-DP-7369 AAGUACAUACGCUACCUUGAUAA 114 GUACAUACGCUACCUUGAUTT 270 AUCAAGGUAGCGUAUGUACTT 426 AL-DP-7466 AAUCGAGCUUUAUAAGAUUAAAA 115 UCGAGCUUUAUAAGAUUAATT 271 UUAAUCUUAUAAAGCUCGATT 427 AL-DP-7491 AACUACCACCAGUUAACUGAGAA 116 CUACCACCAGUUAACUGAGTT 272 CUCAGUUAACUGGUGGUAGTT 428 AL-DP-7482 AAUUUUAUCGAGGCACGUGAUAA 117 UUUUAUCGAGGCACGUGAUTT 273 AUCACGUGCCUCGAUAAAATT 429 AL-DP-7398 AAAUUUUAUCGAGGCACGUGAAA 118 AUUUUAUCGAGGCACGUGATT 274 UCACGUGCCUCGAUAAAAUTT 430 AL-DP-7471 AAAACGUUACUUUCAUGUACUAA 119 AACGUUACUUUCAUGUACUTT 275 AGUACAUGAAAGUAACGUUTT 431 AL-DP-7383 AAAGAAUUCGCAUGUACAGUGAA 120 AGAAUUCGCAUGUACAGUGTT 276 CACUGUACAUGCGAAUUCUTT 432 AL-DP-7367 AAUCACGUAUGCCAAAGGAUUAA 121 UCACGUAUGCCAAAGGAUUTT 277 AAUCCUUUGGCAUACGUGATT 433 AL-DP-7386 AACUUUAAUCCGUGUUAUUGGAA 122 CUUUAAUCCGUGUUAUUGGTT 278 CCAAUAACACGGAUUAAAGTT 434 AL-DP-7525 AAACAUACGCUACCUUGAUGAAA 123 ACAUACGCUACCUUGAUGATT 279 UCAUCAAGGUAGCGUAUGUTT 435 AL-DP-7486 AAUCCUAGUCUUCUGUGUAUGAA 124 UCCUAGUCUUCUGUGUAUGTT 280 CAUACACAGAAGACUAGGATT 436 AL-DP-7539 AACAUUUUAUCGAGGCACGUGAA 125 CAUUUUAUCGAGGCACGUGTT 281 CACGUGCCUCGAUAAAAUGTT 437 AL-DP-7483 AAUUAACUGAUUACUGUAGAUAA 126 UUAACUGAUUACUGUAGAUTT 282 AUCUACAGUAAUCAGUUAATT 438 AL-DP-7503 AACCUUCGUGCAACUGUAGUCAA 127 CCUUCGUGCAACUGUAGUCTT 283 GACUACAGUUGCACGAAGGTT 439 AL-DP-7537 AAUGUUAGGGUACAUACGCUAAA 128 UGUUAGGGUACAUACGCUATT 284 UAGCGUAUGUACCCUAACATT 440 AL-DP-7396 AACACUGUUAGGGUACAUACGAA 129 CACUGUUAGGGUACAUACGTT 285 CGUAUGUACCCUAACAGUGTT 441 AL-DP-7404

AAUGUGGCACUUUUCACCAUAAA 130 UGUGGCACUUUUCACCAUATT 286 UAUGGUGAAAAGUGCCACATT 442 AL-DP-7543 AAAUCACGUAUGCCAMGGAUAA 131 AUCACGUAUGCCAAAGGAUTT 287 AUCCUUUGGCAUACGUGAUTT 443 AL-DP-7379 AAAGGGUACAUACGCUACCUUAA 132 AGGGUACAUACGCUACCUUTT 288 AAGGUAGCGUAUGUACCCUTT 444 AL-DP-7502 AAACUAAGGUGAGACAUUGAUAA 133 ACUAAGGUGAGACAUUGAUTT 289 AUCAAUGUCUCACCUUAGUTT 445 AL-DP-7519 AAUGUCACCUAACGUGGAAUGAA 134 UGUCACCUAACGUGGAAUGTT 290 CAUUCCACGUUAGGUGACATT 446 AL-DP-7506 AAAUAGAAUUCGCAUGUACAGAA 135 AUAGAAUUCGCAUGUACAGTT 291 CUGUACAUGCGAAUUCUAUTT 447 AL-DP-7457 AAUUCGUGACUUGGGAGACUCAA 136 UUCGUGACUUGGGAGACUCTT 292 GAGUCUCCCAAGUCACGAATT 448 AL-DP-7468 AAUAGAAUUCGCAUGUACAGUAA 137 UAGAAUUCGCAUGUACAGUTT 293 ACUGUACAUGCGAAUUCUATT 449 AL-DP-7368 AAUGAGGGCGUUUUAUAUAAUAA 138 UGAGGGCGUUUUAUAUAAUTT 294 AUUAUAUAAAACGCCCUCATT 450 AL-DP-7402 AAACACUGUUAGGGUACAUACAA 139 ACACUGUUAGGGUACAUACTT 295 GUAUGUACCCUAACAGUGUTT 451 AL-DP-7481 AAUUCUCGUUGUUGUUUUAAGAA 140 UUCUCGUUGUUGUUUUAAGTT 296 CUUAAAACAACAACGAGAATT 452 AL-DP-7465 AACUGUUAGGGUACAUACGCUAA 141 CUGUUAGGGUACAUACGCUTT 297 AGCGUAUGUACCCUAACAGTT 453 AL-DP-7496 AAUUUAACUAAGGUGAGACAUAA 142 UUUAACUAAGGUGAGACAUTT 298 AUGUCUCACCUUAGUUAAATT 454 AL-DP-7549 AAUAACAGUCGAAAUCUAGUGAA 143 UAACAGUCGAAAUCUAGUGTT 299 CACUAGAUUUCGACUGUUATT 455 AL-DP-7394

4^ AAUGUGGUCUAAAUACAAUGCAA 144 UGUGGUCUAAAUACAAUGCTT 300 GCAUUGUAUUUAGACCACATT 456 AL-DP-7477 oo AAUCACCUAACGUGGAAUGUGAA 145 UCACCUAACGUGGAAUGUGTT 301 CACAUUCCACGUUAGGUGATT 457 AL-DP-7516 AAUUCUUAGUAUAUGAAAGGAAA 146 UUCUUAGUAUAUGAAAGGATT 302 UCCUUUCAUAUACUAAGAATT 458 AL-DP-7556 AAAAUAGAACGCUACUACCACAA 147 AAUAGAACGCUACUACCACTT 303 GUGGUAGUAGCGUUCUAUUTT 459 AL-DP-7387 AACUGAGGGCGUUUUAUAUAAAA 148 CUGAGGGCGUUUUAUAUAATT 304 UUAUAUAAAACGCCCUCAGTT 460 AL-DP-7524 AAAAAUCGUUCAUUCAUUUACAA 149 AAAUCGUUCAUUCAUUUACTT 305 GUAAAUGAAUGAACGAUUUTT 461 AL-DP-7378 AAUUUUCGUGACUUGGGAGACAA 150 UUUUCGUGACUUGGGAGACTT 306 GUCUCCCAAGUCACGAAAATT 462 AL-DP-7389 AAUCGUGCAACUGUAGUCAUCAA 151 UCGUGCAACUGUAGUCAUCTT 307 GAUGACUACAGUUGCACGATT 463 AL-DP-7384 AAUCAUACAGUAAGCUGACCUAA 152 UCAUACAGUAAGCUGACCUTT 308 AGGUCAGCUUACUGUAUGATT 464 AL-DP-7497 AACGUGCAACUGUAGUCAUCUAA 153 CGUGCAACUGUAGUCAUCUTT 309 AGAUGACUACAGUUGCACGTT 465 AL-DP-7559 AAUGCCAUUAGAAGGGUCUACAA 154 UGCCAUUAGAAGGGUCUACTT 310 GUAGACCCUUCUAAUGGCATT 466 AL-DP-7520 AAUUACAAUAACUGUAUACUGAA 155 UUACAAUAACUGUAUACUGTT 311 CAGUAUACAGUUAUUGUAATT 467 AL-DP-7505 AAUUCGCAUGUACAGUGAACGAA 156 UUCGCAUGUACAGUGAACGTT 312 CGUUCACUGUACAUGCGAATT 468 AL-DP-7460

dsRNA synthesis

Source of reaRents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500A, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2 -0-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2'-O- methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85 - 90⁰C for 3 minutes and cooled to room temperature over a period of 3 - 4 hours. The annealed RNA solution was stored at -20 ⁰C until use. For the synthesis of 3 '-cholesterol-conjugated siRNAs (herein referred to as -Chol-3'), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-l ,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice- cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]- amino} -propionic acid ethyl ester AB

AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0⁰C. It was then followed by the addition of Diethyl-azabutane-l,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50 % EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylrnethyl-arnino]-propionic acid ethyl ester AC

AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]- amino} -propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamϊde at 0⁰C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(l,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,l 1,12,13,14,15,16,17- tetradecahydro- 1 H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]- hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

AD

The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]- propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0⁰C on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).

l-{6-[17-(l,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4₅7,8,9,10,l 1,12,13,14,15,16,17- tetradecahydro- 1 H-cyclopenta[a] phenanthren-3-yloxycarbonylamino]-hexanoyI }-4-oxo- pyrrolidine-3-carboxylic acid ethyl ester AE

AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0⁰C on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5°C during the addition. The stirring was continued for 30 mins at 0⁰C and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH₂PO₄-H₂O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0⁰C and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%). [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-l-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5- dimethyl-hexyl)- 10, 13-dimethyl-2,3,4,7,8,9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro- 1 H- cyclopenta[a]phenanthren-3-yl ester AF

AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b- ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3 x 40 mL). The ^• combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-l-yl}-6- oxo-hexyl)-carbamic acid 17-(l,5-dimethyl-hexyl)-10,13-dimethyl-

2,3,4,7,8,9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro- 1 H-cyclopenta[a]phenanthren-3-yl ester AG

AG

Diol AF ( 1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2 x 5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with IM aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf= 0.5 in 5% MeOHVCHCl₃) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-l-{6-[l 7^(1,5- dimethyl-hexyl)- 10, 13-dimethyl 2,3,4,7,8,9, 10, 1 1 , 12, 13, 14, 15, 16, 17-tetradecahydro-l H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrroIidin-3-yl) ester AH

AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40⁰C overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt%, 30 mL) and water (2 X 20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.

Cholesterol derivatised CPG AI

AI Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5'-12-dodecanoic acid bisdecylamide group (herein referred to as "5'-C32-") or a 5'-cholesteryl derivative group (herein referred to as "5'-Chol-") was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5'-end of the nucleic acid oligomer.

dsRNA expression vectors

In another aspect of the invention, E6AP specific dsRNA molecules that modulate E6AP gene 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, US 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 plasm ids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Mu∑yczka, 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. 150:4104-41 15; U.S. Patent No. 4,868,116; U.S. Patent 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 83:251 1-2515)).

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-Dl - thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit- TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single E6AP gene or multiple E6AP genes 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 E6AP 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. Patent 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.

E6AP siRNA screening in HCT-116 cells

HCT-1 16 cells were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) (Braunschweig, Germany, cat. No. ACC 581) and cultured in McCoys (Biochrom AG, Berlin, Germany, cat. No. FlOl 5) supplemented to contain 10% fetal calf serum (FCS), Penicillin 100 U/ml, Streptomycin 100 μg/ml and 2mM L-Glutamin at 37°C in an atmosphere with 5% CO₂ in a humidified incubator.

For transfection with siRNA, HCT-116 cells were seeded at a density of 2.0 x 10⁴ cells/well in 96-welI plates and transfected directly. Transfection of siRNA (3OnM and 3nM for single dose screen) was carried out with lipofectamine 2000 (Invitrogen) as described by the manufacturer. 24 hours after transfection HCT-116 cells were lysed and E6AP mRNA expression levels were quantified with the Quantigene Explore Kit (Panomics, Inc. (Fremont, CAXformerly Genospectra, Inc.)) according to the standard protocol. E6AP mRNA levels were normalized to GAP-DH mRNA. For each siRNA four individual datapoints were collected. siRNA duplexes unrelated to E6AP gene were used as control. The activity of a given E6AP specific siRNA duplex was expressed as percent E6AP mRNA concentration in treated cells relative to E6AP mRNA concentration in cells treated with the control siRNA duplex.

Table 2 below provides the results. Many active siRNA molecules that target the E6AP gene were identified.

Table 2 Activity of dsRNA targeting E6AP

mean Standard mean Standard duplex activity deviation activity deviation name at 3OnM at 3OnM at 3nM at 3nM

AL-DP-7545 9.35 3.36 14.04 3.82

AL-DP-7558 12.36 3.07 18.49 4.36

AL-DP-7548 12.55 5.85 18.92 4.72

AL-DP-7509 14.42 3.99 19.39 2.71

AL-DP-7492 11.25 2.53 19.61 7.89

AL-DP-7554 14.16 4.56 19.83 5.15

AL-DP-7557 16.00 6.50 19.97 7.04

AL-DP-7476 14.15 7.05 20.21 6.19

AL-DP-7514 24.01 12.46 20.54 6.13

AL-DP-7540 15.61 5.14 21.78 3.95

AL-DP-7397 13.05 5.68 22.03 11.42

AL-DP-7526 15.87 5.65 22.28 5.61

AL-DP-7473 17.22 6.09 22.65 6.64

AL-DP-7478 16.76 9.85 22.69 6.84

AL-DP-7553 23.50 5.15 23.19 3.34

AL-DP-7395 17.30 7.48 23.22 8.88

AL-DP-7522 26.16 10.71 23.51 8.18

AL-DP-7499 14.21 6.15 23.81 12.13

AL-DP-7527 24.11 5.05 23.98 8.89

AL-DP-7544 17.23 5.90 24.03 2.56

AL-DP-7489 23.56 10.21 24.54 7.57

AL-DP-7365 14.54 7.13 24.56 8.90

AL-DP-7390 16.44 6.37 24.74 6.73

AL-DP-7458 14.25 5.11 25.28 6.56

AL-DP-7532 21.47 4.18 25.48 6.40 AL-DP-7546 17.,66 4.28 25.91 7.73

AL-DP-7512 27. 88 6.58 26 .22 5 .07

AL-DP-7470 28. 22 6.50 26 .31 8 .12

AL-DP-7406 20. .23 6.01 26 .62 6. .35

AL-DP-7382 17. 82 7.24 26 .93 9. .30

AL-DP-7547 24. 63 6.66 28 .80 10. .23

AL-DP-7490 25. 94 9.32 28 .95 10. .29

AL-DP-7493 12. 53 5.36 29 .56 12. .54

AL-DP-7529 17. 61 8.36 29 .59 10. ,53

AL-DP-7400 21. 03 14.86 30 .04 12. ,58

AL-DP-7391 26. 74 12.00 30 .06 8. .07

AL-DP-7393 22. 40 9.77 30 .69 8. .51

AL-DP-7511 26. 50 6.02 30 .88 6. ,43

AL-DP-7454 25. 16 14.85 31 .09 8. ,75

AL-DP-7450 20. 09 8.43 32 .10 8. 57

AL-DP-7533 26. 93 5.86 33 .91 5. 52

AL-DP-7485 27. 45 4.36 34 .12 10. 28

AL-DP-7495 28. 51 13.42 34, .45 11. 20

AL-DP-7456 16. 82 5.62 34, .54 10. 30

AL-DP-7538 29. 04 5.12 34, .71 6. 42

AL-DP-7377 22. 98 8.19 35, .31 12. 53

AL-DP-7405 21. 93 10.30 35, .66 15. 22

AL-DP-7392 23. 83 8.93 36. .14 6. 31

AL-DP-7453 25. 78 12.10 36. .98 5. 22

AL-DP-7366 19. 60 7.30 37. .20 13. 88

AL-DP-7534 26. 35 5.24 37. .69 8. 49

AL-DP-7401 28. 74 9.10 37. .75 7. 70

AL-DP-7523 33. 88 6.85 39. .81 9. 45

AL-DP-7555 29. 13 8.87 40. .35 6. 23

AL-DP-7536 32. 33 3.49 41. ,08 8. 00

AL-DP-7371 25. 49 9.83 42. ,19 16. 08

AL-DP-7372 21. 83 12.03 42. .87 17. 78

AL-DP-7370 24. 51 12.64 43. 75 14. 09

AL-DP-7474 32. 57 13.13 44. 40 7. 78

AL-DP-7452 30. 12 12.02 46. 66 9. 19

AL-DP-7498 32. 38 11.81 54. 11 12. 74

AL-DP-7504 15.04 6.39 19. 69 5. 67

AL-DP-7467 19.81 6.42 21. 66 8. 12

AL-DP-7463 26.63 8.84 21. 73 8. 80

AL-DP-7399 15.62 8.32 22. 98 7. 65

AL-DP-7501 17.32 5.24 23. 45 7. 44

AL-DP-7385 17.60 5.11 28. 00 11. 84

AL-DP-7480 21.89 8.21 29. 42 8. 64

AL-DP-7528 26.47 2.94 30. 76 10. 87

AL-DP-7535 26.65 3.13 31. 77 4. 34 AL-DP-7403 24.10 6.21 38.79 14.41

AL-DP-7380 29.84 7.65 40.42 5.72

AL-DP-7469 17.18 7.41 21.13 6.29

AL-DP-7518 15.71 6.00 21.89 6.68

AL-DP-7464 29.18 12.30 22.13 8.99

AL-DP-7560 17.33 4.85 24.84 5.80

AL-DP-7461 30.55 8.26 25.62 9.48

AL-DP-7472 25.17 11.50 26.31 8.61

AL-DP-7459 29.60 7.71 27.27 9.68

AL-DP-7381 17.29 6.63 27.31 7.42

AL-DP-7515 32.18 10.22 29.76 6.01

AL-DP-7517 29.75 6.99 29.87 5.69

AL-DP-7521 28.60 8.06 31.68 5.72

AL-DP-7530 31.09 8.09 31.94 3.36

AL-DP-7388 22.81 3.80 32.28 6.23

AL-DP-7451 22.66 8.92 32.45 8.26

AL-DP-7484 26.77 13.00 32.84 6.95

AL-DP-7376 34.18 14.11 39.93 8.41

AL-DP-7500 32.69 8.47 41.55 13.32

AL-DP-7776 17.91 4.95 21.77 5.04

AL-DP-7777 21.10 -7.60

AL-DP-7510 34.70 5.83

AL-DP-7507 35.11 6.78

AL-DP-7479 35.29 13.76

AL-DP-7542 36.32 5.00

AL-DP-7494 38.34 12.68

AL-DP-7531 38.58 14.26

AL-DP-7373 39.04 16.08

AL-DP-7508 39.95 12.87

AL-DP-7487 40.48 15.20

AL-DP-7375 41.19 15.06

AL-DP-7462 41.61 17.23

AL-DP-7513 41.69 9.15

AL-DP-7455 43.35 12.72

AL-DP-7374 43.37 12.26

AL-DP-7475 43.68 11.45

AL-DP-7369 43.99 15.44

AL-DP-7466 44.27 15.53

AL-DP-7491 45.06 10.32

AL-DP-7482 45.06 12.37

AL-DP-7398 45.79 9.50

AL-DP-7471 46.11 13.53

AL-DP-7383 46.87 20.08 AL-DP-7367 46.96 16.88

AL-DP-7386 47. 46 10.01

AL-DP-7525 49. 60 14.11

AL-DP-7486 49. 64 8.95

AL-DP-7539 49. 97 12.73

AL-DP-7483 49. 97 12.50

AL-DP-7503 51. 28 7.08

AL-DP-7537 53. 19 7.75

AL-DP-7396 54. 11 13.02

AL-DP-7404 54. 96 17.72

AL-DP-7543 55. 48 9.23

AL-DP-7379 55. 82 18.47

AL-DP-7502 56. 15 16.52

AL-DP-7519 56. 15 13.30

AL-DP-7506 57. 24 21.04

AL-DP-7457 57. 30 15.19

AL-DP-7468 57. 83 15.40

AL-DP-7368 59. 38 22.50

AL-DP-7402 59. 57 13.42

AL-DP-7481 60. 17 14.54

AL-DP-7465 61. 44 28.49

AL-DP-7496 61. 65 17.78

AL-DP-7549 61. 90 12.36

AL-DP-7394 61. 94 17.08

AL-DP-7477 63. 20 14.74

AL-DP-7516 67. 72 19.24

AL-DP-7556 69. 49 13.89

AL-DP-7387 72. 14 16.20

AL-DP-7524 72. 52 19.76

AL-DP-7378 73. 44 19.20

AL-DP-7389 73. 74 23.83

AL-DP-7384 76. 45 21.99

AL-DP-7497 77. 66 22.60

AL-DP-7559 78. 86 16.61

AL-DP-7520 85. 45 14.83

AL-DP-7505 86. 86 39.07

AL-DP-7460 100. 95 22.69

Testing of Chemically Modified dsRNA Targeting E6AP

Chemically modified dsRNA were tested to identify their relative abilities to reduce the expression level of mRNA encoding E6AP in a cell. The assay conditions described above for HCT-116 cells were employed. The activity of a given E6AP specific siRNA duplex was expressed as percent E6AP mRNA concentration in treated cells relative to E6AP mRNA concentration in cells treated with the control siRNA duplex.

1. Chemically Modified dsRNA

Table 3 sets forth dsRNA compositions of the invention. In this table the unmodified sequence is followed by the same sequence containing one or more nucleotide modifications.

Table 3

Upper case letters: unmodified ribonucleotide (except for T which is an unmodified deoxyribonucleotide)

Lower case letters: ribonucloetide bearing 2'-O-methyl substituent on ribose moiety s : Indicates position of phosphorothioate internucleoside linkage chol : cholesterol moiety conjugated to 3' ribonucleotide.

'duplex name'means the name of the composition formed by specific hybridization of the indicated sense strand and the indicated antisense strand.

sense strand antisense strand SEQ SEQ ID ID duplex sequence (5' -3') NO: sequence (5 '-3') NO: name

AUACGAUGAAUCUACAAAATT 469 UUUUGUAGAUUCAUCGUAUTT 644 AL-DP-7545 AUACGAUGAAUCUACAAAATST 470 UUUUGUAGAUUCAUCGUAUTST 645 ND-8763 AuAcGAuGAAucuAcAAAATsT 471 UUUUGuAGAUUcAUCGuAUTsT 646 ND-8782 AuAcGAuGAAucuAcAAAATsT 472 uuuuGuAGAuUcAUCGuAUTsT 647 ND-8801 AUACGAUGAAUCUACAAAATTChOl 473 UUUUGUAGAUUCAUCGUAUTST 648 ND-8820

AuAcGAuGAAucuAcAAAATTcho1 474 UUUUGuAGAUUcAUCGuAUTsT 649 ND-8845

AuAcGAuGAAucuAcAAAATTchol 475 uuuuGuAGAuUcAUCGuAUTsT 650 ND-8870

UGACUACAUUCUCAAUAAATT 476 UUUAUUGAGAAUGUAGUCATT 651 AL-DP-7558

UGACUACAUUCUCAAUAAATST 477 UUUAUUGAGAAUGUAGUCATST 652 ND-8764 uGAcuAcAuucucAAuAAATsT 478 UUuAUUGAGAAUGuAGUcATsT 653 ND-8783 uGAcuAcAuucucAAuAAATsT 479 uuuAuuGAGAAuGuAGUcATsT 654 ND-8802

UGACUACAUUCUCAAUAAATTChol 480 UUUAUUGAGAAUGUAGUCATST 655 ND-8821 uGAcuAcAuucucAAuAAATTchol 481 UUuAUUGAGAAUGuAGUcATsT 656 ND-8846 uGAcuAcAuucucAAuAAATTchol 482 uuuAuuGAGAAuGuAGUcATsT 657 ND-8871

AGCCUGCACGAAUGAGUUUTT 483 AAACUCAUUCGUGCAGGCUTT 658 AL-DP-7548

AGCCUGCACGAAUGAGUUUTST 484 AAACUCAUUCGUGCAGGCUTST 659 ND-8765

AGccuGcAcGAAuGAGuuuTsT 485 AAACUcAUUCGUGcAGGCUTsT 660 ND-8784

AGccuGcAcGAAuGAGuuuTsT 486 AAACUcAuUCGuGcAGGCUTsT 661 ND-8803

AGCCUGCACGAAUGAGUUUTTChol 487 AAACUCAUUCGUGCAGGCUTST 662 ND-8822

AGccuGcAcGAAuGAGuuuTTchol 488 AAACUcAUUCGUGcAGGCUTsT 663 ND-8847

AGccuGcAcGAAuGAGuuuTTchol 489 AAACUcAuUCGuGcAGGCUTsT 664 ND-8872

GGAUUGUCGAAAACCACUUTT 490 AAGUGGUUUUCGACAAUCCTT 665 AL-DP-7509

GGAUUGUCGAAAACCACUUTST 491 AAGUGGUUUUCGACAAUCCTST 666 ND-8766

GGAuuGucGAAAAccAcuuTsT 492 AAGUGGUUUUCGACAAUCCTST 667 ND-8785

GGAuuGucGAAAAccAcuuTsT 493 AAGuGGuUuUCGAcAAUCCTsT 668 ND-8804

GGAUUGUCGAAAACCACUUTTChol 494 AAGUGGUUUUCGACAAUCCTST 669 ND-8823

GGAuuGucGAAAAccAcuuTTchol 495 AAGUGGUUUUCGACAAUCCTST 670 ND-8848

GGAuuGucGAAAAccAcuuTTchol 496 AAGuGGuUuUCGAcAAUCCTsT 671 ND-8873

CUCUCGAGAUCCUAAUUAUTT 497 AUAAUUAGGAUCUCGAGAGTT 672 AL-DP-7492

CUCUCGAGAUCCUAAUUAUTST 498 AUAAUUAGGAUCUCGAGAGTST 673 ND-8767 cucucGAGAuccuAAuuAuTsT 499 AuAAUuAGGAUCUCGAGAGTsT 674 ND-8786 cucucGAGAuccuAAuuAuTsT 500 AuAAuUAGGAUCUCGAGAGTsT 675 ND-8805

CUCUCGAGAUCCUAAUUAUTTChOl 501 AUAAUUAGGAUCUCGAGAGTST 676 ND-8824 cucucGAGAuccuAAuuAuTTchol 502 AuAAUuAGGAUCUCGAGAGTsT 677 ND-8849 cucucGAGAuccuAAuuAuTTchol 503 AuAAuUAGGAUCUCGAGAGTsT 678 ND-8874

AUGUGACUUACUUAACAGATT 504 UCUGUUAAGUAAGUCACAUTT 679 AL-DP-7554

AUGUGACUUACUUAACAGATST 505 UCUGUUAAGUAAGUCACAUTST 680 ND-8768

AuGuGAcuuAcuuAAcAGATsT 506 UCUGUuAAGuAAGUcAcAUTsT 681 ND-8787

AuGuGAcuuAcuuAAcAGATsT 507 UCuGuuAAGuAAGUcAcAUTsT 682 ND-8806

AUGUGACUUACUUAACAGATTChol 508 UCUGUUAAGUAAGUCACAUTST 683 ND-8825

AuGuGAcuuAcuuAAcAGATTchol 509 UCUGUuAAGuAAGUcAcAUTsT 684 ND-8850

AuGuGAcuuAcuuAAcAGATTchol 510 UCuGuuAAGuAAGUcAcAUTsT 685 ND-8875

GUAUACUCUCGAGAUCCUATT 511 UAGGAUCUCGAGAGUAUACTT 686 AL-DP-7557 GUAUACUCUCGAGAUCCUATST 512 UAGGAUCUCGAGAGUAUACTST 687 ND-8769

GuAuAcucucGAGAuccuATsT 513 uAGGAUCUCGAGAGuAuACTST 688 ND-8788

GuAuAcucucGAGAuccuATsT 514 uAGGAUCUCGAGAGuAuACTsT 689 ND-8788

GUAUACUCUCGAGAUCCUATTChol 515 UAGGAUCUCGAGAGUAUACTST 690 ND-8826

GuAuAcucucGAGAuccuATTchol 516 UAGGAUCUCGAGAGuAuACTsT 691 ND-8851

GuAuAcucucGAGAuccuATTchol 517 UAGGAUCUCGAGAGuAuACTsT 692 ND-8851

AGGUUACCUACAUCUCAUATT 518 UAUGAGAUGUAGGUAACCUTT 693 AL-DP-7476

AGGUUACCUACAUCUCAUATST 519 UAUGAGAUGUAGGUAACCUTST 694 ND-8770

AGGuuAccuAcAucucAuATsT 520 uAUGAGAUGuAGGuAACCUTsT 695 ND-8789

AGGuuAccuAcAucucAuATsT 521 uAuGAGAuGuAGGuAACCUTsT 696 ND-8808

AGGUUACCUACAUCUCAUATTChol 522 UAUGAGAUGUAGGUAACCUTST 697 ND-8827

AGGuuAccuAcAucucAuATTchol 523 UAUGAGAUGuAGGuAACCUTsT 698 ND-8852

AGGuuAccuAcAucucAuATTchol 524 UAuGAGAuGuAGGuAACCUTsT 699 ND-8877

AGUACUUAUUCAGACCAGATT 525 UCUGGUCUGAAUAAGUACUTT 700 AL-DP-7514

AGUACUUAUUCAGACCAGATST 526 UCUGGUCUGAAUAAGUACUTST 701 ND-8771

AGuAcuuAuucAGAccAGATsT 527 UCUGGUCUGAAUAAGUACUTST 702 ND-8790

AGuAcuuAuucAGAccAGATsT 528 UCuGGUCuGAAuAAGuACUTsT 703 ND-8809

AGUACUUAUUCAGACCAGATTChol 529 UCUGGUCUGAAUAAGUACUTST 704 ND-8828

AGuAcuuAuucAGAccAGATTchol 530 UCUGGUCUGAAUAAGUACUTsT 705 ND-8853

AGuAcuuAuucAGAccAGATTchol 531 UCuGGUCuGAAuAAGuACUTsT 706 ND-8878

AUCCUAAUUAUCUGAAUUUTT 532 AAAUUCAGAUAAUUAGGAUTT 707 AL-DP-7540

AUCCUAAUUAUCUGAAUUUTST 533 AAAUUCAGAUAAUUAGGAUTsT 708 ND-8772

AuccuAAuuAucuGAAuuuTsT 534 AAAUUcAGAuAAUuAGGAUTsT 709 ND-8791

AuccuAAuuAucuGAAuuuTsT 535 AAAuUcAGAuAAuUAGGAUTsT 710 ND-8810

AUCCUAAUUAUCUGAAUUUTTCho1 536 AAAUUCAGAUAAUUAGGAUTST 711 ND-8829

AuccuAAuuAucuGAAuuuTTchol 537 AAAUUcAGAuAAUuAGGAUTsT 712 ND-8854

AuccuAAuuAucuGAAuuuTTchol 538 AAAuUcAGAuAAuUAGGAUTsT 713 ND-8879

AAGGAUAGGUGAUAGCUCATT 539 UGAGCUAUCACCUAUCCUUTT 714 AL-DP-7397

AAGGAUAGGUGAUAGCUCATST 540 UGAGCUAUCACCUAUCCUUTST 715 ND-8731

AAGGAuAGGuGAuAGcucATsT 541 UGAGCuAUcACCuAUCCUUTsT 716 ND-8743

AAGGAuAGGuGAuAGcucATsT 542 uGAGCuAUcACCuAUCCuuTsT 717 ND-8754

AAGGAUAGGUGAUAGCUCATTChol 543 UGAGCUAUCACCUAUCCUUTST 718 ND-8839

AAGGAuAGGuGAuAGcucATTchol 544 UGAGCuAUcACCuAUCCUUTsT 719 ND-8864

AAGGAuAGGuGAuAGcucATTchol 545 UGAGCuAUcACCuAUCCuuTsT 720 ND-8889

GGAAGCCGGAAUCUAGAUUTT 546 AAUCUAGAUUCCGGCUUCCTT 721 AL-DP-7526

GGAAGCCGGAAUCUAGAUUTST 547 AAUCUAGAUUCCGGCUUCCTST 722 ND-8773

GGAAGccGGAAucuAGAuuTsT 548 AAUCuAGAUUCCGGCUUCCTsT 723 ND-8792

GGAAGccGGAAucuAGAuuTST 549 AAUCuAGAuUCCGGCuUCCTsT 724 ND-8811

GGAAGCCGGAAUCUAGAUUTTChol 550 AAUCUAGAUUCCGGCUUCCTST 725 ND-8830 GGAAGccGGAAucuAGAuuTTchol 551 AAUCuAGAUUCCGGCUUCCTsT 726 ND-8855

GGAAGccGGAAucuAGAuuTTchol 552 AAUCuAGAuUCCGGCuUCCTsT 727 ND-8880

UGCUUCGAAGUGCUUGAAATT 553 UUUCAAGCACUUCGAAGCATT 728 AL-DP-7473

UGCUUCGAAGUGCUUGAAATST 554 UUUCAAGCACUUCGAAGCATST 729 ND-8774 uGcuucGAAGuGcuuGAAATsT 555 UUUcAAGcACUUCGAAGcATsT 730 ND-8793 uGcuucGAAGuGcuuGAAATsT 556 uuUcAAGcACuUCGAAGcATsT 731 ND-8812

UGCUUCGAAGUGCUUGAAATTChol 557 UUUCAAGCACUUCGAAGCATST 732 ND-8831 uGcuucGAAGuGcuuGAAATTchol 558 UUUcAAGcACUUCGAAGcATsT 733 ND-8856 uGcuucGAAGuGcuuGAAATTchol 559 uuUcAAGcACuUCGAAGcATsT 734 ND-8881

UGGAUUGUCGAAAACCACUTT 560 AGUGGUUUUCGACAAUCCATT 735 AL-DP-7478

UGGAUUGUCGAAAACCACUTST 561 AGUGGUUUUCGACAAUCCATST 736 ND-8775 uGGAuuGucGAAAAccAcuTsT 562 AGUGGUUUUCGACAAUCCATsT 737 ND-8794 uGGAuuGucGAAAAccAcuTsT 563 AGuGGuuuUCGAcAAUCcATsT 738 ND-8813

UGGAUUGUCGAAAACCACUTTCho1 564 AGUGGUUUUCGACAAUCCATST 739 ND-8832 uGGAuuGucGAAAAccAcuTTchol 565 AGUGGUUUUCGAcAAUCcATsT 740 ND-8857 uGGAuuGucGAAAAccAcuTTchol 566 AGuGGuuuUCGAcAAUCcATsT 741 ND-8882

CGGCUAGAGAUGAUCGCUATT 567 UAGCGAUCAUCUCUAGCCGTT 742 AL-DP-7553

CGGCUAGAGAUGAUCGCUATST 568 UAGCGAUCAUCUCUAGCCGTST 743 ND-8776 cGGcuAGAGAuGAucGcuATsT 569 uAGCGAUcAUCUCuAGCCGTsT 744 ND-8795 cGGcuAGAGAuGAucGcuATsT 570 uAGCGAUcAUCUCuAGCCGTsT 745 ND-8795

CGGCUAGAGAUGAUCGCUATTChol 571 UAGCGAUCAUCUCUAGCCGTST 746 ND-8833 cGGcuAGAGAuGAucGcuATTchol 572 UAGCGAUcAUCUCuAGCCGTsT 747 ND-8858 cGGcuAGAGAuGAucGcuATTchol 573 UAGCGAUcAUCUCuAGCCGTsT 748 ND-8858

ACAGUCGAAAUCUAGUGAATT 574 UUCACUAGAUUUCGACUGUTT 749 AL-DP-7395

ACAGUCGAAAUCUAGUGAATST 575 UUCACUAGAUUUCGACUGUTST 750 ND-8730

AcAGucGAAAucuAGuGAATsT 576 UUcACuAGAUUUCGACUGUTsT 751 ND-8742

AcAGucGAAAucuAGuGAATsT 577 uucACuAGAuuUCGACuGUTsT 752 ND-8753

ACAGUCGAAAUCUAGUGAATTChol 578 UUCACUAGAUUUCGACUGUTST 753 ND-8840

AcAGucGAAAucuAGuGAATTchol 579 UUCACUAGAUUUCGACUGUTsT 754 ND-8865

AcAGucGAAAucuAGuGAATTchol 580 -uucACuAGAuuUCGACuGUTsT 755 ND-8890

CUCGAGAUCCUAAUUAUCUTT 581 AGAUAAUUAGGAUCUCGAGTT 756 AL-DP-7499

CUCGAGAUCCUAAUUAUCUTsT 582 AGAUAAUUAGGAUCUCGAGTsT 757 ND-8777 cucGAGAuccuAAuuAucuTsT 583 AGAuAAUuAGGAUCUCGAGTsT 758 ND-8796 cucGAGAuccuAAuuAucuTsT 584 AGAUAAUUAGGAUCUCGAGTST 759 ND-8815

CUCGAGAUCCUAAUUAUCUTTChol 585 AGAUAAUUAGGAUCUCGAGTsT 760 ND-8834 cucGAGAuccuAAuuAucuTTchol 586 AGAuAAUuAGGAUCUCGAGTsT 761 ND-8859 cucGAGAuccuAAuuAucuTTchol 587 AGAuAAuUAGGAUCUCGAGTsT 762 ND-8884

CACCUAACGUGGAAUGUGATT 588 UCACAUUCCACGUUAGGUGTT 763 AL-DP-7365

CACCUAACGUGGAAUGUGATST 589 UCACAUUCCACGUUAGGUGTST 764 ND-8724 cAccuAAcGuGGAAuGuGATsT 590 UcAcAUUCcACGUuAGGUGTsT 765 ND-8736 cAccuAAcGuGGAAuGuGATsT 591 UcAcAuuCcACGuuAGGuGTsT 766 ND-8748

CACCUAACGUGGAAUGUGATTChol 592 UCACAUUCCACGUUAGGUGTST 767 ND-8841 cAccuAAcGuGGAAuGuGATTchol 593 UcAcAUUCcACGUuAGGUGTsT 768 ND-8866 cAccuAAcGuGGAAuGuGATTchol 594 UcAcAuuCcACGuuAGGuGTsT 769 ND-8891

AAUCGUUCAUUCAUUUACATT 595 UGUAAAUGAAUGAACGAUUTT 770 AL-DP-7390

AAUCGUUCAUUCAUUUACATsT 596 UGUAAAUGAAUGAACGAUUTST 771 ND-8727

AAucGuucAuucAuuuAcATsT 597 UGuAAAUGAAUGAACGAUUTsT 772 ND-8739

AAucGuucAuucAuuuAcATsT 598 uGuAAAuGAAuGAACGAuuTST 773 ND-8750

AAUCGUUCAUUCAUUUACATTChol 599 UGUAAAUGAAUGAACGAUUTsT 774 ND-8842

AAucGuucAuucAuuuAcATTchol 600 UGUAAAUGAAUGAACGAUUTsT 775 ND-8867

AAucGuucAuucAuuuAcATTchol 601 UGuAAAuGAAuGAACGAuuTsT 776 ND-8892

AACUUUUCGUGACUUGGGATT 602 UCCCAAGUCACGAAAAGUUTT 777 AL-DP-7382

AACUUUUCGUGACUUGGGATST 603 UCCCAAGUCACGAAAAGUUTST 778 ND-8726

AAcuuuucGuGAcuuGGGATsT 604 UCCcAAGUcACGAAAAGUUTsT 779 ND-8738

AAcuuuucGuGAcuuGGGATsT 605 UCCcAAGUcACGAAAAGuuTsT 780 ND-8749

AACUUUUCGUGACUUGGGATTChol 606 UCCCAAGUCACGAAAAGUUTsT 781 ND-8843

AAcuuuucGuGAcuuGGGATTchol 607 UCCcAAGUcACGAAAAGUUTsT 782 ND-8868

AAcuuuucGuGAcuuGGGATTchol 608 UCCcAAGUcACGAAAAGuuTsT 783 ND-8893

AACAGUCGAAAUCUAGUGATT 609 UCACUAGAUUUCGACUGUUTT 784 AL-DP-7393

AACAGUCGAAAUCUAGUGATST 610 UCACUAGAUUUCGACUGUUTST 785 ND-8729

AAcAGucGAAAucuAGuGATsT 611 UcACuAGAUUUCGACUGUUTsT 786 ND-8741

AAcAGucGAAAucuAGuGATsT 612 UcACuAGAuuUCGACuGuuTsT 787 ND-8752

AACAGUCGAAAUCUAGUGATTChol 613 UCACUAGAUUUCGACUGUUTST 788 ND-8844

AAcAGucGAAAucuAGuGATTchol 614 UcACuAGAUUUCGACUGUUTsT 789 ND-8869

AAcAGucGAAAucuAGuGATTchol 615 UcACuAGAuuUCGACuGuuTsT 790 ND-8894

ACGAAUGAGUUUUGUGCUUTT 616 AAGCACAAAACUCAUUCGUTT 791 AL-DP-7366

ACGAAUGAGUUUUGUGCUUTST 617 AAGCACAAAACUCAUUCGUTST 792 ND-8778

AcGAAuGAGuuuuGuGcuuTsT 618 AAGcAcAAAACUcAUUCGUTsT 793 ND-8797

AcGAAuGAGuuuuGuGcuuTsT 619 AAGcAcAAAACUcAuUCGUTsT 794 ND-8816

ACGAAUGAGUUUUGUGCUUTTChol 620 AAGCACAAAACUCAUUCGUTST 795 ND-8835

AcGAAuGAGuuuuGuGcuuTTchol 621 AAGcAcAAAACUcAUUCGUTsT 796 ND-8860

AcGAAuGAGuuuuGuGcuuTTchol 622 AAGcAcAAAACUcAuUCGUTsT 797 ND-8885

AAUUCGCAUGUACAGUGAATT 623 UUCACUGUACAUGCGAAUUTT 798 AL-DP-7371

AAUUCGCAUGUACAGUGAATST 624 UUCACUGUACAUGCGAAUUTST 799 ND-8779

AAuucGcAuGuAcAGuGAATsT 625 UUcACUGuAcAUGCGAAUUTsT 800 ND-8798

AAuucGcAuGuAcAGuGAATsT 626 uUcACuGuAcAuGCGAAuUTST 801 ND-8817

AAUUCGCAUGUACAGUGAATTChol 627 UUCACUGUACAUGCGAAUUTST 802 ND-8836

AAuucGcAuGuAcAGuGAATTchol 628 UUcACUGuAcAUGCGAAUUTsT 803 ND-8861 AAuucGcAuGuAcAGuGAATTchol 629 uUcACuGuAcAuGCGAAuUTsT 804 ND-8886

AAUAGAAUUCGCAUGUACATT 630 UGUACAUGCGAAUUCUAUUTT 805 AL-DP-7372

AAUAGAAUUCGCAUGUACATST 631 UGUACAUGCGAAUUCUAUUTST 806 ND-8780

AAuAGAAuucGcAuGuAcATsT 632 UGuAcAUGCGAAUUCuAUUTsT 807 ND-8799

AAuAGAAuucGcAuGuAcATsT 633 uGuAcAuGCGAAuUCuAuUTsT 808 ND-8818

AAUAGAAUUCGCAUGUACATTChol 634 UGUACAUGCGAAUUCUAUUTST 809 ND-8837

AAuAGAAuucGcAuGuAcATTchol 635 UGuAcAUGCGAAUUCuAUUTsT 810 ND-8862

AAuAGAAuucGcAuGuAcATTchol 636 uGuAcAuGCGAAuUCuAuUTsT 811 ND-8887

UGGUAACCCAAUGAUGUAUTT 637 AUACAUCAUUGGGUUACCATT 812 AL-DP-7370

UGGUAACCCAAUGAUGUAUTST 638 AUACAUCAUUGGGUUACCATST 813 ND-8781 uGGuAAcccAAuGAuGuAuTsT 639 AuAcAUcAUUGGGUuACcATsT 814 ND-8800 uGGuAAcccAAuGAuGuAuTsT 640 AuAcAUcAuUGGGuUACcATsT 815 ND-8819

UGGUAACCCAAUGAUGUAUTTChol 641 AUACAUCAUUGGGUUACCATST 816 ND-8838 uGGuAAcccAAuGAuGuAuTTchol 642 AuAcAUcAUUGGGUuACcATsT 817 ND-8863 uGGuAAcccAAuGAuGviAuTTchol 643 AuAcAUcAuUGGGuUACcATsT 818 ND-8888 cucGAGAuccuAAuuAucuTsT 1748 AGAuAAuuAGGAUCUCGAGTst 1749 ND-9300

Table 4 sets forth the results of testing of dsRNA listed in Table 3.

Table 4

Mean Mean activity activity remaining remaining after after

Duplex 3OnM Standard 10OpM Standard name treatment deviation treatment deviation

AL-DP-7545 6.74 1.80 18.41 4.14

ND-8763 6.28 1.79 21.38 5.93

ND-8782 7.21 1.59 23.76 7.49

ND-8801 18.52 2.58 51.10 12.01

ND-8820 9.26 1.88 58.34 10.61

ND-8845 34.08 7.03 69.28 14.97

ND-8870 30.96 5.97 77.58 12.41

AL-DP-7558 11.93 1.66 25.71 3.57

ND-8764 8.97 1.53 25.81 7.79

ND-8783 27.33 3.10 51.35 6.52

ND-8802 28.82 4.39 91.90 14.32

ND-8821 8.96 2.36 71.92 10.68 ND-8846 75.94 17.07 88.87 7.90

ND-8871 58.02 9.96 91.79 13.92

AL-DP-7548 11.23 1.92 35.58 6.27

ND-8765 8.24 1.01 45.42 10.63

ND-8784 25.07 4.28 68.74 7.10

ND-8803 45.89 10.22 97.46 12.87

ND-8822 11.59 2.94 75.22 17.11

ND-8847 64.96 9.30 100.47 16.50

ND-8872 78.50 14.11 86.77 5.96

AL-DP-7509 17.62 2.26 21.58 2.98

ND-8766 15.26 1.22 25.45 2.92

ND-8785 19.66 3.35 43.13 4.50

ND-8804 21.66 2.34 50.36 8.66

ND-8823 15.66 2.09 48.14 4.88

ND-8848 27.84 3.58 95.42 20.53

ND-8873 29.97 3.32 91.79 13.36

AL-DP-7492 11.09 1.19 19.22 3.29

ND-8767 11.90 1.73 20.65 2.66

ND-8786 11.69 1.72 19.78 2.74

ND-8805 14.97 1.46 26.41 6.08

ND-8824 11.53 1.51 43.76 5.00

ND-8849 25.37 11.97 43.95 10.44

ND-8874 16.84 2.99 53.87 6.12

AL-DP-7554 15.01 1.22 23.48 4.39

ND-8768 14.46 1.30 26.79 4.77

ND-8787 15.20 2.47 24.76 4.44

ND-8806 15.01 2.02 33.77- 10.43

ND-8825 17.00 3, 82 72.33 14.34

ND-8850 29.25 7, 49 93.94 19.23

ND-8875 23.33 3, 94 79.79 9.03

AL-DP-7557 13.10 1, 34 22.30 8.07

ND-8769 11.17 1, 10 24.91 4.44

ND-8788 21.84 2.02 60.20 10.58

ND-8788 23.53 1.55 69.43 13.87

ND-8826 12.81 1.35 50.68 10.86

ND-8851 36.41 3.49 116.14 48.06

ND-8851 36.42 5.05 100.91 26.50

AL-DP-7476 17.11 2.75 25.33 7.43

ND-8770 13.36 1.65 30.58 8.25 ND-8789 46.06 6.35 76.12 14.80

ND-8808 43.15 5.55 98.81 21.90

ND-8827 14.76 2.03 56.08 13.96

ND-8852 70.35 13.51 107.70 22.62

ND-8877 58.73 8.08 90.83 10.87

AL-DP-7514 15.63 2.76 18.89 0.67

ND-8771 14.96 1.69 23.31 10.62

ND-8790 15.91 1.57 31.71 2.88

ND-8809 16.79 2.80 36.42 5.40

ND-8828 14.61 2. 09 53.50 8.13

ND-8853 34.20 4, 88 81.95 16.33

ND-8878 26.63 2.95 87.21 33.73

AL-DP-7540 18.18 3. 06 32.59 5.25

ND-8772 19.31 2 99 36.01 5.41

ND-8791 35.43 4.60 55.34 7.39

ND-8810 17.83 2.64 25.48 7.36

ND-8829 18.93 3.20 68.53 14.55

ND-8854 50.71 6.95 89.19 9.26

ND-8879 21.76 5.10 62.43 16.86

AL-DP-7397 17.10 2.37 22.44 4.36

ND-8731 17.09 2.86 31.25 8.34

ND-8743 15.89 2.29 27.33 4.67

ND-8754 19.53 2, 97 41.57 9.22

ND-8839 18.18 2, 95 66.39 13.77

ND-8864 19.51 3 79 59.13 5.60

ND-8889 19.91 2.30 92.91 14.85

AL-DP-7526 17.67 2.32 41.09 7.63

ND-8773 15.59 1.57 42.07 6.55

ND-8792 19.42 2.08 46.87 6.99

ND-8811 34.56 7.82 72.57 9.85

ND-8830 19.49 3.09 69.87 7.25

ND-8855 27.49 4.52 85.38 13.45

ND-8880 38.04 6.41 87.78 13.97

AL-DP-7473 15.81 2.07 31.86 6.43

ND-8774 15.81 2.61 30.89 8.60

ND-8793 14.04 1.44 25.98 2.91

ND-8812 21.16 3.28 49.59 8.12

ND-8831 19.07 3.60 75.06 16.79

ND-8856 17.86 5.51 65.08 11.14 ND-8881 28.56 6.18 83.97 12.49

AL-DP-7478 16.41 2.58 33.38 6.20

ND-8775 16.14 1.63 31.52 2.92

ND-8794 19.05 3.19 24.88 3.34

ND-8813 17.04 2.34 26.53 3.90

ND-8832 16.40 2.16 66.67 15.18

ND-8857 26.53 6.20 69.13 9.30

ND-8882 20.04 2.43 68.67 8.67

AL-DP-7553 20.83 2.66 28.97 4.93

ND-8776 21.10 2, 76 29.95 5.15

ND-8795 26.00 3, 54 79.53 11.78

ND-8795 25.14 3 95 80.83 12.02

ND-8833 21.76 3.23 52.28 7.24

ND-8858 33.25 7, 09 92.18 20.40

ND-8858 31.50 5, 36 84.22 13.01

AL-DP-7395 18.01 2.33 25.01 4.17

ND-8730 18.63 2.22 35.55 6.30

ND-8742 18.04 2.92 29.24 6.48

ND-8753 19.03 3.21 50.35 10.66

ND-8840 24.81 3.87 81.78 17.12

ND-8865 27.65 3.29 72.55 12.44

ND-8890 22.03 1. 60 105.32 26.89

AL-DP-7499 12.40 1.94 25.24 3.83

ND-8777 12.78 2 14 25.07 6.35

ND-8796 11.28 0.83 21.19 2.68

ND-8815 10.85 1.12 27.56 7.33

ND-8834 9.88 1.77 48.81 8.56

ND-8859 38.05 5.09 56.68 8.15

ND-8884 38.13 7.42 75.98 15.04

AL-DP-7365 15.72 2.57 23.60 5.58

ND-8724 14.88 2.37 27.95 11.09

ND-8736 71.51 11.99 81.07 19.08

ND-8748 71.98 14.80 82.12 16.76

ND-8841 18.39 3.01 66.82 19.67

ND-8866 79.40 15.36 80.86 15.81

ND-8891 73.79 17.04 86.53 21.21

AL-DP-7390 17.45 3.14 30.46 4.87

ND-8727 17.98 3.47 44.60 4.60

ND-8739 23.47 4.83 53.99 8.89 ND-8750 25.98 3.55 83.20 10.09

ND-8842 21.10 2.77 109.29 34.23

ND-8867 44.74 4.83 91.06 22.68

ND-8892 57.70 9.50 96.07 23.52

AL-DP-7382 16.90 3.54 30.39 3.91

ND-8726 17.17 3.84 38.93 6.26

ND-8738 19.51 2.77 41.20 3.80

ND-8749 17.03 3.66 34.11 8.30

ND-8843 26.36 4.99 83.57 8.12

ND-8868 26.78 3.25 88.44 7.96

ND-8893 22.70 2.05 86.71 12.41

AL-DP-7393 24.38 3.02 38.04 7.48

ND-8729 29.07 4.34 59.65 11.35

ND-8741 68.38 7.91 87.12 8.74

ND-8752 50.68 7.27 86.26 11.15

ND-8844 36.14 5.29 102.26 16.83

ND-8869 71.02 12.42 97.57 17.41

ND-8894 52.86 8.43 106.24 17.77

AL-DP-7366 18.69 2 .05 44.08 7.35

ND-8778 18.46 2.08 41.29 5.42

ND-8797 15.49 2.21 36.71 5.29

ND-8816 13.61 1.76 33.13 6.21

ND-8835 22.00 3.84 76.17 11.70

ND-8860 17.81 4.03 68.48 8.32

ND-8885 15.76 2.33 70.03 10.95

AL-DP-7371 18.77 2.20 52.94 11.86

ND-8779 19.88 2.86 56.27 9.68

ND-8798 24.79 4.89 59.87 8.65

ND-8817 26.06 2.89 85.76 15.79

ND-8836 33.17 7.60 87.15 20.65

ND-8861 78.78 18.21 88.22 14.97

ND-8886 70.66 10.62 96.55 14.35

AL-DP-7372 19.23 3.22 46.80 10.62

ND-8780 19.94 0.97 62.29 12.87

ND-8799 42.73 4.67 81.61 10.68

ND-8818 88.99 6.53 104.90 8.74

ND-8837 25.18 3.70 99.41 12.93

ND-8862 79.80 15.30 92.36 10.28

ND-8887 78.27 16.96 92.55 14.48 AL-DP-7370 20.04 1.52 46.57 9.68

ND-8781 16.68 1.83 62.43 12.82

ND-8800 41.69 5.44 77.39 12.71

ND-8819 35.98 3.15 78.56 17.49

ND-8838 21.49 3.65 84.71 30.30

ND-8863 59.44 19.74 91.91 17.80

ND-8888 . 49.74 16.09 97.57 15.46

Design of siRNA Targeting HPV El Gene Expression

Table 5 sets forth dsRNA compositions of the invention.

Table 5

Upper case letters: unmodified ribonucleotide (except for T which is an unmodified deoxyribonucleotide)

Lower case letters: ribonucloetide bearing 2'-O-methyl substituent on ribose moiety s : Indicates position of phosphorothioate internucleoside linkage chol : cholesterol moiety conjugated to 3' ribonucleotide.

'duplex name'means the name of the composition formed by specific hybridization of the indicated sense strand and the indicated antisense strand.

Target sequence of mRNA antisense strand from HPV El reference Sense strand (guide sequence)

Ul sequence (sequence of SEQ (target sequence) SEQ having double TT SEQ total 19mer target site ID. having double TT ID. overhang ID. duplex + AA at both ends) NO. overhang, (5 '-3/ )_j NO. (5'-3') NO. name

AAAAAUCAACGUGUUGCGAUUAA 819 AAAUCAACGUGUUGCGAUUTT 945 AAUCGCAACACGUUGAUUUTT 1141 ND-9061

AAGAGCCUCCAAAAUUGCGUAAA 820 GAGCCUCCAAAAUUGCGUATT 946 UACGCAAUUUUGGAGGCUCTT 1142 ND-9062

AAUCAACGUGUUGCGAUUGGUAA 821 UCAACGUGUUGCGAUUGGUTT 947 ACCAAUCGCAACACGUUGATT 1143 ND-9063

AAUCCAAAAUUGCGUAGUACAAA 822 UCCAAAAUUGCGUAGUACATT 948 UGUACUACGCAAUUUUGGATT 1144 ND-.9064

AAAAUCAACGUGUUGCGAUUGAA 823 AAUCAACGUGUUGCGAUUGTT 949 CAAUCGCAACACGUUGAUUTT 1145 ND-9065

AACCUCCAAAAUUGCGUAGUAAA 824 CCUCCAAAAUUGCGUAGUATT 950 UACUACGCAAUUUUGGAGGTT 1146 ND-9066

AAAGAGCCUCCAAAAUUGCGUAA 825 AGAGCCUCCAAAAUUGCGUTT 951 ACGCAAUUUUGGAGGCUCUTT 1147 ND-9067

AACAACGUGUUGCGAUUGGUGAA 826 CAACGUGUUGCGAUUGGUGTT 952 CACCAAUCGCAACACGUUGTT 1148 ND-9068

AAAUAGAUGUGAUAGGGUAGAAA 827 AUAGAUGUGAUAGGGUAGATT 953 UCUACCCUAUCACAUCUAUTT 1149 ND-9069

AAGGGAAGAGGGUACGGGAUGAA 828 GGGAAGAGGGUACGGGAUGTT 954 CAUCCCGUACCCUCUUCCCTT 1150 ND-9070

AAAGAUUAAGUUUGCACGAGGAA 829 AGAUUAAGUUUGCACGAGGTT 955 CCUCGUGCAAACUUAAUCUTT 1151 ND-9071

AAGGUAUCAAGGUGUAGAGUUAA 830 GGUAUCAAGGUGUAGAGUUTT 956 AACUCUACACCUUGAUACCTT 1152 ND-9072

AAACUUAGUGAUAUUAGUGGAAA 831 ACUUAGUGAUAUUAGUGGATT 957 UCCACUAAUAUCACUAAGUTT 1153 ND-9073 AAGAGAUUAUUUGAAAGCGAAAA 832 GAGAUUAUUUGAAAGCGAATT 958 UUCGCUUUCAAAUAAUCUCTT 1154 ND-9074 AAAACACCAUGUAGUCAGUAUAA 833 AACACCAUGUAGUCAGUAUTT 959 AUACUGACUACAUGGUGUUTT 1155 ND-9075 AAAGCCUCCAAAAUUGCGUAGAA 834 AGCCUCCAAAAUUGCGUAGTT 960 CUACGCAAUUUUGGAGGCUTT 1156 ND-9076 AAGCCUCCAAAAUUGCGUAGUAA 835 GCCUCCAAAAUUGCGUAGUTT 961 ACUACGCAAUUUUGGAGGCTT 1157 ND-9077 AAGUGUAUGGAGACACGCCAGAA 836 GUGUAUGGAGACACGCCAGTT 962 CUGGCGUGUCUCCAUACACTT 1158 ND-9078 AAGUACAAUGGGCCUACGAUAAA 837 GUACAAUGGGCCUACGAUATT 963 UAUCGUAGGCCCAUUGUACTT 1159 ND-9079 AAUACAAUGGGCCUACGAUAAAA 838 UACAAUGGGCCUACGAUAATT 964 UUAUCGUAGGCCCAUUGUATT 1160 ND-9080 AAUGACAUAGUAGACGAUAGUAA 839 UGACAUAGUAGACGAUAGUTT 965 ACUAUCGUCUACUAUGUCATT 1161 ND-9081 AAGACAUAGUAGACGAUAGUGAA 840 GACAUAGUAGACGAUAGUGTT 966 CACUAUCGUCUACUAUGUCTT 1162 ND-9082 AAACUCUUUGCCAACGUUUAAAA 841 ACUCUUUGCCAACGUUUAATT 967 UUAAACGUUGGCAAAGAGUTT 1163 ND-9083 AAAUAAUGACAUAGUAGACGAAA 842 AUAAUGACAUAGUAGACGATT 968 UCGUCUACUAUGUCAUUAUTT 1164 ND-9084 AAAAGUAUUUGGGUAGUCCACAA 843 AAGUAUUUGGGUAGUCCACTT 969 GUGGACUACCCAAAUACUUTT 1165 ND-9085 AAACGUGUUGCGAUUGGUGUAAA 844 ACGUGUUGCGAUUGGUGUATT 970 UACACCAAUCGCAACACGUTT 1166 ND-9086 AACGAAAGUAUUUGGGUAGUCAA 845 CGAAAGUAUUUGGGUAGUCTT 971 GACUACCCAAAUACUUUCGTT 1167 ND-9087 AACUCCAAAAUUGCGUAGUACAA 846 CUCCAAAAUUGCGUAGUACTT 972 GUACUACGCAAUUUUGGAGTT 1168 ND-9088 AAUGGUACAAUGGGCCUACGAAA 847 UGGUACAAUGGGCCUACGATT 973 UCGUAGGCCCAUUGUACCATT 1169 ND-9089 AAUAAUGACAUAGUAGACGAUAA 848 UAAUGACAUAGUAGACGAUTT 974 AUCGUCUACUAUGUCAUUATT 1170 ND-9090 AAACAUAGUAGACGAUAGUGAAA 849 ACAUAGUAGACGAUAGUGATT 975 UCACUAUCGUCUACUAUGUTT 1171 ND-9091 AAGUGUAGACAUUAUAAACGAAA 850 GUGUAGACAUUAUAAACGATT 976 UCGUUUAUAAUGUCUACACTT 1172 ND-9092 AAUAGACAUUAUAAACGAGCAAA 851 UAGACAUUAUAAACGAGCATT 977 UGCUCGUUUAUAAUGUCUATT 1173 ND-9093 AAUUGCGAUUGGUGUAUUGCUAA 852 UUGCGAUUGGUGUAUUGCUTT 978 AGCAAUACACCAAUCGCAATT 1174 ND-9094 AAUUGGCAGACACUAAUAGUAAA 853 UUGGCAGACACUAAUAGUATT 979 UACUAUUAGUGUCUGCCAATT 1175 ND-9095 AAUUCAGAAUUAGUAAGACCAAA 854 UUCAGAAUUAGUAAGACCATT 980 UGGUCUUACUAAUUCUGAATT 1176 ND-9096 AAGGAGAUUAUUUGAAAGCGAAA 855 GGAGAUUAUUUGAAAGCGATT 981 UCGCUUUCAAAUAAUCUCCTT 1177 ND-9097 AAACCAUGUAGUCAGUAUAGUAA 856 ACCAUGUAGUCAGUAUAGUTT 982 ACUAUACUGACUACAUGGUTT 1178 ND-9098 AAGAAGAGGGUACGGGAUGUAAA 857 GAAGAGGGUACGGGAUGUATT 983 UACAUCCCGUACCCUCUUCTT 1179 ND-9099 AAAUAAAUCAACGUGUUGCGAAA 858 AUAAAUCAACGUGUUGCGATT 984 UCGCAACACGUUGAUUUAUTT 1180 ND-9100

AACGUGUUGCGAUUGGUGUAUAA 859 CGUGUUGCGAUUGGUGUAUTT 985 AUACACCAAUCGCAACACGTT 1181 ND-9101 AAUGGGCCUACGAUAAUGACAAA 860 UGGGCCUACGAUAAUGACATT 986 UGUCAUUAUCGUAGGCCCATT 1182 ND-9102 AAUGCAUAUUACUAUAUGGUGAA 861 UGCAUAUUACUAUAUGGUGTT 987 CACCAUAUAGUAAUAUGCATT 1183 ND-9103 AACCAGAUUAAGUUUGCACGAAA 862 CCAGAUUAAGUUUGCACGATT 988 UCGUGCAAACUUAAUCUGGTT 1184 ND-9104 AAAGAUUAUUUGAAAGCGAAGAA 863 AGAUUAUUUGAAAGCGAAGTT 989 CUUCGCUUUCAAAUAAUCUTT 1185 ND-9105 AAGUUACAGGUAGAAGGGCGCAA 864 GUUACAGGUAGAAGGGCGCTT 990 GCGCCCUUCUACCUGUAACTT 1186 ND-9106 AAGCCAAAAUAGGUAUGUUAGAA 865 GCCAAAAUAGGUAUGUUAGTT 991 CUAACAUACCUAUUUUGGCTT 1187 ND-9107 AAGCAUAGACCAUUGGUACAAAA 866 GCAUAGACCAUUGGUACAATT 992 UUGUACCAAUGGUCUAUGCTT 1188 ND-9108 AAAAAUUGCGUAGUACAGCAGAA 867 AAAUUGCGUAGUACAGCAGTT 993 CUGCUGUACUACGCAAUUUTT 1189 ND-9109 AAAGUAUUUGGGUAGUCCACUAA 868 AGUAUUUGGGUAGUCCACUTT 994 AGUGGACUACCCAAAUACUTT 1190 ND-9110 AAAUGACAUAGUAGACGAUAGAA 869 AUGACAUAGUAGACGAUAGTT 995 CUAUCGUCUACUAUGUCAUTT 1191 ND-9111 AAGGUAGUCCACUUAGUGAUAAA 870 GGUAGUCCACUUAGUGAUATT 996 UAUCACUAAGUGGACUACCTT 1192 ND-9112 AAAGCAUAGACCAUUGGUACAAA 871 AGCAUAGACCAUUGGUACATT 997 UGUACCAAUGGUCUAUGCUTT 1193 ND-9113 AAUUUCAGAAUUAGUAAGACCAA 872 UUUCAGAAUUAGUAAGACCTT 998 GGUCUUACUAAUUCUGAAATT 1194 ND-9114 AAUGCGAUUGGUGUAUUGCUGAA 873 UGCGAUUGGUGUAUUGCUGTT 999 CAGCAAUACACCAAUCGCATT 1195 ND-9115

AACCAAAAUUGCGUAGUACAGAA 874 CCAAAAUUGCGUAGUACAGTT 1000 CUGUACUACGCAAUUUUGGTT 1196 ND-9116 AAGACAGCACAUGCGUUGUUUAA 875 GACAGCACAUGCGUUGUUUTT 1001 AAACAACGCAUGUGCUGUCTT 1197 ND-9117 AAUGUUACAGGUAGAAGGGCGAA 876 UGUUACAGGUAGAAGGGCGTT 1002 CGCCCUUCUACCUGUAACATT 1198 ND-9118

AAAGACAAUAAUAUUAGUCCUAA 877 AGACAAUAAUAUUAGUCCUTT 1003 AGGACUAAUAUUAUUGUCUTT 1199 ND-9119 AAAUGAUUAUUUAACACAGGCAA 878 AUGAUUAUUUAACACAGGCTT 1004 GCCUGUGUUAAAUAAUCAUTT 1200 ND-9120 AAAGAUGUGAUAGGGUAGAUGAA 879 AGAUGUGAUAGGGUAGAUGTT 1005 CAUCUACCCUAUCACAUCUTT 1201 ND-9121 AACUGCAAGGGUCUGUAAUAUAA 880 CUGCAAGGGUCUGUAAUAUTT 1006 AUAUUACAGACCCUUGCAGTT 1202 ND-9122 AAUCAGAUGACGAGAACGAAAAA 881 UCAGAUGACGAGAACGAAATT 1007 UUUCGUUCUCGUCAUCUGATT 1203 ND-9123 AACACCAUGUAGUCAGUAUAGAA 882 CACCAUGUAGUCAGUAUAGTT 1008 CUAUACUGACUACAUGGUGTT 1204 ND-9124 AAAGACAGCACAUGCGUUGUUAA 883 AGACAGCACAUGCGUUGUUTT 1009 AACAACGCAUGUGCUGUCUTT 1205 ND-9125 AAAGAGGGUACGGGAUGUAAUAA 884 AGAGGGUACGGGAUGUAAUTT- 1010 AUUACAUCCCGUACCCUCUTT 1206 ND-9126 AAAAAAGUAAUAAAUCAACGUAA 885 AAAAGUAAUAAAUCAACGUTT 1011 ACGUUGAUUUAUUACUUUUTT 1207 ND-9127 AAAAAGCAUAGACCAUUGGUAAA 886 AAAGCAUAGACCAUUGGUATT 1012 UACCAAUGGUCUAUGCUUUTT 1208 ND-9128

AAUUGUACAUUUGAAUUAUCAAA 887 UUGUACAUUUGAAUUAUCATT 1013 UGAUAAUUCAAAUGUACAATT 1209 ND-9129 AAGUAAAGCAUAGACCAUUGGAA 888 GUAAAGCAUAGACCAUUGGTT 1014 CCAAUGGUCUAUGCUUUACTT 1210 ND-9130

AAAucAAcGuGuuGcGAuuTsT 1015 AAUCGcAAcACGUUGAUUUTsT 1211 ND-9131

GAGccuccAAAAuuGcGuATsT 1016 uACGcAAUUUUGGAGGCUCTsT 1212 ND-9132 ucAAcGuGuuGcGAuuGGuTsT 1017 ACcAAUCGcAAcACGUUGATsT 1213 ND-9133 uccAAAAuuGcGuAGuAcATsT 1018 UGuACuACGcAAUUUUGGATsT 1214 ND-9134

AAucAAcGuGuuGcGAuuGTsT 1019 cAAUCGcAAcACGUUGAUUTsT 1215 ND-9135 ccuccAAAAuuGcGuAGuATsT 1020 uACuACGcAAUUUUGGAGGTsT 1216 ND-9136

AGAGccuccAAAAuuGcGuTsT 1021 ACGcAAUUUUGGAGGCUCUTsT 1217 ND-9137 cAAcGuGuuGcGAuuGGuGTsT 1022 cACcAAUCGcAAcACGUUGTsT 1218 ND-9138

AuAGAuGuGAuAGGGuAGATsT 1023 UCuACCCuAUcAcAUCuAUTsT 1219 ND-9139

GGGAAGAGGGuAcGGGAuGTsT 1024 cAUCCCGuACCCUCUUCCCTsT 1220 ND-9140

AGAuuAAGuuuGcAcGAGGTsT 1025 CCUCGUGcAAACUuAAUCUTsT 1221 ND-9141

OO GGuAucAAGGuGuAGAGuuTsT 1026 AACUCuAcACCUUGAuACCTsT 1222 ND-9142

AcuuAGuGAuAuuAGuGGATsT 1027 UCcACuAAuAUcACuAAGUTsT 1223 ND-9143

GAGAuuAuuuGAAAGcGAATsT 1028 UUCGCUUUcAAAuAAUCUCTsT 1224 ND-9144

AAcAccAuGuAGucAGuAuTsT 1029 AuACUGACuAcAUGGUGUUTsT 1225 ND-9145

AGccuccAAAAuuGcGuAGTsT 1030 CuACGcAAUUUUGGAGGCUTsT 1226 ND-9146

GccuccAAAAuuGcGuAGuTsT 1031 ACuACGcAAUUUUGGAGGCTsT 1227 ND-9147

GuGuAuGGAGAcAcGccAGTsT 1032 CUGGCGUGUCUCcAuAcACTsT 1228 ND-9148

GuAcAAuGGGccuAcGAuATsT 1033 uAUCGuAGGCCcAUUGuACTsT 1229 ND-9149 uAcAAuGGGccuAcGAuAATsT 1034 UuAUCGuAGGCCcAUUGuATsT 1230 ND-9150 uGAcAuAGuAGAcGAuAGuTsT 1035 ACuAUCGUCuACuAUGUcATsT 1231 ND-9151

GAcAuAGuAGAcGAuAGuGTsT 1036 cACuAUCGUCuACuAUGUCTsT 1232 ND-9152

AcucuuuGccAAcGuuuAATsT 1037 UuAAACGUUGGcAAAGAGUTsT 1233 ND-9153

AuAAuGAcAuAGuAGAcGATsT 1038 UCGUCuACuAUGUcAUuAUTsT 1234 ND-9154

AAGuAuuuGGGuAGuccAcTsT 1039 GUGGACuACCcAAAuACUUTsT 1235 ND-9155

AcGuGuuGcGAuuGGuGuATsT 1040 uAcACcAAUCGcAAcACGUTsT 1236 ND-9156

cGAAAGuAuuuGGGuAGucTsT 1041 GACuACCcAAAuACUUUCGTsT 1237 ND-9157 cuccAAAAuuGcGuAGuAcTsT 1042 GuACuACGcAAUUUUGGAGTsT 1238 ND-9158 uGGuAcAAuGGGccuAcGATsT 1043 UCGuAGGCCcAUUGuACcATsT 1239 ND-9159 uAAuGAcAuAGuAGAcGAuTsT 1044 AUCGUCuACuAUGUcAUuATsT 1240 ND-9160

AcAuAGuAGAcGAuAGuGATsT 1045 UcACuAUCGUCuACuAUGUTsT 1241 ND-9161

GuGuAGAcAuuAuAAAcGATsT 1046 UCGUUuAuAAUGUCuAcACTsT 1242 ND-9162 uAGAcAuuAuAAAcGAGcATsT 1047 UGCUCGUUuAuAAUGUCuATsT 1243 ND-9163 uuGcGAuuGGuGuAuuGcuTsT 1048 AGcAAuAcACcAAUCGcAATsT 1244 ND-9164 uuGGcAGAcAcuAAuAGuATsT 1049 uACuAUuAGUGUCUGCcAATsT 1245 ND-9165 uucAGAAuuAGuAAGAccATsT 1050 UGGUCUuACuAAUUCUGAATsT 1246 ND-9166

GGAGAuuAuuuGAAAGcGATsT 1051 UCGCUUUcAAAuAAUCUCCTsT 1247 ND-9167

AccAuGuAGucAGuAuAGuTsT 1052 ACuAuACUGACuAcAUGGUTsT 1248 ND-9168

GAAGAGGGuAcGGGAuGuATsT 1053 uAcAUCCCGuACCCUCUUCTsT 1249 ND-9169

AuAAAucAAcGuGuuGcGATsT 1054 UCGcAAcACGUUGAUϋuAUTsT 1250 ND-9170 cGuGuuGcGAuuGGuGuAuTsT 1055 AuAcACcAAUCGcAAcACGTsT 1251 ND-9171 uGGGccuAcGAuAAuGAcATsT 1056 UGUcAUuAUCGuAGGCCcATsT 1252 ND-9172 uGcAuAuuAcuAuAuGGuGTsT 1057 cACcAuAuAGuAAuAUGcATST 1253 ND-9173

CCAGAuuAAGuuuGcAcGATsT 1058 UCGUGcAAACUuAAUCUGGTsT 1254 ND-9174

AGAuuAuuuGAAAGcGAAGTsT 1059 CUUCGCUUϋcAAAuAAUCUTsT 1255 ND-9175

GuuAcAGGuAGAAGGGcGcTsT 1060 GCGCCCUUCuACCUGuAACTsT 1256 ND-9176

GccAAAAuAGGuAuGuuAGTsT 1061 CuAAcAuACCuAUUUUGGCTsT 1257 ND-9177

GcAuAGAccAuuGGuAcAATsT 1062 UUGuACcAAUGGUCuAUGCTsT 1258 ND-9178

AAAuuGcGuAGuAcAGcAGTsT 1063 CUGCUGuACuACGcAAUUUTsT 1259 ND-9179

AGuAuuuGGGuAGuccAcuTsT 1064 AGUGGACuACCcAAAuACUTsT 1260 ND-9180

AuGAcAuAGuAGAcGAuAGTsT 1065 CuAUCGUCuACuAUGUcAUTsT 1261 ND-9181

GGuAGuccAcuuAGuGAuATsT 1066 uAUcACuAAGUGGACuACCTsT 1262 ND-9182

AGcAuAGAccAuuGGuAcATsT 1067 UGuACcAAUGGUCuAUGCUTsT 1263 ND-9183 uuucAGAAuuAGuAAGAccTsT 1068 GGUCUuACuAAUUCUGAAATsT 1264 ND-9184

uGcGAuuGGuGuAuuGcuGTsT 1069 cAGcAAuAcACcAAUCGcATsT 1265 ND-9185 ccAAAAuuGcGuAGuAcAGTsT 1070 CUGuACuACGcAAUUUUGGTsT 1266 ND-9186

GAcAGcAcAuGcGuuGuuuTsT 1071 AAAcAACGcAUGUGCUGUCTsT 1267 ND-9187 uGuuAcAGGuAGAAGGGcGTsT 1072 CGCCCUUCuACCUGuAAcATsT 1268 ND-9188

AGAcAAuAAuAuuAGuccuTsT 1073 AGGACuAAuAUuAUUGUCUTsT 1269 ND-9189

AuGAuuAuuuAAcAcAGGcTsT 1074 GCCUGUGUuAAAuAAUcAUTsT 1270 ND-9190

AGAuGuGAuAGGGuAGAuGTsT 1075 cAUCuACCCuAUcAcAUCUTsT 1271 ND-9191 cuGcAAGGGucuGuAAuAuTsT 1076 AuAUuAcAGACCCUUGcAGTsT 1272 ND-9192 ucAGAuGAcGAGAAcGAAATsT 1077 UUUCGUUCUCGUcAUCUGATsT 1273 ND-9193 cAccAuGuAGucAGuAuAGTsT 1078 CuAuACUGACuAcAUGGUGTsT 1274 ND-9194

AGAcAGcAcAuGcGuuGuuTsT 1079 AAcAACGcAUGUGCUGUCUTST 1275 ND-9195

AGAGGGuAcGGGAuGuAAuTsT 1080 AUuAcAUCCCGuACCCUCUTsT 1276 ND-9196

OO AAAAGUAAUAAAUcAAcGuTsT 1081 ACGUUGAUUuAUuACUUUUTsT 1277 ND-9197

O AAAGcAuAGAccAuuGGuATsT 1082 uACcAAUGGUCuAUGCUUUTsT 1278 ND-9198 uuGuAcAuuuGAAuuAucATsT 1083 UGAuAAUUcAAAUGuAcAATsT 1279 ND-9199

GuAAAGcAuAGAccAuuGGTsT 1084 CcAAUGGUCuAUGCUUuACTsT 1280 ND-9200

AAAUAUCAAAUAUUAGUGAAGAA 889 AUAUCAAAUAUUAGUGAAGTT 1085 CUUCACUAAUAUUUGAUAUTT 1281 AL-DP-8042 AAUAUCAAAUAUUAGUGAAGUAA 890 UAUCAAAUAUUAGUGAAGUTT 1086 ACUUCACUAAUAUUUGAUATT 1282 AL-DP-8043 AAGGGUAUGGCAAUACUGAAGAA 891 GGGUAUGGCAAUACUGAAGTT 1087 CUUCAGUAUUGCCAUACCCTT 1283 AL-DP-8044 AACAACGUUUAAAUGUGUGUCAA 892 CAACGUUUAAAUGUGUGUCTT 1088 GACACACAUUUAAACGUUGTT 1284 AL-DP-8045 AAAACGUUUAAAUGUGUGUCAAA 893 AACGUUUAAAUGUGUGUCATT 1089 UGACACACAUUUAAACGUUTT 1285 AL-DP-8046 AAGAAAACGAUGGAGACUCUUAA 894 GAAAACGAUGGAGACUCUUTT 1090 AAGAGUCUCCAUCGUUUUCTT 1286 AL-DP-8047 AAGCGGGUAUGGCAAUACUGAAA 895 GCGGGUAUGGCAAUACUGATT 1091 UCAGUAUUGCCAUACCCGCTT 1287 AL-DP-8048 AACGGGUAUGGCAAUACUGAAAA 896 CGGGUAUGGCAAUACUGAATT 1092 UUCAGUAUUGCCAUACCCGTT 1288 AL-DP-8049 AAUUAUAAACGAGCAGAAAAAAA 897 UUAUAAACGAGCAGAAAAATT 1093 UUUUUCUGCUCGUUUAUAATT 1289 AL-DP-8050 AAACAAUGUGUAGACAUUAUAAA 898 ACAAUGUGUAGACAUUAUATT 1094 UAUAAUGUCUACACAUUGUTT 1290 AL-DP-8051 AAAUUAUAAACGAGCAGAAAAAA 899 AUUAUAAACGAGCAGAAAATT 1095 UUUUCUGCUCGUUUAUAAUTT 1291 AL-DP-8052 AAUGUGUAGACAUUAUAAACGAA 900 UGUGUAGACAUUAUAAACGTT 1096 CGUUUAUAAUGUCUACACATT 1292 AL-DP-8053

AAUGUGUGUCAGGACAAAAUAAA 901 UGUGUGUCAGGACAAAAUATT 1097 UAUUUUGUCCUGACACACATT 1293 AL-DP-8054 AAACAUUAUAAACGAGCAGAAAA 902 ACAUUAUAAACGAGCAGAATT 1098 UUCUGCUCGUUUAUAAUGUTT 1294 AL-DP-8055 AAAGACAGCGGGUAUGGCAAUAA 903 AGACAGCGGGUAUGGCAAUTT 1099 AUUGCCAUACCCGCUGUCUTT 1295 AL-DP-8056 AAGACAGCGGGUAUGGCAAUAAA 904 GACAGCGGGUAUGGCAAUATT 1100 UAUUGCCAUACCCGCUGUCTT 1296 AL-DP-8057 AAACAGCGGGUAUGGCAAUACAA 905 ACAGCGGGUAUGGCAAUACTT 1101 GUAUUGCCAUACCCGCUGUTT 1297 AL-DP-8058 AAAGCGGGUAUGGCAAUACUGAA 906 AGCGGGUAUGGCAAUACUGTT 1102 CAGUAUUGCCAUACCCGCUTT 1298 AL-DP-8059 AAAACAAUGUGUAGACAUUAUAA 907 AACAAUGUGUAGACAUUAUTT 1103 AUAAUGUCUACACAUUGUUTT 1299 AL-DP-8060 AACAUUAUAAACGAGCAGAAAAA 908 CAUUAUAAACGAGCAGAAATT 1104 UUUCUGCUCGUUUAUAAUGTT 1300 AL-DP-8061 AAACGUUUAAAUGUGUGUCAGAA 909 ACGUUUAAAUGUGUGUCAGTT 1105 CUGACACACAUUUAAACGUTT 1301 AL-DP-8062 AAAAAUGUGUGUCAGGACAAAAA 910 AAAUGUGUGUCAGGACAAATT 1106 UUUGUCCUGACACACAUUUTT 1302 AL-DP-8063 AAGGUUCUAAAACGAAAGUAUAA 911 GGUUCUAAAACGAAAGUAUTT 1107 AUACUUUCGUUUUAGAACCTT 1303 AL-DP-8064 AAAUGUGUAGACAUUAUAAACAA 912 AUGUGUAGACAUUAUAAACTT 1108 GUUUAUAAUGUCUACACAUTT 1304 AL-DP-8065 oo AAAGUAAGACCAUUUAAAAGUAA 913 AGUAAGACCAUUUAAAAGUTT 1109 ACUUUUAAAUGGUCUUACUTT 1305 AL-DP-8066 AAUAGUAAGACCAUUUAAAAGAA 914 UAGUAAGACCAUUUAAAAGTT 1110 CUUUUAAAUGGUCUUACUATT 1306 AL-DP-8067 AAGAAUUAGUAAGACCAUUUAAA 915 GAAUUAGUAAGACCAUUUATT 1111 UAAAUGGUCUUACUAAUUCTT 1307 AL-DP-8068 AAAAUUAGUAAGACCAUUUAAAA 916 AAUUAGUAAGACCAUUUAATT 1112 UUAAAUGGUCUUACUAAUUTT 1308 AL-DP-8069 AAAUUAGUAAGACCAUUUAAAAA 917 AUUAGUAAGACCAUUUAAATT 1113 UUUAAAUGGUCUUACUAAUTT 1309 AL-DP-8070 AAUUAGUAAGACCAUUUAAAAAA 918 UUAGUAAGACCAUUUAAAATT 1114 UUUUAAAUGGUCUUACUAATT 1310 AL-DP-8071 AAAAUACUGAAGUGGAAACUCAA 919 AAUACUGAAGUGGAAACUCTT 1115 GAGUUUCCACUUCAGUAUUTT 1311 AL-DP-8072 AAAUACUGAAGUGGAAACUCAAA 920 AUACUGAAGUGGAAACUCATT 1116 UGAGUUUCCACUUCAGUAUTT 1312 AL-DP-8073 AACAAUACUGAAGUGGAAACUAA 921 CAAUACUGAAGUGGAAACUTT 1117 AGUUUCCACUUCAGUAUUGTT 1313 AL-DP-8074 AAUACUGAAGUGGAAACUCAGAA 922 UACUGAAGUGGAAACUCAGTT 1118 CUGAGUUUCCACUUCAGUATT 1314 AL-DP-8075 AAACUGAAGUGGAAACUCAGCAA 923 ACUGAAGUGGAAACUCAGCTT 1119 GCUGAGUUUCCACUUCAGUTT 1315 AL-DP-8076 AACUGAAGUGGAAACUCAGCAAA 924 CUGAAGUGGAAACUCAGCATT 1120 UGCUGAGUUUCCACUUCAGTT 1316 AL-DP-8077 AAUGAAGUGGAAACUCAGCAGAA 925 UGAAGUGGAAACUCAGCAGTT 1121 CUGCUGAGUUUCCACUUCATT 1317 AL-DP-8078 AAGAAGUGGAAACUCAGCAGAAA 926 GAAGUGGAAACUCAGCAGATT 1122 UCUGCUGAGUUUCCACUUCTT 1318 AL-DP-8079 AAAAGUGGAAACUCAGCAGAUAA 927 AAGUGGAAACUCAGCAGAUTT 1123 AUCUGCUGAGUUUCCACUUTT 1319 AL-DP-8080 AAAGUGGAAACUCAGCAGAUGAA 928 AGUGGAAACUCAGCAGAUGTT 1124 CAUCUGCUGAGUUUCCACUTT 1320 AL-DP-8081

AAAUGGCAAUACUGAAGUGGAAA 929 AUGGCAAUACUGAAGUGGATT 1125 UCCACUUCAGUAUUGCCAUTT 1321 AL-DP-8082 AAAAAUCCUUUUUCUCAAGGAAA 930 AAAUCCUUUUUCUCAAGGATT 1126 UCCUUGAGAAAAAGGAUUUTT 1322 AL-DP-8083 AAUCCUUUUUCUCAAGGACGUAA 931 UCCUUUUUCUCAAGGACGUTT 1127 ACGUCCUUGAGAAAAAGGATT 1323 AL-DP-8084

AAAUCCUUUUUCUCAAGGACGAA 932 AUCCUUUUUCUCAAGGACGTT 1128 CGUCCUUGAGAAAAAGGAUTT 1324 AL-DP-8085 AAGGCAAUACUGAAGUGGAAAAA 933 GGCAAUACUGAAGUGGAAATT 1129 UUUCCACUUCAGUAUUGCCTT 1325 AL-DP-8086

AACUUUUUCUCAAGGACGUGGAA 934 CUUUUUCUCAAGGACGUGGTT 1130 CCACGUCCUUGAGAAAAAGTT 1326 AL-DP-8087 AACCUUUUUCUCAAGGACGUGAA 935 CCUUUUUCUCAAGGACGUGTT 1131 CACGUCCUUGAGAAAAAGGTT 1327 AL-DP-8088 AAUUUUUCUCAAGGACGUGGUAA 936 UUUUUCUCAAGGACGUGGUTT 1132 ACCACGUCCUUGAGAAAAATT 1328 AL-DP-8089 AAUGGAAAUCCUUUUUCUCAAAA 937 UGGAAAUCCUUUUUCUCAATT 1133 UUGAGAAAAAGGAUUUCCATT 1329 AL-DP-8090 AAGGAAAUCCUUUUUCUCAAGAA 938 GGAAAUCCUUUUUCUCAAGTT 1134 CUUGAGAAAAAGGAUUUCCTT 1330 AL-DP-8091 AAGAAAUCCUUUUUCUCAAGGAA 939 GAAAUCCUUUUUCUCAAGGTT 1135 CCUUGAGAAAAAGGAUUUCTT 1331 AL-DP-8092 AAAAUCCUUUUUCUCAAGGACAA 940 AAUCCUUUUUCUCAAGGACTT 1136 GUCCUUGAGAAAAAGGAUUTT 1332 AL-DP-8093 AAUAUGGCAAUACUGAAGUGGAA 941 UAUGGCAAUACUGAAGUGGTT 1137 CCACUUCAGUAUUGCCAUATT 1333 AL-DP-8094

CO AAUGGCAAUACUGAAGUGGAAAA 942 UGGCAAUACUGAAGUGGAATT 1138 UUCCACUUCAGUAUUGCCATT 1334 AL-DP-8095 AAGCAAUACUGAAGUGGAAACAA 943 GCAAUACUGAAGUGGAAACTT 1139 GUUUCCACUUCAGUAUUGCTT 1335 AL-DP-8096 AAAAUGUGUAGACAUUAUAAAAA 944 AAUGUGUAGACAUUAUAAATT 1140 UUUAUAAUGUCUACACAUUTT 1336 AL-DP-8097

Testing of siRNA Targeting HPV £1 Gene Expression

Unmodified and chemically modified dsRNA were tested to identify their relative abilities to reduce the expression level of mRNA encoding HPV El gene in a cell.

The assay conditions employed were as follows: C33A cells were obtained from ATCC. Sequences encoding HPV 16 E6 and El were cloned into the pNAS-055 vector (Husken et al,. Nucleic Acids Research, 31:elO2, 2003), for expression as YFP fusion transcripts. The resulting plasmids were transfected into C33A cells, and stable lines expressing these fusion transcripts were derived by Zeocin selection, as per the manufacturer's protocol (Invitrogen). For transfection with siRNA against HPVl 6 E6 or HPVl 6 El, respective cells were seeded at a density of 2.0 x 10⁴ cells/well in 96-well plates and transfected directly. Transfection of siRNA (3OnM, 3nM or 300pm as indicated) was carried out in a single dose with lipofectamine 2000® (Invitrogen) as described by the manufacturer.

24 hours after transfection,cells were lysed and fusion YFP mRNA expression levels were quantified with the Quantigene Explore Kit (Panomics, Inc. (Fremont, CA)(formerly Genospectra, Inc.)) using a probe directed against YFP, according to the standard protocol. Fusion- YFP mRNA levels were normalized to GAP-DH mRNA. For each siRNA four individual datapoints were collected. siRNA duplexes unrelated to the HPVl 6 El or E6 genes were used as control. The activity of a given siRNA duplex was expressed as percent fusion- YFP mRNA concentration in treated cells relative to concentration of the same transcript in cells treated with the control siRNA duplex.

Table 6 shows the results of testing the El dsRNA of the invention.

%mRNA remaining after

Duplex treatement dsRNA at 30OpM S. D.

ND-9061 41.45 10.69

ND-9062 30.67 10.43

ND-9063 61.87 22.99

ND-9064 40.79 22.73

ND-9065 68.58 28.46

ND-9066 23.51 7.60

ND-9067 37.13 13.60

ND-9068 34.50 17.21

ND-9069 40.61 12.42

ND-9070 32.61 8.73

ND-9071 30.68 11.65

ND-9072 24.38 7.47

ND-9073 76.28 15.06

ND-9074 29.11 10.42

ND-9075 27.20 11.56

ND-9076 42.06 17.88

ND-9077 51.19 9.09

ND-9078 43.42 16.63

ND-9079 25.79 4.85

ND-9080 29.33 5.67

ND-9081 36.66 4.51

ND-9082 48.67 10.47

ND-9083 39.51 12.70

ND-9084 44.28 7.54

ND-9085 55.73 9.77

ND-9086 28.90 7.93

ND-9087 28.88 5.47

ND-9088 45.35 11.67

ND-9089 49.13 12.46

ND-9090 41.76 5.88

ND-9091 31.35 9.16 ND-9092 23.79 8.74

ND-9093 47. 62 9.89

ND-9094 91. 33 29.84

ND-9095 43. 33 8.69

ND-9096 63. 53 11.44

ND-9097 30. 51 4.48

ND-9098 40. 76 10.57

ND-9099 37. 61 9.94

ND-9100 106. 18 30.69

ND-9101 37. 75 16.37

ND-9102 41. 98 14.66

ND-9103 98. 17 14.30

ND-9104 29. 61 11.44

ND-9105 29. 71 6.48

ND-9106 51. 42 14.12

ND-9107 78. 38 28.72

ND-9108 34. 69 4.19

ND-9109 97. 63 14.18

ND-9110 47. 58 7.48

ND-9111 65. 14 15.02

ND-9112 30. 24 7.33

ND-9113 31. 69 10.80

ND-9114 108. 54 7.17

ND-9115 87. 16 14.74

ND-9116 56. 35 14.69

ND-9117 33. 79 8.42

ND-9118 65. 12 19.60

ND-9119 33. 37 12.37

ND-9120 70. 98 18.74

ND-9121 39. 37 10.06

ND-9122 33. 24 14.79

ND-9123 20. 37 7.53

ND-9124 30. 47 5.18

ND-9125 26. 22 5.56

ND-9126 29. 86 5.15

ND-9127 84. 95 22.37

ND-9128 35. 14 6.10

ND-9129 49. 41 15.75

ND-9130 51. 54 12.31 ND-9131 45.51 7.96

ND-9132 81.48 16.52

ND-9133 46.79 13.27

ND-9134 63.22 32.12

ND-9135 118.82 19.88

ND-9136 47.83 12.16

ND-9137 65.11 15.44

ND-9138 92.31 36.27

ND-9139 42.01 10.70

ND-9140 40.54 7.24

ND-9141 101.31 24.39

ND-9142 33.83 7.06

ND-9143 86.43 16.50

ND-9144 33.94 11.74

ND-9145 41.93 12.85

ND-9146 118.24 29.81

ND-9147 69.90 30.13

ND-9148 40.74 6.28

ND-9149 65.26 10.10

ND-9150 36.62 4.85

ND-9151 27.83 4.48

ND-9152 88.99 9.86

ND-9153 66.45 33.75

ND-9154 45.42 8.86

ND-9155 63.55 8.36

ND-9156 53.00 7.71

ND-9157 32.74 7.39

ND-9158 102.06 26.87

ND-9159 59.47 10.16

ND-9160 31.23 7.52

ND-9161 84.78 36.89

ND-9162 24.83 5.17

ND-9163 26.64 5.90

ND-9164 77.97 10.06

ND-9165 59.95 25.75

ND-9166 69.74 8.15

ND-9167 23.04 5.43

ND-9168 46.16 12.02

ND-9169 62.24 11.73 ND-9170 92.69 14.72

ND-9171 46.55 6.56

ND-9172 49.39 16.23

ND-9173 98.36 37.53

ND-9174 44.90 13.73

ND-9175 69.98 18.22

ND-9176 60.73 13.02

ND-9177 70.93 10.18

ND-9178 62.53 7.70

ND-9179 76.68 31.77

ND-9180 66.35 10.48

ND-9181 78.42 12.70

ND-9182 72.09 28.88

ND-9183 58.97 28.59

ND-9184 97.06 8.62

ND-9185 85.29 16.92

ND-9186 77.52 18.17

ND-9187 60.16 36.16

ND-9188 58.61 39.92

ND-9189 69.35 30.11

ND-9190 71.87 36.13

ND-9191 81.64 18.99

ND-9192 52.76 14.33

ND-9193 25.18 8.23

ND-9194 50.69 12.78

ND-9195 40.01 10.21

ND-9196 47.41 15.85

ND-9197 94.68 24.60

ND-9198 103.12 27.52

ND-9199 50.82 15.18

ND-9200 97.72 24.20

AL-DP-8042 111.14 34.54

AL-DP-8043 131.44 38.69

AL-DP-8044 28.60 11.52

AL-DP-8045 120.81 36.35

AL-DP-8046 93.19 17.57

AL-DP-8047 66.27 5.06

AL-DP-8048 33.70 8.18

AL-DP-8049 34.31 7.16 AL-DP-8050 60.60 19.36

AL-DP-8051 66.49 12.36

AL-DP-8052 45.46 12.49

AL-DP-8053 121.92 29.06

AL-DP-8054 45.00 4.56

AL-DP-8055 51.64 9.55

AL-DP-8056 35.51 4.67

AL-DP-8057 45.89 8.82

AL-DP-8058 38.47 4.44

AL-DP-8059 34.97 7.85

AL-DP-8060 66.44 14.39

AL-DP-8061 52.17 12.80

AL-DP-8062 100.52 25.88

AL-DP-8063 43.83 8.22

AL-DP-8064 26.25 5.84

AL-DP-8065 107.74 32.53

AL-DP-8066 94.13 13.45

AL-DP-8067 107.09 17.49

AL-DP-8068 48.99 10.40

AL-DP-8069 68.14 19.39

AL-DP-8070 60.42 11.52

AL-DP-8071 71.76 13.75

AL-DP-8072 62.25 6.16

AL-DP-8073 31.33 7.21

AL-DP-8074 47.97 11.55

AL-DP-8075 51.35 14.67

AL-DP-8076 50.40 17.25

AL-DP-8077 38.99 8.15

AL-DP-8078 50.93 11.54

AL-DP-8079 32.27 10.82

AL-DP-8080 33.91 10.48

AL-DP-8081 31.45 6.72

AL-DP-8082 26.41 7.99

AL-DP-8083 86.75 6.66

AL-DP-8084 112.73 25.79

AL-DP-8085 112.33 22.53

AL-DP-8086 39.84 12.22

AL-DP-8087 104.24 29.47

AL-DP-8088 59.29 13.99 AL-DP-8089 114.08 24.06

AL-DP-8090 35.69 6.75

AL-DP-8091 47.28 12.14

AL-DP-8092 92.85 19.28

AL-DP-8093 102.59 15.83

AL-DP-8094 87.51 18.86

AL-DP-8095 27J99 8.27

AL-DP-8096 31.74 7.52

AL-DP-8097 40.29 9.18

Design of dsRNA Targeting HPV E6 Gene Expression

Table 7 sets forth dsRNA compositions of the invention.

Table 7

Upper case letters: unmodified ribonucleotide (except for T which is an unmodified deoxyribonucleotide)

Lower case letters: ribonucloetide bearing 2'-O-methyl substituent on ribose moiety s : Indicates position of phosphorothioate internucleoside linkage chol : cholesterol moiety conjugated to 3' ribonucleotide.

'duplex name'means the name of the composition formed by specific hybridization of the indicated sense strand and the indicated antisense strand.

AAUCGGUGGACCGGUCGAUGUAA 1336 UCGGUGGACCGGUCGAUGUTT 1424 ACAUCGACCGGUCCACCGATT 1586 ND-8899

<-O

AAGGUCGGUGGACCGGUCGAUAA 1337 GGUCGGUGGACCGGUCGAUTT 1425 AUCGACCGGUCCACCGACCTT 1587 ND-8900

AACGGUGGACCGGUCGAUGUAAA 1338 CGGUGGACCGGUCGAUGUATT 1426 UACAUCGACCGGUCCACCGTT 1588 ND-8901

AAGUCGGUGGACCGGUCGAUGAA 1339 GUCGGUGGACCGGUCGAUGTT 1427 CAUCGACCGGUCCACCGACTT 1589 ND-8902

AAAUCAUCAAGAACACGUAGAAA 1340 AUCAUCAAGAACACGUAGATT 1428 UCUACGUGUUCUUGAUGAUTT 1590 ND-8903

AACAACAGUUACUGCGACGUGAA 1341 CAACAGUUACUGCGACGUGTT 1429 CACGUCGCAGUAACUGUUGTT 1591 ND-8904

AACAAUACAACAAACCGUUGUAA 1342 CAAUACAACAAACCGUUGUTT 1430 ACAACGGUUUGUUGUAUUGTT 1592 ND-8905

AAGCUGCAAACAACUAUACAUAA 1343 GCUGCAAACAACUAUACAUTT 1431 AUGUAUAGUUGUUUGCAGCTT 1593 ND-8906

AAGGUGGACCGGUCGAUGUAUAA 1344 GGUGGACCGGUCGAUGUAUTT 1432 AUACAUCGACCGGUCCACCTT 1594 ND-8907

AAAAAUUAGUGAGUAUAGACAAA 1345 AAAUUAGUGAGUAUAGACATT 1433 UGUCUAUACUCACUAAUUUTT 1595 ND-8908

AAUCAUCAAGAACACGUAGAGAA 1346 UCAUCAAGAACACGUAGAGTT 1434 CUCUACGUGUUCUUGAUGATT 1596 ND-8909

AAAUACAACAAACCGUUGUGUAA 1347 AUACAACAAACCGUUGUGUTT 1435 ACACAACGGUUUGUUGUAUTT 1597 ND-8910

AAUGGACCGGUCGAUGUAUGUAA 1348 UGGACCGGUCGAUGUAUGUTT 1436 ACAUACAUCGACCGGUCCATT 1598 ND-8911

AAUACAACAAACCGUUGUGUGAA 1349 UACAACAAACCGUUGUGUGTT 1437 CACACAACGGUUUGUUGUATT 1599 ND-8912

AAAGAUUCCAUAAUAUAAGGGAA 1350 AGAUUCCAUAAUAUAAGGGTT 1438 CCCUUAUAUUAUGGAAUCUTT 1600 ND-8913

AACAAGCAACAGUUACUGCGAAA 1351 CAAGCAACAGUUACUGCGATT 1439 UCGCAGUAACUGUUGCUUGTT 1601 ND-8914

AAGUUAAUUAGGUGUAUUAACAA 1352 GUUAAUUAGGUGUAUUAACTT 1440 GUUAAUACACCUAAUUAACTT 1602 ND-8915

AAUUUGCUUUUCGGGAUUUAUAA 1353 UUUGCUUUUCGGGAUUUAUTT 1441 AUAAAUCCCGAAAAGCAAATT 1603 ND-8916

AAACUUUGCUUUUCGGGAUUUAA 1354 ACUUUGCUUUUCGGGAUUUTT 1442 AAAUCCCGAAAAGCAAAGUTT 1604 ND-8917

AACUGCAAACAACUAUACAUGAA 1355 CUGCAAACAACUAUACAUGTT 1443 CAUGUAUAGUUGUUUGCAGTT 1605 ND-8918

AAAUGACUUUGCUUUUCGGGAAA 1356 AUGACUUUGCUUUUCGGGATT 1444 UCCCGAAAAGCAAAGUCAUTT 1606 ND-8919

AACGACCCAGAAAGUUACCACAA 1357 CGACCCAGAAAGUUACCACTT 1445 GUGGUAACUUUCUGGGUCGTT 1607 ND-8920

AAUUACUGCGACGUGAGGUAUAA 1358 UUACUGCGACGUGAGGUAUTT 1446 AUACCUCACGUCGCAGUAATT 1608 ND-8921

AAGUUACUGCGACGUGAGGUAAA 1359 GUUACUGCGACGUGAGGUATT 1447 UACCUCACGUCGCAGUAACTT 1609 ND-8922

<£>

M AAUGCGACGUGAGGUAUAUGAAA 1360 UGCGACGUGAGGUAUAUGATT 1448 UCAUAUACCUCACGUCGCATT 1610 ND-8923

AAGUCGAUGUAUGUCUUGUUGAA 1361 GUCGAUGUAUGUCUUGUUGTT 1449 CAACAAGACAUACAUCGACTT 1611 ND-8924

AACGACGUGAGGUAUAUGACUAA 1362 CGACGUGAGGUAUAUGACUTT 1450 AGUCAUAUACCUCACGUCGTT 1612 ND-8925

AAGACUUUGCUUUUCGGGAUUAA 1363 GACUUUGCUUUUCGGGAUUTT 1451 AAUCCCGAAAAGCAAAGUCTT 1613 ND-8926

AAUUAGGUGUAUUAACUGUCAAA 1364 UUAGGUGUAUUAACUGUCATT 1452 UGACAGUUAAUACACCUAATT 1614 ND-8927

AAUUACCACAGUUAUGCACAGAA 1365 UUACCACAGUUAUGCACAGTT 1453 CUGUGCAUAACUGUGGUAATT 1615 ND-8928

AAGCAACAGUUACUGCGACGUAA 1366 GCAACAGUUACUGCGACGUTT 1454 ACGUCGCAGUAACUGUUGCTT 1616 ND-8929

AAUGCUUUUCGGGAUUUAUGCAA 1367 UGCUUUUCGGGAUUUAUGCTT 1455 GCAUAAAUCCCGAAAAGCATT 1617 ND-8930

AAUUAGUGAGUAUAGACAUUAAA 1368 UUAGUGAGUAUAGACAUUATT 1456 UAAUGUCUAUACUCACUAATT 1618 ND-8931

AAUAAUUAGGUGUAUUAACUGAA 1369 UAAUUAGGUGUAUUAACUGTT 1457 CAGUUAAUACACCUAAUUATT 1619 ND-8932

AAGAUGUAUGUCUUGUUGCAGAA 1370 GAUGUAUGUCUUGUUGCAGTT 1458 CUGCAACAAGACAUACAUCTT 1620 ND-8933

AACCGGUCGAUGUAUGUCUUGAA 1371 CCGGUCGAUGUAUGUCUUGTT 1459 CAAGACAUACAUCGACCGGTT 1621 ND-8934

AAGGAGCGACCCAGAAAGUUAAA 1372 GGAGCGACCCAGAAAGUUATT 1460 UAACUUUCUGGGUCGCUCCTT 1622 ND-8935

AAGAGCGACCCAGAAAGUUACAA 1373 GAGCGACCCAGAAAGUUACTT 1461 GUAACUUUCUGGGUCGCUCTT 1623 ND-8936

AAUGAGUAUAGACAUUAUUGUAA 1374 UGAGUAUAGACAUUAUUGUTT 1462 ACAAUAAUGUCUAUACUCATT 1624 ND-8937

AAAAUACAACAAACCGUUGUGAA 1375 AAUACAACAAACCGUUGUGTT 1463 CACAACGGUUUGUUGUAUUTT 1625 ND-8938

AAGUAUGUCUUGUUGCAGAUCAA 1376 GUAUGUCUUGUUGCAGAUCTT 1464 GAUCUGCAACAAGACAUACTT 1626 ND-8939

AACUUUGCUUUUCGGGAUUUAAA 1377 CUUUGCUUUUCGGGAUUUATT 1465 UAAAUCCCGAAAAGCAAAGTT 1627 ND-8940

AAAUUAGUGAGUAUAGACAUUAA 1378 AUUAGUGAGUAUAGACAUUTT 1466 AAUGUCUAUACUCACUAAUTT 1628 ND-8941

AAAAGAUUCCAUAAUAUAAGGAA 1379 AAGAUUCCAUAAUAUAAGGTT 1467 CCUUAUAUUAUGGAAUCUUTT 1629 ND-8942

AAGGUCGAUGUAUGUCUUGUUAA 1380 GGUCGAUGUAUGUCUUGUUTT 1468 AACAAGACAUACAUCGACCTT 1630 ND-8943

AACAUCAAGAACACGUAGAGAAA 1381 CAUCAAGAACACGUAGAGATT 1469 UCUCUACGUGUUCUUGAUGTT 1631 ND-8944

AAAACAGUUACUGCGACGUGAAA 1382 AACAGUUACUGCGACGUGATT 1470 UCACGUCGCAGUAACUGUUTT 1632 ND-8945

CD u> AAACAGUUACUGCGACGUGAGAA 1383 ACAGUUACUGCGACGUGAGTT 1471 CUCACGUCGCAGUAACUGUTT 1633 ND-8946

AAGUGUGAUUUGUUAAUUAGGAA 1384 GUGUGAUUUGUUAAUUAGGTT 1472 CCUAAUUAACAAAUCACACTT 1634 ND-8947

AAAUCAAGAACACGUAGAGAAAA 1385 AUCAAGAACACGUAGAGAATT 1473 UUCUCUACGUGUUCUUGAUTT 1635 ND-8948

AAUUUCGGGAUUUAUGCAUAGAA 1386 UUUCGGGAUUUAUGCAUAGTT 1474 CUAUGCAUAAAUCCCGAAATT 1636 ND-8949

AAACCCACAGGAGCGACCCAGAA 1387 ACCCACAGGAGCGACCCAGTT 1475 CUGGGUCGCUCCUGUGGGUTT 1637 ND-8950

AAAGAUGGGAAUCCAUAUGCUAA 1388 AGAUGGGAAUCCAUAUGCUTT 1476 AGCAUAUGGAUUCCCAUCUTT 1638 ND-8951

AAUAGUGAGUAUAGACAUUAUAA 1389 UAGUGAGUAUAGACAUUAUTT 1477 AUAAUGUCUAUACUCACUATT 1639 ND-8952

AAUGUGUGAUUUGUUAAUUAGAA 1390 UGUGUGAUUUGUUAAUUAGTT 1478 CUAAUUAACAAAUCACACATT 1640 ND-8953

AAUUAAUUAGGUGUAUUAACUAA 1391 UUAAUUAGGUGUAUUAACUTT 1479 AGUUAAUACACCUAAUUAATT 1641 ND-8954

AAAUAUGACUUUGCUUUUCGGAA 1392 AUAUGACUUUGCUUUUCGGTT 1480 CCGAAAAGCAAAGUCAUAUTT 1642 ND-8955

AACGGUCGAUGUAUGUCUUGUAA 1393 CGGUCGAUGUAUGUCUUGUTT 1481 ACAAGACAUACAUCGACCGTT 1643 ND-8956

AACAGGACCCACAGGAGCGACAA 1394 CAGGACCCACAGGAGCGACTT 1482 GUCGCUCCUGUGGGUCCUGTT 1644 ND-8957

AAUUUUCGGGAUUUAUGCAUAAA 1395 UUUUCGGGAUUUAUGCAUATT 1483 UAUGCAUAAAUCCCGAAAATT 1645 ND-8958

AAAAACAACUAUACAUGAUAUAA 1396 AAACAACUAUACAUGAUAUTT 1484 AUAUCAUGUAUAGUUGUUUTT 1646 ND-8959

AAUCCAUAUGCUGUAUGUGAUAA 1397 UCCAUAUGCUGUAUGUGAUTT 1485 AUCACAUACAGCAUAUGGATT 1647 ND-8960

AAUAUUCUAAAAUUAGUGAGUAA 1398 UAUUCUAAAAUUAGUGAGUTT 1486 ACUCACUAAUUUUAGAAUATT 1648 ND-8961

AAUAUGGAACAACAUUAGAACAA 1399 UAUGGAACAACAUUAGAACTT 1487 GUUCUAAUGUUGUUCCAUATT 1649 ND-8962

AAGUCUUGUUGCAGAUCAUCAAA 1400 GUCUUGUUGCAGAUCAUCATT 1488 UGAUGAUCUGCAACAAGACTT 1650 ND-8963

AAUAUUAACUGUCAAAAGCCAAA 1401 UAUUAACUGUCAAAAGCCATT 1489 UGGCUUUUGACAGUUAAUATT 1651 ND-8964

AAACCAAAAGAGAACUGCAAUAA 1402 ACCAAAAGAGAACUGCAAUTT 1490 AUUGCAGUUCUCUUUUGGUTT 1652 ND-8965

AAAAUUAGUGAGUAUAGACAUAA 1403 AAUUAGUGAGUAUAGACAUTT 1491 AUGUCUAUACUCACUAAUUTT 1653 ND-8966

AACAGAUCAUCAAGAACACGUAA 1404 CAGAUCAUCAAGAACACGUTT 1492 ACGUGUUCUUGAUGAUCUGTT 1654 ND-8967

AAUAUGCAUAGUAUAUAGAGAAA 1405 UAUGCAUAGUAUAUAGAGATT 1493 UCUCUAUAUACUAUGCAUATT 1655 ND-8968

AAAGAGAUGGGAAUCCAUAUGAA 1406 AGAGAUGGGAAUCCAUAUGTT 1494 CAUAUGGAUUCCCAUCUCUTT 1656 ND-8969

AAAGUGAGUAUAGACAUUAUUAA 1407 AGUGAGUAUAGACAUUAUUTT 1495 AAUAAUGUCUAUACUCACUTT 1657 ND-8970

AAUUCUAAAAUUAGUGAGUAUAA 1408 UUCUAAAAUUAGUGAGUAUTT 1496 AUACUCACUAAUUUUAGAATT 1658 ND-8971

AAAUGCAUAGUAUAUAGAGAUAA 1409 AUGCAUAGUAUAUAGAGAUTT 1497 AUCUCUAUAUACUAUGCAUTT 1659 ND-8972 ucGGuGGAccGGucGAuGuTsT 1498 AcAUCGACCGGUCcACCGATsT 1660 ND-8987

GGucGGuGGAccGGucGAuTsT 1499 AUCGACCGGUCcACCGACCTsT 1661 ND-8988 cGGuGGAccGGucGAuGuATsT 1500 uAcAUCGACCGGUCcACCGTsT 1662 ND-8989

GucGGuGGAccGGucGAuGTsT 1501 cAUCGACCGGUCcACCGACTsT 1663 ND-8990

AucAucAAGAAcAcGuAGATsT 1502 UCuACGUGUUCUUGAUGAUTsT 1664 ND-8991 cAAcAGuuAcuGcGAcGuGTsT 1503 cACGUCGcAGuAACUGUUGTsT 1665 ND-8992 cAAuAcAAcAAAccGuuGuTsT 1504 AcAACGGUUUGUUGuAUUGTsT 1666 ND-8993

GcuGcAAAcAAcuAuAcAuTsT 1505 AUGuAuAGUUGUUUGcAGCTsT 1667 ND-8994

GGuGGAccGGucGAuGuAuTsT 1506 AuAcAUCGACCGGUCcACCTsT 1668 ND-8995

AAAuuAGuGAGuAuAGAcATsT 1507 UGUCuAuACUcACuAAUϋUTsT 1669 ND-8996 ucAucAAGAAcAcGuAGAGTsT 1508 CUCuACGUGUUCUUGAUGATsT 1670 ND-8997

AuAcAAcAAAccGuuGuGuTsT 1509 AcAcAACGGUUUGUUGuAUTsT 1671 ND-8998 uGGAccGGucGAuGuAuGuTsT 1510 AcAuAcAUCGACCGGUCcATsT 1672 ND-8999 uAcAAcAAAccGuuGuGuGTsT 1511 cAcAcAACGGUUUGUUGuATsT 1673 ND-9000

AGAuuccAuAAuAuAAGGGTsT 1512 CCCUuAuAUuAUGGAAUCUTsT 1674 ND-9001 cAAGcAAcAGuuAcuGcGATsT 1513 UCGcAGuAACUGUUGCUUGTsT 1675 ND-9002

GuuAAuuAGGuGuAuuAAcTsT 1514 GUuAAuAcACCuAAUuAACTsT 1676 ND-9003 uuuGcuuuucGGGAuuuAuTsT 1515 AuAAAUCCCGAAAAGcAAATsT 1677 ND-9004 to AcuuuGcuuuucGGGAuuuTsT 1516 AAAUCCCGAAAAGcAAAGUTsT 1678 ND-9005

^ cuGcAAAcAAcuAuAcAuGTsT 1517 cAUGuAuAGUUGUUUGcAGTsT 1679 ND-9006

AuGAcuuuGcuuuucGGGATsT 1518 UCCCGAAAAGcAAAGUcAUTsT 1680 ND-9007 cGAcccAGAAAGuuAccAcTsT 1519 GUGGuAACUUUCUGGGUCGTsT 1681 ND-9008 uuAcuGcGAcGuGAGGuAuTsT 1520 AuACCϋcACGUCGcAGuAATsT 1682 ND-9009

GuuAcuGcGAcGuGAGGuATsT 1521 uACCUcACGUCGcAGuAACTsT 1683 ND-9010 uGcGAcGuGAGGuAuAuGATsT 1522 UcAuAuACCUcACGUCGcATsT 1684 ND-9011

GucGAuGuAuGucuuGuuGTsT 1523 cAAcAAGAcAuAcAUCGACTsT 1685 ND-9012 cGAcGuGAGGuAuAuGAcuTsT 1524 AGUcAuAuACCUcACGUCGTsT 1686 ND-9013

GAcuuuGcuuuucGGGAuuTsT 1525 AAUCCCGAAAAGcAAAGUCTsT 1687 ND-9014

UuAGGuGuAuuAAcuGucATsT 1526 UGAcAGUuAAuAcACCuAATsT 1688 ND-9015 uuAccAcAGuuAuGcAcAGTsT 1527 CUGUGcAuAACUGUGGuAATsT 1689 ND- 9016

GcAAcAGuuAcuGcGAcGuTsT 1528 ACGUCGcAGuAACUGUUGCTsT 1690 ND- 9017

uGcuuuucGGGAuuuAuGcTsT 1529 GcAuAAAUCCCGAAAAGcATsT 1691 ND-9018 uuAGuGAGuAuAGAcAuuATsT 1530 uAAUGUCuAuACUcACuAATsT 1692 ND-9019 uAAuuAGGuGuAuuAAcuGTsT 1531 cAGUuAAuAcACCuAAUuATsT 1693 ND-9020

GAuGuAuGucuuGuuGcAGTsT 1532 CUGcAAcAAGAcAuAcAUCTsT 1694 ND-9021 ccGGucGAuGuAuGucuuGTsT 1533 cAAGAcAuAcAUCGACCGGTsT 1695 ND-9022

GGAGcGAcccAGAAAGuuATsT 1534 uAACUUUCUGGGUCGCUCCTsT 1696 ND-9023

GAGcGAcccAGAAAGuuAcTsT 1535 GuAACUUUCUGGGUCGCUCTsT 1697 ND-9024 uGAGuAuAGAcAuuAuuGuTsT 1536 AcAAuAAUGUCuAuACUcATsT 1698 ND-9025

AAuAcAAcAAAccGuuGuGTsT 1537 cAcAACGGUUUGUUGuAUUTsT 1699 ND-9026

GuAuGucuuGuuGcAGAucTsT 1538 GAUCUGcAAcAAGAcAuACTsT 1700 ND-9027 cuuuGcuuuucGGGAuuuATsT 1539 uAAAUCCCGAAAAGcAAAGTsT 1701 ND-9028 io AuuAGuGAGuAuAGAcAuuTsT 1540 AAUGUCuAuACUcACuAAUTsT 1702 ND-9029

AAGAuuccAuAAuAuAAGGTsT 1541 CCUuAuAUuAUGGAAUCUUTsT 1703 ND-9030

GGucGAuGuAuGucuuGuuTsT 1542 AAcAAGAcAuAcAUCGACCTsT 1704 ND-9031 cAucAAGAAcAcGuAGAGATsT 1543 UCUCuACGUGUUCUUGAUGTsT 1705 ND-9032

AAcAGuuAcuGcGAcGuGATsT 1544 UcACGUCGcAGuAACUGUUTsT 1706 ND-9033

AcAGuuAcuGcGAcGuGAGTsT 1545 CUcACGUCGcAGuAACUGUTsT 1707 ND-9034

GuGuGAuuuGuuAAuuAGGTsT 1546 CCuAAUuAAcAAAUcAcACTsT 1708 ND-9035

AucAAGAAcAcGuAGAGAATsT 1547 UUCUCuACGUGUUCUUGAUTsT 1709 ND-9036 uuucGGGAuuuAuGcAuAGTsT 1548 CuAUGcAuAAAUCCCGAAATsT 1710 ND-9037

AcccAcAGGAGcGAcccAGTsT 1549 CuGGGUCGCUCCuGuGGGUTsT 1711 ND-9038

AGAuGGGAAuccAuAuGcuTsT 1550 AGcAuAUGGAUUCCcAUCUTsT 1712 ND-9039 uAGuGAGuAuAGAcAuuAuTsT 1551 AuAAUGUCuAuACUcACuATsT 1713 ND-9040

uGuGuGAuuuGuuAAuuAGTsT 1552 CuAAUuAAcAAAUcAcAcATsT 1714 ND-9041 uuAAuuAGGuGuAuuAAcuTsT 1553 AGUuAAuAcACCuAAUUAATsT 1715 ND-9042

AuAuGAcuuuGcuuuucGGTsT 1554 CCGAAAAGcAAAGUcAuAUTsT 1716 ND-9043 cGGucGAuGuAuGucuuGuTsT 1555 AcAAGAcAuAcAUCGACCGTsT 1717 ND-9044 cAGGAcccAcAGGAGcGAcTsT 1556 GUCGCUCCuGuGGGUCCUGTsT 1718 ND-9045 uuuucGGGAuuuAuGcAuATsT 1557 uAUGcAuAAAUCCCGAAAATsT 1719 ND-9046

AAAcAAcuAuAcAuGAuAuTsT 1558 AuAUcAUGuAuAGUUGUUUTsT 1720 ND-9047 uccAuAuGcuGuAuGuGAuTsT 1559 AUcAcAuAcAGcAuAUGGATsT 1721 ND-9048 uAuucuAAAAuuAGuGAGuTsT 1560 ACUcACuAAUUUuAGAAuATsT 1722 ND-9049 uAuGGAAcAAcAuuAGAAcTsT 1561 GUUCuAAUGUUGUUCcAuATsT 1723 ND-9050

CD GucuuGuuGcAGAucAucATsT 1562 UGAUGAUCUGcAAcAAGACTsT 1724 ND-9051 uAuuAAcuGucAAAAGccATsT 1563 UGGCUUUUGAcAGUuAAuATST 1725 ND-9052

AccAAAAGAGAAcuGcAAuTsT 1564 AUUGcAGUUCUCUUUUGGUTsT 1726 ND-9053

AAuuAGuGAGuAuAGAcAuTsT 1565 AUGUCuAuACUcACuAAUUTsT 1727 ND-9054 cAGAucAucAAGAAcAcGuTsT 1566 ACGuGUUCUuGAuGAUCuGTsT 1728 ND-9055 uAuGcAuAGuAuAuAGAGATsT 1567 UCUCuAuAuACuAUGcAuATsT 1729 ND-9056

AGAGAuGGGAAuccAuAuGTST 1568 cAuAUGGAUUCCcAUCUCUTST 1730 ND-9057

AGuGAGuAuAGAcAuuAuuTsT 1569 AAuAAUGUCuAuACUcACUTsT 1731 ND-9058 uucuAAAAuuAGuGAGuAuTsT 1570 AuACUcACuAAUUUuAGAATsT 1732 ND-9059

AuGcAuAGuAuAuAGAGAuTsT 1571 AUCUCuAuAuACuAUGcAUTsT 1733 ND-9060

AAGUGAUUUGUUAAUUAGGUGAA 1410 GUGAUUUGUUAAUUAGGUGTT 1572 CACCUAAUUAACAAAUCACTT 1734 AL-DP-7778

AAUGAUUUGUUAAUUAGGUGUAA 1411 UGAUUUGUUAAUUAGGUGUTT 1573 ACACCUAAUUAACAAAUCATT 1735 AL-DP-7779

AAGAUUUGUUAAUUAGGUGUAAA 1412 GAUUUGUUAAUUAGGUGUATT 1574 UACACCUAAUUAACAAAUCTT 1736 AL-DP-7780

AAAUUUGUUAAUUAGGUGUAUAA 1413 AUUUGUUAAUUAGGUGUAUTT 1575 AUACACCUAAUUAACAAAUTT 1737 AL-DP-7781

AAUGUGAUUUGUUAAUUAGGUAA 1414 UGUGAUUUGUUAAUUAGGUTT 1576 ACCUAAUUAACAAAUCACATT 1738 AL-DP-7782

AAUGUAUGGAACAACAUUAGAAA 1415 UGUAUGGAACAACAUUAGATT 1577 UCUAAUGUUGUUCCAUACATT 1739 AL-DP-7783

AAGUAUGGAACAACAUUAGAAAA 1416 GUAUGGAACAACAUUAGAATT 1578 UUCUAAUGUUGUUCCAUACTT 1740 AL-DP-7784

AAUGUGUACUGCAAGCAACAGAA 1417 UGUGUACUGCAAGCAACAGTT 1579 CUGUUGCUUGCAGUACACATT 1741 AL-DP-7803

AAACUGCGACGUGAGGUAUAUAA 1418 ACUGCGACGUGAGGUAUAUTT 1580 AUAUACCUCACGUCGCAGUTT 1742 7804

AAGAGGUAUAUGACUUUGCUUAA 1419 GAGGUAUAUGACUUUGCUUTT 1581 AAGCAAAGUCAUAUACCUCTT 1743 AL-DP-7805

AAAUGCUGUAUGUGAUAAAUGAA 1420 AUGCUGUAUGUGAUAAAUGTT 1582 CAUUUAUCACAUACAGCAUTT 1744 AL-DP-7807

AAUUUAUUCUAAAAUUAGUGAAA 1421 UUUAUUCUAAAAUUAGUGATT 1583 UCACUAAUUUUAGAAUAAATT 1745 AL-DP-7808

AACUGCGACGUGAGGUAUAUGAA 1422 CUGCGACGUGAGGUAUAUGTT 1584 CAUAUACCUCACGUCGCAGTT 1746 AL-DP-7810

AAACCGUUGUGUGAUUUGUUAAA 1423 ACCGUUGUGUGAUUUGUUATT 1585 UAACAAAUCACACAACGGUTT 1747 AL-DP-7812

CD CO

Testing of siRNA Targeting HPV E6 Gene Expression

Unmodified and chemically modified dsRNA were tested to identify their relative abilities to reduce the expression level of mRNA encoding HPV E6 gene in a cell.

The assay conditions employed were as follows: C33A cells were obtained from ATCC. Sequences encoding HPVl 6 E6 and El were cloned into the pNAS-055 vector (Husken et al,. Nucleic Acids Research, 31:elO2, 2003), for expression as YFP fusion transcripts. The resulting plasmids were transfected into C33A cells, and stable lines expressing these fusion transcripts were derived by Zeocin selection, as per the manufacturer's protocol (ϊnvitrogen). For transfection with siRNA against HPV 16 E6 or HPVl 6 El, respective cells were seeded at a density of 2.0 x 10⁴ cells/well in 96-well plates and transfected directly. Transfection of siRNA (3OnM, 3nM or 300pm as indicated) was carried out in a single dose with lipofectamine 2000® (Invitrogen) as described by the manufacturer.

24 hours after transfection,cells were lysed and fusion YFP mRNA expression levels were quantified with the Quantigene Explore Kit (Panomics, Inc. (Fremont, CA)(formerly Genospectra, Inc.)) using a probe directed against YFP, according to the standard protocol. Fusion- YFP mRNA levels were normalized to GAP-DH mRNA. For each siRNA four individual datapoints were collected. siRNA duplexes unrelated to the HPV 16 El or E6 genes were used as control. The activity of a given siRNA duplex was expressed as percent fusion- YFP mRNA concentration in treated cells relative to concentration of the same transcript in cells treated with the control siRNA duplex.

Table 8 shows the results of testing the E6 dsRNA of the invention. Table 8

Mean Mean activity activity remaining remaining after 30 after 300 duplex nM pM name treatment sd treatment sd

ND-8899 15.23 3.19 31.29 9.57

ND-8900 11.61 2.88 26.80 10.23

ND-8901 10.88 3.54 24.77 5.19

ND-8902 20.19 7.36 43.46 6.89

ND-8903 10.38 .2.51 22.95 5.47

ND-8904 13.71 4.67 22.11 5.50

ND-8905 13.81 4.29 24.69 4.62

ND-8906 8.35 2.17 24.23 6.62

ND-8907 13.88 3.12 36.94 6.13

ND-8908 14.47 3.48 45.15 7.92

ND-8909 19.99 3.67 49.36 9.80

ND-8910 36.96 9.77 74.18 15.82

ND-8911 18.66 4.19 45.51 6.82

ND-8912 47.42 6.99 76.11 12.97

ND-8913 55.53 16.75 76.63 15.44

ND-8914 9.69 2.50 19.91 6.63

ND-8915 49.02 7.97 93.38 6.83

ND-8916 11.88 2.94 49.78 8.49

ND-8917 14.00 2.04 50.36 8.31

ND-8918 13.70 3.43 29.01 6.51

ND-8919 10.31 2.44 42.51 10.89

ND-8920 10.29 2.72 25.20 9.73

ND-8921 20.23 3.71 37.17 10.15

ND-8922 11.64 2.31 24.95 8.99

ND-8923 12.43 1.97 24.39 8.13 ND-8924 15.19 4.52 32.09 7.01 ND-8925 14.24 1.87 34.21 5.61 ND-8926 10.17 2.85 19.04 3.68 ND-8927 20.77 4.89 41.40 9.23 ND-8928 95.02 20.87 92.24 15.19 ND-8929 17.51 5.27 19.86 6.81 ND-8930 13.58 2.65 61.16 11.03 ND-8931 13.78 2.00 37.55 8.11 ND-8932 105.07 21.10 91.19 12.68 ND-8933 14.88 3.07 43.06 8.64 ND-8934 13.03 3.75 24.32 5.92 ND-8935 13.19 2.88 21.87 4.17 ND-8936 10.04 1.94 21.98 6.92 ND-8937 15.39 3.44 42.70 11.35 ND-8938 55.90 5.56 93.49 10.41 ND-8939 11.51 2.04 29.57 10.38 ND-8940 12.80 2.94 29.67 6.73 ND-8941 19.46 2.91 43.13 3.64 ND-8942 96.02 29.93 85.34 4.57 ND-8943 13.44 3.90 19.03 5.18 ND-8944 14.35 2 09 20.03 4.68 ND-8945 11.45 1 98 23.80 8.36 ND-8946 15.43 2.27 43.12 13.34 ND-8947 13.32 2.20 53.58 18.04 ND-8948 12.85 3.18 23.22 6.79 ND-8949 86.23 23.43 75.99 7.17 ND-8950 29.49 7.99 47.19 14.70 ND-8951 10.51 2.85 20.21 6.23 ND-8952 12.10 2.74 28.82 9.06 ND-8953 41.13 11.23 77.64 9.46 ND-8954 46.52 8.41 81.61 14.93 ND-8955 38.40 8.46 83.38 16.32 ND-8956 12.13 2.23 21.94 9.54 ND-8957 28.39 6.18 53.84 12.53

ND-8958 36.41 9.92 55.67 8.77

ND-8959 33.95 11.23 63.63 5.53

ND-8960 13.63 2.39 26.49 9.24

ND-8961 80.42 18.39 89.13 8.61

ND-8962 33.00 4.18 82.57 9.01

ND-8963 16.67 1.85 28.39 9.50

ND-8964 14.17 2.58 43.74 14.37

ND-8965 17.23 5.15 48.03 10.97

ND-8966 23.01 5.10 53.86 10.10

ND-8967 18.68 5.13 23.30 6.20

ND-8968 10.99 1.83 28.22 9.33

ND-8969 13.75 2.67 32.62 10.53

ND-8970 11.02 2.68 29.14 10.73

ND-8971 21.71 3.11 57.75 10.54

ND-8972 17.10 3.20 52.10 10.84

ND-8987 40.36 7.25 91.12 9.07

ND-8988 20.54 5.27 30.76 12.23

ND-8989 36.60 7.23 74.41 8.61

ND-8990 17.55 8.33 61.02 11.13

ND-8991 11.29 2.87 19.03 5.98

ND-8992 14.49 3.37 44.53 14.74

ND-8993 18.45 5.75 48.07 6.79

ND-8994 13.16 1.80 25.92 7.94

ND-8995 52.21 5.93 90.43 4.86

ND-8996 32.77 6.96 57.54 7.12

ND-8997 14.45 1.50 20.63 4.28

ND-8998 137.83 33.37 90.09 14.65

ND-8999 82.01 13.74 85.69 10.39

ND-9000 69.77 21.32 83.16 14.34

ND-9001 54.71 18.91 74.70 8.87

ND-9002 12.15 2.05 22.98 6.98

ND-9003 76.52 11.49 98.54 7.10 ND-9004 62.23 16.29 84.38 8.22

ND-9005 38.12 6.77 64.57 6.57

ND-9006 12.96 3.15 26.03 4.76

ND-9007 18.24 4.88 42.16 7.87

ND-9008 21.06 4.60 20.01 6.00

ND-9009 35.15 5.62 79.96 7.01

ND-9010 13.71 2.83 53.80 12.21

ND-9011 38.04 3.56 60.45 10.19

ND-9012 ^'44.63 37.28 67.30 8.30

ND-9013 13.31 1.81 31.12 6.40

ND-9014 12.69 3.66 27.50 7.48

ND-9015 16.26 3.61 21.18 4.80

ND-9016 29.49 8.14 66.50 15.07

ND-9017 16.98 2.22 27.17 7.64

ND-9018 35.62 7.31 86.49 7.60

ND-9019 23.48 2.57 60.66 13.05

ND-9020 113.04 21.57 88.75 12.94

ND-9021 38.45 5.44 68.21 9.53

ND-9022 14.21 2.86 53.78 13.38

ND-9023 21.84 3.72 41.95 11.93

ND-9024 117.68 33.94 86.00 6.55

ND-9025 86.38 19.82 81.09 9.82

ND-9026 113.52 9.02 95.62 10.60

ND-9027 13.61 2.09 51.98 15.63

ND-9028 14.49 4.02 45.08 11.80

ND-9029 20.16 3.25 39.00 8.28

ND-9030 104.95 34.72 76.74 10.03

ND-9031 19.90 6.09 26.32 9.90

ND-9032 16.43 3.38 19.10 5.44

ND-9033 100.99 24.54 86.16 11.95

ND-9034 13.77 2.84 33.36 13.56

ND-9035 13.54 1.58 57.07 19.24

ND-9036 12.91 3.20 21.78 6.03 ND-9037 30.90 8.30 74.12 12.35

ND-9038 121.49 24.79 87.65 7.07

ND-9039 10.19 3.13 23.32 9.60

ND-9040 11.45 2.34 22.86 8.27

ND-9041 33.73 8.63 82.99 13.62

ND-9042 18.21 3.81 60.07 13.85

ND-9043 36.15 3.87 71.81 12.23

ND-9044 13.77 3.59 30.27 10.81

ND-9045 56.81 19.55 85.99 9.99

ND-9046 26.03 6.18 51.21 10.14

ND-9047 100.23 24.53 85.98 5.59

ND-9048 21.82 4.07 44.44 12.82

ND-9049 82.93 21.46 87.79 7.07

ND-9050 18.51 3.33 40.70 10.96

ND-9051 22.80 3.37 42.44 14.86

ND-9052 12.61 3.78 37.58 13.35

ND-9053 19.88 4.32 53.11 3.23

ND-9054 33.65 8.32 59.71 6.42

ND-9055 22.61 7.41 27.44 7.04

ND-9056 16.61 3.38 34.34 13.22

ND-9057 25.51 6.29 51.45 10.10

ND-9058 27.60 4.56 54.99 13.52

ND-9059 23.83 4.36 84.76 13.88

ND-9060 17.12 3.29 44.54 15.68

AL-DP-7778 19.35 8.95 63.31 14.21

AL-DP-7779 41.30 9.51 65.96 7.82

AL-DP-7780 24.01 7.52 59.43 8.85

AL-DP-7781 13.69 3.41 53.58 9.31

AL-DP-7782 31.35 5.31 65.84 10.41

AL-DP-7783 14.46 2.85 38.92 10.30

AL-DP-7784 13.52 1.52 25.09 7.89

AL-DP-7803 39.68 4.75 66.72 11.32

7804 12.56 3.96 26.81 6.28 AL-DP-7805 13.92 2.22 35.87 8.95

AL-DP-7807 35.54 4.95 70.94 11.01

AL-DP-7808 81.47 9.77 96.18 10.87

AL-DP-7810 15.14 2.12 37.66 16.19

AL-DP-7812 12.89 1.99 25.18 12.05

Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the instant disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.

Claims

CLAIMSWe claim:

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human E6AP gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding E6AP, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said E6AP, inhibits expression of said E6AP gene by at least 40%.

2. The dsRNA of claim 1, wherein said first sequence is selected from the group consisting of Table 1 and said second sequence is selected from the group consisting of Table 1.

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

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

5. The dsRNA of claims 3 or 4, 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.

6. The dsRNA of claims 3 or 4, wherein said modified nucleotide is chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

7. The dsRNA of claims 3 or 4, wherein said first sequence is selected from the group consisting of Table 1 and said second sequence is selected from the group consisting of Table 1.

8. The dsRNA of claims 6 or 7, wherein said first sequence is selected from the group consisting of Table 1 and said second sequence is selected from the group consisting of Table 1.

9. A cell comprising the dsRNA of claim 1.

10. A pharmaceutical composition for inhibiting the expression of the E6AP gene in an organism, comprising a dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding E6AP, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said E6AP, inhibits expression of said E6AP gene by at least 20%.

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

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

13. A method for inhibiting the expression of the E6AP gene in a cell, the method comprising:

(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 and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding E6AP, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said E6AP, inhibits expression of said E6AP gene 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 the E6AP gene, thereby inhibiting expression of the E6AP gene in the cell.

14. A method of treating, preventing or managing pathological processes mediated by HPV infection comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA, wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding E6AP, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said E6AP, inhibits expression of said E6AP gene by at least 40%.

15. A vector for inhibiting the expression of the E6AP gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA, wherein one of the strands of said dsRNA is substantially complementary to at least a part of a mRNA encoding E6AP and wherein said dsRNA is less than 30 base pairs in length and wherein said dsRNA, upon contact with a cell expressing said E6AP, inhibits the expression of said E6AP gene by at least 40%.

16. A cell comprising the vector of claim 16.

17. A double-stranded ribonucleic acid (dsRNA) for reducing the expression level of a human E6AP gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding E6AP, and wherein said dsRNA, upon contact with a cell expressing said E6AP, reduces the expression level of said E6AP gene.

18. The dsRNA of claim 17, wherein said contact reduces the expression level of said E6AP gene by at least 40%.

19. The dsRNA of claim 17, wherein said contact is performed in vitro at 30 nM or less.

20. A pharmaceutical composition for reducing the expression level of the E6AP gene in an organism, comprising the dsRNA of claim 17 and a pharmaceutically acceptable carrier.

21. A method of treating an HPV associated disorder comprising administering to a patient in need of such treatment, a therapeutically effective amount of a dsRNA of claim 17.

22. A method of treating an E6AP-associated disorder comprising administering to a patient in need of such treatment, a therapeutically effective amount of a dsRNA of claim 17.

23. A dsRNA selected from among those listed on Table 3.

24. A pharmaceutical composition comprising the dsRNA of claim 23.

25. A method of treating an HPV associated disorder comprising administering to a patient in need of such treatment a therapeutically effective amount of a dsRNA of claim 23.

26. A dsRNA selected from among those listed on Table 5.

I l l

27. A pharmaceutical composition comprising the dsRNA of claim 26.

28. A method of treating an HPV associated disorder comprising administering to a patient in need of such treatment a therapeutically effective amount of a dsRNA of claim 26.

29. A dsRNA selected from among those listed on Table 7.

30. A pharmaceutical composition comprising the dsRNA of claim 29.

31. A method of treating an HPV associated disorder comprising administering to a patient in need of such treatment a therapeutically effective amount of a dsRNA of claim 29.

32. A pharmaceutical composition comprising at least two dsRNA selected from among the dsRNA of claim 2, claim 23, claim 26 and claim 29.

33. A method of treating an HPV associated disorder comprising administering to a patient in need of such treatment a therapeutically effective amount of the pharmaceutical composition of claim 32.

34. The dsRNA of claim 26 or claim 29, wherein said dsRNA comprises at least one modified nucleotide.

35. The dsRNA of claim 34, 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.

36. The dsRNA of claim 34, wherein said modified nucleotide is chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.