dsRNA COMPOSITIONS AND METHODS FOR TREATING HPV INFECTIONS

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: Sl -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 HSEL (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. HIV 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 HTV 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 respond to other treatment may have to be removed by surgery. Genital warts have been known to return following physical removal; in these instances ointerferon has been directly injected into these warts. However, a. -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): el51.

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 E6 AP 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'-alkyl-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 human E6AP gene, 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 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 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 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 (Scheffner, M. et al., 1990. Cell. 63:1129-1136; 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 HPV16 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 maybe 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 E3 A (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, NMJ30839.1, and NM 000462.2.

As used herein, "El" refers to the human papillomavirus type 16 (HPV 16) 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 OO 1526, 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 inM PIPES pH 6.4, 1 mM EDTA, 5O ⁰ C or 7O ⁰ C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be

encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes of the invention.

"Complementary" sequences, as used herein, may also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.

The terms "complementary", "fully complementary" and "substantially complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is "substantially complementary to at least part of a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary

to a contiguous portion of the mRNA of interest (e.g., encoding 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'-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) . _nnn . — • 100%

(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.

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.

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 lthe 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, 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, 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 can readily be made using the reference sequence and the target sequence provided.

In addition, the RNAi agents provided in Table 1 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 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 dsRN A 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 nig/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 Sc 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 Iigand 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-chloroethyl)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: 1116-8). 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-(hexadecyI)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, 75, 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 maybe 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-niolecule 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 incorporation of a 2'-O-methyl, 2'-0-ethyl, 2'-O-propyl, 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, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2'-O- methyl, 2'-0-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-Modifier C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the 5'-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'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5 ⁽ to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free-acid forms are also included.

Representative 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 internucleoside 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 CH ₂ 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. RY. 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, 10:11 1; 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,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a

polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such 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 3 sets out patterns of chemical modifications preferred for use with the duplex dsRNA set out in Table 1 of the invention.

Table 3

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-lip id 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-[l,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 Morrissey, 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., ribavirin, 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 HPV 16 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. Thus the invention contemplates the use of 2, 3, 4, 5 or more dsRNA duplexes selected from among Table 1 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.

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, papilloadenocystomas, papilloma neuropathicum, 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 I-III), 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- 11 , 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 £ene

siRNA design was carried out to identify siRNAs targeting human ubiquitin protein ligase E3A (ube3 A, also referred to as E6AP). Human mRNA sequences to E6AP representing different isoforms (NM l 30838.1, NMJ 30839.1, 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-specifϊc off-target potential, the following assumptions were made:

1) complementarity to a target gene in positions 2 to 9 (counting 5' to 3') of a strand (seed region) maybe 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 19mer 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 in descending order according to the off-target score and sequences containing stretches with more than 3 Gs in a row were excluded from selection.

The 327 siRNAs with an off-target score of >=3 were selected and synthesized (Table 1).

Table 1 dsRNA targeting E6AP

GUGAUAGCUCACAGGGAGA 1 GUGAUAGCUCACAGGGAGATT 328 UCUCCCUGUGAGCUAUCACTT 655 ND-10118

CUCAGCUUACCUUGAGAAC 2 CUCAGCUUACCUUGAGAACTT 329 GUUCUCAAGGUAAGCUGAGTT 656 ND-10119

GAGGUUUUCAUAUGGUGAC 3 GAGGUUUUCAUAUGGUGACTT 330 GUCACCAUAUGAAAACCUCTT 657 ND-10120

AAUCCUGCAGACUUGAAGA 4 AAUCCUGCAGACUUGAAGATT 331 UCUUCAAGUCUGCAGGAUUTT 658 ND-10121

GGUGAUAGCUCACAGGGAG 5 GGUGAUAGCUCACAGGGAGTT 332 CUCCCUGUGAGCUAUCACCTT 659 ND-10122

AAAUGGUUUACUAUGCAAA 6 AAAUGGUUUACUAUGCAAATT 333 UUUGCAUAGUAAACCAUUUTT 660 ND-10123

CAGUUAACUGAGGGCUGUG 7 CAGUUAACUGAGGGCUGUGTT 334 CACAGCCCUCAGUUAACUGTT 661 ND-10124

ACUACAUUCUCAAUAAAUC 8 ACUACAUUCUCAAUAAAUCTT 335 GAUUUAUUGAGAAUGUAGUTT 662 ND-10125

CAUCCCUCCAAGAAAGGAG 9 CAUCCCUCCAAGAAAGGAGTT 336 CUCCUUUCUUGGAGGGAUGTT 663 ND-10126

GAUUAGGGAGUUCUGGGAA 10 GAUUAGGGAGUUCUGGGAATT 337 UUCCCAGAACUCCCUAAUCTT 664 ND-10127

UGAUAGCUCACAGGGAGAC 11 UGAUAGCUCACAGGGAGACTT 338 GUCUCCCUGUGAGCUAUCATT 665 ND-10128

UCCACAGUCCUGAAUAUCU 12 UCCACAGUCCUGAAUAUCUTT 339 AGAUAUUCAGGACUGUGGATT 666 ND-10129

CUCCACAGUCCUGAAUAUC 13 CUCCACAGUCCUGAAUAUCTT 340 GAUAUUCAGGACUGUGGAGTT 667 ND-10130

UGGGUCUGGCUAUUUACAA 14 UGGGUCUGGCUAUUUACAATT 341 UUGUAAAUAGCCAGACCCATT 668 ND-10131

AAAAUCCUGCAGACUUGAA 15 AAAAUCCUGCAGACUUGAATT 342 UUCAAGUCUGCAGGAUUUUTT 669 ND-10132

AAAAUGGUUUACUAUGCAA 16 AAAAUGGUUUACUAUGCAATT 343 UUGCAUAGUAAACCAUUUUTT 670 ND-10133

CAAAGGAUUUGGCAUGCUG 17 CAAAGGAUUUGGCAUGCUGTT 344 CAGCAUGCCAAAUCCUUUGTT 671 ND-10134

CACGAAUGAGUUUUGUGCU 18 CACGAAUGAGUUUUGUGCUTT 345 AGCACAAAACUCAUUCGUGTT 672 ND-10135

CUUUUCGUGACUUGGGAGA 19 CUUUUCGUGACUUGGGAGATT 346 UCUCCCAAGUCACGAAAAGTT 673 ND-10136

UGGUCUAAAUACAAUGCAG 20 UGGUCUAAAUACAAUGCAGTT 347 CUGCAUUGUAUUUAGACCATT 674 ND-10137

GUGGUCUAAAUACAAUGCA 21 GUGGUCUAAAUACAAUGCATT 348 UGCAUUGUAUUUAGACCACTT 675 ND-10138

AGGUGAUAGCUCACAGGGA 22 AGGUGAUAGCUCACAGGGATT 349 UCCCUGUGAGCUAUCACCUTT 676 ND-10-139

CCUCGAGCUUUAUAAGAUU 23 CCUCGAGCUUUAUAAGAUUTT 350 AAUCUUAUAAAGCUCGAGGTT 677 ND-10140

GAGAACUCGAAAGGUGCCC 24 GAGAACUCGAAAGGUGCCCTT 351 GGGCACCUUUCGAGUUCUCTT 678 ND-10141

ACCAGUUAACUGAGGGCUG 25 ACCAGUUAACUGAGGGCUGTT 352 CAGCCCUCAGUUAACUGGUTT 679 ND-10142

CCUGCACGAAUGAGUUUUG 26 CCUGCACGAAUGAGUUUUGTT 353 CAAAACUCAUUCGUGCAGGTT 680 ND-10143

CCCUCGAGCUUUAUAAGAU 27 CCCUCGAGCUUUAUAAGAUTT 354 AUCUUAUAAAGCUCGAGGGTT 681 ND-10144

CUUACCUUGAGAACUCGAA 28 CUUACCUUGAGAACUCGAATT 355 UUCGAGUUCUCAAGGUAAGTT 682 ND-10145

CUGUGGUCUAAAUACAAUG 29 CUGUGGUCUAAAUACAAUGTT 356 CAUUGUAUUUAGACCACAGTT 683 ND-10146

UCAGACUGUGGUCUAAAUA 3O UCAGACUGUGGUCUAAAUATT 357 UAUUUAGACCACAGUCUGATT 684 ND-10147

UUGAGAACUCGAAAGGUGC 31 UUGAGAACUCGAAAGGUGCTT 358 GCACCUUUCGAGUUCUCAATT 685 ND-10148

AGCUUAGUUCAAGGACAGC 32 AGCUUAGUUCAAGGACAGCTT 359 GCUGUCCUUGAACUAAGCUTT 686 ND-10149

CAGCUUAGUUCAAGGACAG 33 CAGCUUAGUUCAAGGACAGTT 360 CUGUCCUUGAACUAAGCUGTT 687 ND-10150

GACUUACUUAACAGAAGAG 34 GACUUACUUAACAGAAGAGTT 361 CUCUUCUGUUAAGUAAGUCTT 688 ND-101S1

UUCAUAUGGUGACCAAUGA 35 UUCAUAUGGUGACCAAUGATT 362 UCAUUGGUCACCAUAUGAATT 689 ND-10152

CAUAUGGUGACCAAUGAAU 36 CAUAUGGUGACCAAUGAAUTT 363 AUUCAUUGGUCACCAUAUGTT 690 ND-10153

AUAGAAAUCUCCACAGUCC 37 AUAGAAAUCUCCACAGUCCTT 364 GGACUGUGGAGAUUUCUAUTT 691 ND-10154

AUUCUGACUACAUUCUCAA 38 AUUCUGACUACAUUCUCAATT 365 UUGAGAAUGUAGUCAGAAUTT 692 ND-10155

GCACGAAUGAGUUUUGUGC 39 GCACGAAUGAGUUUUGUGCTT 366 GCACAAAACUCAUUCGUGCTT 693 ND-10156

GCAUUGGUACAGAGCUUCC 40 GCAUUGGUACAGAGCUUCCTT 367 GGAAGCUCUGUACCAAUGCTT 694 ND-10157

CACCAGUUAACUGAGGGCU 41 CACCAGUUAACUGAGGGCUTT 368 AGCCCUCAGUUAACUGGUGTT 695 ND-10158

GCUUACCUUGAGAACUCGA 42 GCUUACCUUGAGAACUCGATT 369 UCGAGUUCUCAAGGUAAGCTT 696 ND-10159

UUACCUUGAGAACUCGAAA 43 UUACCUUGAGAACUCGAAATT 370 UUUCGAGUUCUCAAGGUAATT 697 ND-10160

GAUUUGGCAUGCUGUAAAA 44 GAUUUGGCAUσCUGUAAAATT 371 UUUUACAGCAUGCCAAAUCTT 698 ND-10161

UGCUUGAAAAUGGUUUACU 45 UGCUUGAAAAUGGUUUACUTT 372 AGUAAACCAUUUUCAAGCATT 699 ND-10162

UGCACGAAUGAGUUUUGUG 46 UGCACGAAUGAGUUUUGUGTT 373 CACAAAACUCAUUCGUGCATT 700 ND-10163

UUUCGUGACUUGGGAGACU 47 UUUCGUGACUUGGGAGACUTT 374 AGUCUCCCAAGUCACGAAATT 701 ND-10164

UGAUCAGACUGUGGUCUAA 48 UGAUCAGACUGUGGUCUAATT 375 UUAGACCACAGUCUGAUCATT 702 ND-10165

UUCUGACUACAUUCUCAAU 49 UUCUGACUACAUUCUCAAUTT 376 AUUGAGAAUGUAGUCAGAATT 703 ND-10166

GUGCUUGAAAAUGGUUUAC 50 GUGCUUGAAAAUGGUUUACTT 377 GUAAACCAUUUUCAAGCACTT 704 ND-10167

UUAUCUGAAUUUGUUCAUU 51 UUAUCUGAAUUUGUUCAUUTT 378 AAUGAACAAAUUCAGAUAATT 705 ND-10168

AAUAGAAAUCUCCACAGUC 52 AAUAGAAAUCUCCACAGUCTT 379 GACUGUGGAGAUUUCUAUUTT 706 ND-10169

GACUGUGGUCUAAAUACAA 53 GACUGUGGUCUAAAUACAATT 380 UUGUAUUUAGACCACAGUCTT 707 ND-10170

CUACAUUCUCAAUAAAUCA 54 CUACAUUCUCAAUAAAUCATT 381 UGAUUUAUUGAGAAUGUAGTT 708 ND-10171

AGAUGUGACUUACUUAACA 55 AGAUGUGACUUACUUAACATT 382 UGUUAAGUAAGUCACAUCUTT 709 ND-10172

AUUUGGCAUGCUGUAAAAC 56 AUUUGGCAUGCUGUAAAACTT 383 GUUUUACAGCAUGCCAAAUTT 710 ND-10173

GUACAUUUUCCCAUGGUUG 57 GUACAUUUUCCCAUGGUUGTT 384 CAACCAUGGGAAAAUGUACTT 711 ND-10174

UCGUAAUGGAGAAUAGAAA 58 UCGUAAUGGAGAAUAGAAATT 385 UUUCUAUUCUCCAUUACGATT 712 ND-10175

CUUAGUUCAAGGACAGCAG 59 CUUAGUUCAAGGACAGCAGTT 386 CUGCUGUCCUUGAACUAAGTT 713 ND-10176

GUCUAAAUACAAUGCAGAC 60 GUCUAAAUACAAUGCAGACTT 387 GUCUGCAUUGUAUUUAGACTT 714 ND-10177

UGUACAUUUUCCCAUGGUU 61 UGUACAUUUUCCCAUGGUUTT 388 AACCAUGGGAAAAUGUACATT 715 ND-10178

UUGGUGUUAAAACCCUGGA 62 UUGGUGUUAAAACCCUGGATT 389 UCCAGGGUUUUAACACCAATT 716 ND-10179

UUUUCCCAUGGUUGUCUAC 63 UUUUCCCAUGGUUGUCUACTT 390 GUAGACAACCAUGGGAAAATT 717 ND-10180

UUUAUUAAUGAACCACUGA 64 UUUAUUAAUGAACCACUGATT 391 UCAGUGGUUCAUUAAUAAATT 718 ND-10181

GGGUCUACACCAGAUUGCU 65 GGGUCUACACCAGAUUGCUTT 392 AGCAAUCUGGUGUAGACCCTT 719 ND-10182

CUGACUACAUUCUCAAUAA 66 CUGACUACAUUCUCAAUAATT 393 UUAUUGAGAAUGUAGUCAGTT 720 ND-10183

GCCCAAGGAAAACUGAUCA 67 GCCCAAGGAAAACUGAUCATT 394 UGAUCAGUUUUCCUUGGGCTT 721 ND-10184

AUCAGACUGUGGUCUAAAU 68 AUCAGACUGUGGUCUAAAUTT 395 AUUUAGACCACAGUCUGAUTT 722 ND-10185

GAAUUCGCAUGUACAGUGA 69 GAAUUCGCAUGUACAGUGATT 396 UCACUGUACAUGCGAAUUCTT 723 ND-10186

GAUCGCUAUGGAAAAUCCU 70 GAUCGCUAUGGAAAAUCCUTT 397 AGGAUUUUCCAUAGCGAUCTT 724 ND-10187

AUCGCUAUGGAAAAUCCUG 71 AUCGCUAUGGAAAAUCCUGTT 398 CAGGAUUUUCCAUAGCGAUTT 725 ND-10188

UUAGGGAGUUCUGGGAAAU 72 UUAGGGAGUUCUGGGAAAUTT 399 AUUUCCCAGAACUCCCUAATT 726 ND-10189

GAUUAUAGCCAAAAAUGGC 73 GAUUAUAGCCAAAAAUGGCTT 400 GCCAUUUUUGGCUAUAAUCTT 727 ND-10190

AGAUGAUCGCUAUGGAAAA 74 AGAUGAUCGCUAUGGAAAATT 401 UUUUCCAUAGCGAUCAUCUTT 728 ND-10191

AGAUAUCACAGACAGAUCU 75 AGAUAUCACAGACAGAUCUTT 402 AGAUCUGUCUGUGAUAUCUTT 729 ND-10192

GGUAUGUUCACAUACGAUG 76 GGUAUGUUCACAUACGAUGTT 403 CAUCGUAUGUGAACAUACCTT 730 ND-10193

ACAAUAGAAUUCGCAUGUA 77 ACAAUAGAAUUCGCAUGUATT 404 UACAUGCGAAUUCUAUUGUTT 731 ND-10194

GGCUAGAGAUGAUCGCUAU 78 GGCUAGAGAUGAUCGCUAUTT 405 AUAGCGAUCAUCUCUAGCCTT 732 ND-10195

CUGUCCAACUUUUCUUCGU 79 CUGUCCAACUUUUCUUCGUTT 406 ACGAAGAAAAGUUGGACAGTT 733 ND-10196

AUGCACUUGUCCGGCUAGA 80 AUGCACUUGUCCGGCUAGATT 407 UCUAGCCGGACAAGUGCAUTT 734 ND-10197

AAUCCAGAUAUUGGUAUGU 81 AAUCCAGAUAUUGGUAUGUTT 408 ACAUACCAAUAUCUGGAUUTT 735 ND-10198

UCCGGCUAGAGAUGAUCGC 82 UCCGGCUAGAGAUGAUCGCTT 409 GCGAUCAUCUCUAGCCGGATT 736 ND-10199

GAUUGUCGAAAACCACUUA 83 GAUUGUCGAAAACCACUUATT 410 UAAGUGGUUUUCGACAAUCTT 737 ND-10200

UGAUCGCUAUGGAAAAUCC 84 UGAUCGCUAUGGAAAAUCCTT 411 GGAUUUUCCAUAGCGAUCATT 738 ND-10201

AUGAUCGCUAUGGAAAAUC 85 AUGAUCGCUAUGGAAAAUCTT 412 GAUUUUCCAUAGCGAUCAUTT 739 ND-10202

CUUAUAUGUGGAAGCCGGA 86 CUUAUAUGUGGAAGCCGGATT 413 UCCGGCUUCCACAUAUAAGTT 740 ND-10203

GAUAUCACAGACAGAUCUU 87 GAUAUCACAGACAGAUCUUTT 414 AAGAUCUGUCUGUGAUAUCTT 741 ND-10204

ACUUAUUCAGACCAGAAGA 88 ACUUAUUCAGACCAGAAGATT 415 UCUUCUGGUCUGAAUAAGUTT 742 ND-10205

UGUCCAACUUUUCUUCGUA 89 UGUCCAACUUUUCUUCGUATT 416 UACGAAGAAAAGUUGGACATT 743 ND-10206

UUAUAUGUGGAAGCCGGAA 90 UUAUAUGUGGAAGCCGGAATT 417 UUCCGGCUUCCACAUAUAATT 744 ND-10207

AGCAGUUGAAUCCAUAUUU 91 AGCAGUUGAAUCCAUAUUUTT 418 AAAUAUGGAUUCAACUGCUTT 745 ND-10208

UGCAAAUGUAGUGGGAGGG 92 UGCAAAUGUAGUGGGAGGGTT 419 CCCUCCCACUACAUUUGCATT 746 ND-10209

GAUGCACUUGUCCGGCUAG 93 GAUGCACUUGUCCGGCUAGTT 420 CUAGCCGGACAAGUGCAUCTT 747 ND-10210

GAUGAUGCACUUGUCCGGC 94 GAUGAUGCACUUGUCCGGCTT 421 GCCGGACAAGUGCAUCAUCTT 748 ND-10211

ACUUGUCCGGCUAGAGAUG 95 ACUUGUCCGGCUAGAGAUGTT 422 CAUCUCUAGCCGGACAAGUTT 749 ND-10212

AAGGUUACCUACAUCUCAU 96 AAGGUUACCUACAUCUCAUTT 423 AUGAGAUGUAGGUAACCUUTT 750 ND-10213

AGAUGAUUAUAGCCAAAAA 97 AGAUGAUUAUAGCCAAAAATT 424 UUUUUGGCUAUAAUCAUCUTT 751 ND-10214

GACUUGGGAGACUCUCACC 98 GACUUGGGAGACUCUCACCTT 425 GGUGAGAGUCUCCCAAGUCTT 752 ND-10215

ACUUUUCUUCGUAUGGAUA 99 ACUUUUCUUCGUAUGGAUATT 426 UAUCCAUACGAAGAAAAGUTT 753 ND-10216

AAAGGUUACCUACAUCUCA 100 AAAGGUUACCUACAUCUCATT 427 UGAGAUGUAGGUAACCUUUTT 754 ND-10217

GACAAUAGAAUUCGCAUGU 101 GACAAUAGAAUUCGCAUGUTT 428 ACAUGCGAAUUCUAUUGUCTT 755 ND-10218

UUUACAAUAACUGUAUACU 102 UUUACAAUAACUGUAUACUTT 429 AGUAUACAGUUAUUGUAAATT 756 ND-10219

UUACUCUGAUUGGCAUAGU 103 UUACUCUGAUUGGCAUAGUTT 430 ACUAUGCCAAUCAGAGUAATT 757 ND-10220

CUGGCUAUUUACAAUAACU 104 CUGGCUAUUUACAAUAACUTT 431 AGUUAUUGUAAAUAGCCAGTT 758 ND-10221

GUAUGUUCACAUACGAUGA 105 GUAUGUUCACAUACGAUGATT 432 UCAUCGUAUGUGAACAUACTT 759 ND-10222

AGUCAUAAGCAAUGAAUUU 106 AGUCAUAAGCAAUGAAUUUTT 433 AAAUUCAUUGCUUAUGACUTT 760 ND-10223

GUUGAAUCCAUAUUUGAGA 107 GUUGAAUCCAUAUUUGAGATT 434 UCUCAAAUAUGGAUUCAACTT 761 ND-10224

UUAAUGCAAAACUCUGUGA 108 UUAAUGCAAAACUCUσUGATT 435 UCACAGAGUUUUGCAUUAATT 762 ND-10225

UGCACUUGUCCGGCUAGAG 109 UGCACUUGUCCGGCUAGAGTT 436 CUCUAGCCGGACAAGUGCATT 763 ND-10226

CCCAAUGAUGUAUGAUCUA 110 CCCAAUGAUGUAUGAUCUATT 437 UAGAUCAUACAUCAUUGGGTT 764 ND-10227

UACUUAUUCAGACCAGAAG 111 UACUUAUUCAGACCAGAAGTT 438 CUUCUGGUCUGAAUAAGUATT 765 ND-10228

AUAUUGGUAUGUUCACAUA 112 AUAUUGGUAUGUUCACAUATT 439 UAUGUGAACAUACCAAUAUTT 766 ND-10229

AAACUCUGUGAUCCUCAUC 113 AAACUCUGUGAUCCUCAUCTT 440 GAUGAGGAUCACAGAGUUUTT 767 ND-10230

UUCCAGAUAUCACAGACAG 114 UUCCAGAUAUCACAGACAGTT 441 CUGUCUGUGAUAUCUGGAATT 768 ND-10231

GAUCACUUUCCAGAUAUCA 115 GAUCACUUUCCAGAUAUCATT 442 UGAUAUCUGGAAAGUGAUCTT 769 ND-10232

AUCACUUUCCAGAUAUCAC 116 AUCACUUUCCAGAUAUCACTT 443 GUGAUAUCUGGAAAGUGAUTT 770 ND-10233

CACUUUCCAGAUAUCACAG 117 CACUUUCCAGAUAUCACAGTT 444 CUGUGAUAUCUGGAAAGUGTT 771 ND-10234

CCAGACACAGAAAGGUUAC 118 CCAGACACAGAAAGGUUACTT 445 GUAACCUUUCUGUGUCUGGTT 772 ND-10235

AAUAACUGUAUACUGGAUσ 119 AAUAACUGUAUACUGGAUGTT 446 CAUCCAGUAUACAGUUAUUTT 773 ND-10236

UCACUUUCCAGAUAUCACA 120 UCACUUUCCAGAUAUCACATT 447 UGUGAUAUCUGGAAAGUGATT 774 ND-10237

UUUAUGACAUGUCCCUUUA 121 UUUAUGACAUGUCCCUUUATT 448 UAAAGGGACAUGUCAUAAATT 775 ND-10238

AGACACAGAAAGGUUACCU 122 AGACACAGAAAGGUUACCUTT 449 AGGUAACCUUUCUGUGUCUTT 776 ND-10239

CCAAAGGAUUUGGCAUGCU 123 CCAAAGGAUUUGGCAUGCUTT 450 AGCAUGCCAAAUCCUUUGGTT 777 ND-10240

CCUACAUCUCAUACUUGCU 124 CCUACAUCUCAUACUUGCUTT 451 AGCAAGUAUGAGAUGUAGGTT 778 ND-10241

CCCAGACACAGAAAGGUUA 125 CCCAGACACAGAAAGGUUATT 452 UAACCUUUCUGUGUCUGGGTT 779 ND-10242

AUAAAAUUCCAAUUACAAA 126 AUAAAAUUCCAAUUACAAATT 453 UUUGUAAUUGGAAUUUUAUTT 780 ND-10243

AUUAUCGUAAUGGAGAAUA 127 AUUAUCGUAAUGGAGAAUATT 454 UAUUCUCCAUUACGAUAAUTT 781 ND-10244

AGUUAGACGUGACCAUAUC 128 AGUUAGACGUGACCAUAUCTT 455 GAUAUGGUCACGUCUAACUTT 782 ND-10245

AUCGUAAUGGAGAAUAGAA 129 AUCGUAAUGGAGAAUAGAATT 456 UUCUAUUCUCCAUUACGAUTT 783 ND-10246

UUCGUAUGGAUAAUAAUGC 130 UUCGUAUGGAUAAUAAUGCTT 457 GCAUUAUUAUCCAUACGAATT 784 ND-10247

ACGUAUCACAAUGUAUACU 131 ACGUAUCACAAUGUAUACUTT 458 AGUAUACAUUGUGAUACGUTT 785 ND-10248

CAUCACGUAUGCCAAAGGA 132 CAUCACGUAUGCCAAAGGATT 459 UCCUUUGGCAUACGUGAUGTT 786 ND-10249

UGUCGAAAACCACUUAUCC 133 UGUCGAAAACCACUUAUCCTT 460 GGAUAAGUGGUUUUCGACATT 787 ND-10250

ACGAUGAAUCUACAAAAUU 134 ACGAUGAAUCUACAAAAUUTT 461 AAUUUUGUAGAUUCAUCGUTT 788 ND-10251

UUACAACGGGCACAGACAG 135 UUACAACGGGCACAGACAGTT 462 CUGUCUGUGCCCGUUGUAATT 789 ND-10252

GAAAUCGUUCAUUCAUUUA 136 GAAAUCGUUCAUUCAUUUATT 463 UAAAUGAAUGAACGAUUUCTT 790 ND-10253

UCUUCGUAUGGAUAAUAAU 137 UCUUCGUAUGGAUAAUAAUTT 464 AUUAUUAUCCAUACGAAGATT 791 ND-10254

CACAUACGAUGAAUCUACA 138 CACAUACGAUGAAUCUACATT 465 UGUAGAUUCAUCGUAUGUGTT 792 ND-10255

UCGAAGUGCUUGAAAAUGG 139 UCGAAGUGCUUGAAAAUGGTT 466 CCAUUUUCAAGCACUUCGATT 793 ND-10256

GUCGAAAACCACUUAUCCC 140 GUCGAAAACCACUUAUCCCTT 467 GGGAUAAGUGGUUUUCGACTT 794 ND-10257

UCACAUACGAUGAAUCUAC 141 UCACAUACGAUGAAUCUACTT 468 GUAGAUUCAUCGUAUGUGATT 795 ND-10258

ACGAAGAAUCACUGUUCUC 142 ACGAAGAAUCACUGUUCUCTT 469 GAGAACAGUGAUUCUUCGUTT 796 ND-10259

ACUCUCGAGAUCCUAAUUA 143 ACUCUCGAGAUCCUAAUUATT 470 UAAUUAGGAUCUCGAGAGUTT 797 ND-10260

UGUAUACUGGAUGUACAUU 144 UGUAUACUGGAUGUACAUUTT 471 AAUGUACAUCCAGUAUACATT 798 ND-10261

UGACCAUAUCAUAGAUGAU 145 UGACCAUAUCAUAGAUGAUTT 472 AUCAUCUAUGAUAUGGUCATT 799 ND-10262

UGCUUAUAUGUGGAAGCCG 146 UGCUUAUAUGUGGAAGCCGTT 473 CGGCUUCCACAUAUAAGCATT 800 ND-10263

UAUACCAGGGACUCUGUUC 147 UAUACCAGGGACUCUGUUCTT 474 GAACAGAGUCCCUGGUAUATT 801 ND-10264

GUACUUAUUCAGACCAGAA 148 GUACUUAUUCAGACCAGAATT 475 UUCUGGUCUGAAUAAGUACTT 802 ND-10265

AUUGGCAUAGUACUGGGUC 149 AUUGGCAUAGUACUGGGUCTT 476 GACCCAGUACUAUGCCAAUTT 803 ND-10266

CUGUAUACUGGAUGUACAU 150 CUGUAUACUGGAUGUACAUTT 477 AUGUACAUCCAGUAUACAGTT 804 ND-10267

UUUUCUUCGUAUGGAUAAU 151 UUUUCUUCGUAUGGAUAAUTT 478 AUUAUCCAUACGAAGAAAATT 805 ND-10268

UCGGAGAGGUUUUCAUAUG 152 UCGGAGAGGUUUUCAUAUGTT 479 CAUAUGAAAACCUCUCCGATT 806 ND-10269

GGGUCAGUUUACUCUGAUU 153 GGGUCAGUUUACUCUGAUUTT 480 AAUCAGAGUAAACUGACCCTT 807 ND-10270

GGUCAGUUUACUCUGAUUG 154 GGUCAGUUUACUCUGAUUGTT 481 CAAUCAGAGUAAACUGACCTT 808 ND-10271

GAAUCUCCCUUAAAGUACU 155 GAAUCUCCCUUAAAGUACUTT 482 AGUACUUUAAGGGAGAUUCTT 809 ND-10272

CCAAUGAAUCUCCCUUAAA 156 CCAAUGAAUCUCCCUUAAATT 483 UUUAAGGGAGAUUCAUUGGTT 810 ND-10273

GACGUGACCAUAUCAUAGA 157 GACGUGACCAUAUCAUAGATT 484 UCUAUGAUAUGGUCACGUCTT 811 ND-10274

ACGUGACCAUAUCAUAGAU 158 ACGUGACCAUAUCAUAGAUTT 485 AUCUAUGAUAUGGUCACGUTT 812 ND-10275

GUUCACAUACGAUGAAUCU 159 GUUCACAUACGAUGAAUCUTT 486 AGAUUCAUCGUAUGUGAACTT 813 ND-10276

UUUAACAGUCGAAAUCUAG 160 UUUAACAGUCGAAAUCUAGTT 487 CUAGAUUUCGACUGUUAAATT 814 ND-10277

GACGGUGGCUAUACCAGGG 161 GACGGUGGCUAUACCAGGGTT 488 CCCUGGUAUAGCCACCGUCTT 815 ND-10278

UCACAAUGUAUACUCUCGA 162 UCACAAUGUAUACUCUCGATT 489 UCGAGAGUAUACAUUGUGATT 816 ND-10279

AUUUAACAGUCGAAAUCUA 163 AUUUAACAGUCGAAAUCUATT 490 UAGAUUUCGACUGUUAAAUTT 817 ND-10280

CCUGGAUUGUCGAAAACCA 164 CCUGGAUUGUCGAAAACCATT 491 UGGUUUUCGACAAUCCAGGTT 818 ND-10281

AAGAUGUGACUUACUUAAC 165 AAGAUGUGACUUACUUAACTT 492 GUUAAGUAAGUCACAUCUUTT 819 ND-10282

CAGACCAGAUUCGGAGAAU 166 CAGACCAGAUUCGGAGAAUTT 493 AUUCUCCGAAUCUGGUCUGTT 820 ND-10283

CUUCGUAUGGAUAAUAAUG 167 CUUCGUAUGGAUAAUAAUGTT 494 CAUUAUUAUCCAUACGAAGTT 821 ND-10284

GACGUAUCACAAUGUAUAC 168 GACGUAUCACAAUGUAUACTT 495 GUAUACAUUGUGAUACGUCTT 822 ND-10285

UCGUAUGGAUAAUAAUGCA 169 UCGUAUGGAUAAUAAUGCATT 496 UGCAUUAUUAUCCAUACGATT 823 ND-10286

CAUACGAUGAAUCUACAAA 170 CAUACGAUGAAUCUACAAATT 497 UUUGUAGAUUCAUCGUAUGTT 824 ND-10287

GACUUGACGUAUCACAAUG 171 GACUUGACGUAUCACAAUGTT 498 CAUUGUGAUACGUCAAGUCTT 825 ND-10288

GCUUUUCGGAGAGGUUUUC 172 GCUUUUCGGAGAGGUUUUCTT 499 GAAAACCUCUCCGAAAAGCTT 826 ND-10289

GGAAAUCGUUCAUUCAUUU 173 GGAAAUCGUUCAUUCAUUUTT 500 AAAUGAAUGAACGAUUUCCTT 827 ND-10290

UCGAAAACCACUUAUCCCU 174 UCGAAAACCACUUAUCCCUTT 501 AGGGAUAAGUGGUUUUCGATT 828 ND-10291

UGUCAAUCUUUAUUCUGAC 175 UGUCAAUCUUUAUUCUGACTT 502 GUCAGAAUAAAGAUUGACATT 829 ND-10292

ACCACUUAUCCCUUUUGAA 176 ACCACUUAUCCCUUUUGAATT 503 UUCAAAAGGGAUAAGUGGUTT 830 ND-10293

GCUUCGAAGUGCUUGAAAA 177 GCUUCGAAGUGCUUGAAAATT 504 UUUUCAAGCACUUCGAAGCTT 831 ND-10294

UUGUCAAUCUUUAUUCUGA 178 UUGUCAAUCUUUAUUCUGATT 505 UCAGAAUAAAGAUUGACAATT 832 ND-10295

UAGACGUGACCAUAUCAUA 179 UAGACGUGACCAUAUCAUATT 5OS UAUGAUAUGGUCACGUCUATT 833 ND-10296

CUGCUUCGAAGUGCUUGAA 180 CUGCUUCGAAGUGCUUGAATT 507 UUCAAGCACUUCGAAGCAGTT 834 ND-10297

CAAGGAUAGGUGAUAGCUC 181 CAAGGAUAGGUGAUAGCUCTT 508 GAGCUAUCACCUAUCCUUGTT 835 ND-10298

UGCACUUGUAUAUUUGUCA 182 UGCACUUGUAUAUUUGUCATT 509 UGACAAAUAUACAAGUGCATT 836 ND-10299

CGUAUCACAAUGUAUACUC 183 CGUAUCACAAUGUAUACUCTT 510 GAGUAUACAUUGUGAUACGTT 837 ND-10300

CGCAUGUACAGUGAACGAA 184 CGCAUGUACAGUGAACGAATT 511 UUCGUUCACUGUACAUGCGTT 838 ND-10301

AUUAGAAGGGUCUACACCA 185 AUUAGAAGGGUCUACACCATT 512 UGGUGUAGACCCUUCUAAUTT 839 ND-10302

UUUGGCAUGCUGUAAAACA 186 UUUGGCAUGCUGUAAAACATT 513 UGUUUUACAGCAUGCCAAATT 840 ND-10303

CCCUUAAAGUACUUAUUCA 187 CCCUUAAAGUACUUAUUCATT 514 UGAAUAAGUACUUUAAGGGTT 841 ND-10304

ACAAUGUAUACUCUCGAGA 188 ACAAUGUAUACUCUCGAGATT 515 UCUCGAGAGUAUACAUUGUTT 842 ND-10305

UGACGGUGGCUAUACCAGG 189 UGACGGUGGCUAUACCAGGTT 516 CCUGGUAUAGCCACCGUCATT 843 ND-10306

UCGAGAUCCUAAUUAUCUG 190 UCGAGAUCCUAAUUAUCUGTT 517 CAGAUAAUUAGGAUCUCGATT 844 ND-10307

UGAGGGUCAGUUUACUCUG 191 UGAGGGUCAGUUUACUCUGTT 518 CAGAGUAAACUGACCCUCATT 845 ND-10308

AUCUCCCUUAAAGUACUUA 192 AUCUCCCUUAAAGUACUUATT 519 UAAGUACUUUAAGGGAGAUTT 846 ND-10309

GAAUUGCUUAUAUGUGGAA 193 GAAUUGCUUAUAUGUGGAATT 520 UUCCACAUAUAAGCAAUUCTT 847 ND-10310

CUCAAGGAUAGGUGAUAGC 194 CUCAAGGAUAGGUGAUAGCTT 521 GCUAUCACCUAUCCUUGAGTT 848 ND-10311

AAAACAGUUCAAGGCUUUU 195 AAAACAGUUCAAGGCUUUUTT 522 AAAAGCCUUGAACUGUUUUTT 849 ND-10312

AGGGAGACAACAAUUUGCA 196 AGGGAGACAACAAUUUGCATT 523 UGCAAAUUGUUGUCUCCCUTT 850 ND-10313

UGUGACUUGACGUAUCACA 197 UGUGACUUGACGUAUCACATT 524 UGUGAUACGUCAAGUCACATT 851 ND-10314

AAUGUGACUUGACGUAUCA 198 AAUGUGACUUGACGUAUCATT 525 UGAUACGUCAAGUCACAUUTT 852 ND-10315

CAAGGCUUUUCGGAGAGGU 199 CAAGGCUUUUCGGAGAGGUTT 526 ACCUCUCCGAAAAGCCUUGTT 853 ND-10316

UCGCAUGUACAGUGAACGA 200 UCGCAUGUACAGUGAACGATT 527 UCGUUCACUGUACAUGCGATT 854 ND-10317

CUAACGUGGAAUGUGACUU 201 CUAACGUGGAAUGUGACUUTT 528 AAGUCACAUUCCACGUUAGTT 855 ND-10318

CACUUAUCCCUUUUGAAGA 202 CACUUAUCCCUUUUGAAGATT 529 UCUUCAAAAGGGAUAAGUGTT 856 ND-10319

AGGACUAGGAAAAUUAAAG 203 AGGACUAGGAAAAUUAAAGTT 530 CUUUAAUUUUCCUAGUCCUTT 857 ND-10320

AGUGAACGAAGAAUCACUG 204 AGUGAACGAAGAAUCACUGTT 531 CAGUGAUUCUUCGUUCACUTT 858 ND-10321

CCAUAUCAUAGAUGAUGCA 205 CCAUAUCAUAGAUGAUGCATT 532 UGCAUCAUCUAUGAUAUGGTT 859 ND-10322

CAACGGGCACAGACAGAGC 206 CAACGGGCACAGACAGAGCTT 533 GCUCUGUCUGUGCCCGUUGTT 860 ND-10323

ACUUAUCCCUUUUGAAGAG 207 ACUUAUCCCUUUUGAAGAGTT 534 CUCUUCAAAAGGGAUAAGUTT 861 ND-10324

UGGAUAAUAAUGCAGCAGC 208 UGGAUAAUAAUGCAGCAGCTT 535 GCUGCUGCAUUAUUAUCCATT 862 ND-10325

GAAUUUAACAGUCGAAAUC 209 GAAUUUAACAGUCGAAAUCTT 536 GAUUUCGACUGUUAAAUUCTT 863 ND-10326

AGAUUUAUUGGAGUAUGAA 210 AGAUUUAUUGGAGUAUGAATT 537 UUCAUACUCCAAUAAAUCUTT 864 ND-10327

CGAAGAAUCACUGUUCUCU 211 CGAAGAAUCACUGUUCUCUTT 538 AGAGAACAGUGAUUCUUCGTT 865 ND-10328

ACUGAGGGUCAGUUUACUC 212 ACUGAGGGUCAGUUUACUCTT 539 GAGUAAACUGACCCUCAGUTT 866 ND-10329

CAGUUUACAACGGGCACAG 213 CAGUUUACAACGGGCACAGTT 540 CUGUGCCCGUUGUAAACUGTT 867 ND-10330

UGAAUUGCUUAUAUGUGGA 214 UGAAUUGCUUAUAUGUGGATT 541 UCCACAUAUAAGCAAUUCATT 868 ND-10331

ACUCAAAGUUAGACGUGAC 215 ACUCAAAGUUAGACGUGACTT 542 GUCACGUCUAACUUUGAGUTT 869 ND-10332

GUGACUUGACGUAUCACAA 216 GUGACUUGACGUAUCACAATT 543 UUGUGAUACGUCAAGUCACTT 870 ND-10333

AUGAAUUUAACAGUCGAAA 217 AUGAAUUUAACAGUCGAAATT 544 UUUCGACUGUUAAAUUCAUTT 871 ND-10334

AUAUGACGGUGGCUAUACC 218 AUAUGACGGUGGCUAUACCTT 545 GGUAUAGCCACCGUCAUAUTT 872 ND-10335

AACGAAGAAUCACUGUUCU 219 AACGAAGAAUCACUGUUCUTT 546 AGAACAGUGAUUCUUCGUUTT 873 ND-10336

GCCAUUAGAAGGGUCUACA 220 GCCAUUAGAAGGGUCUACATT 547 UGUAGACCCUUCUAAUGGCTT 874 ND-10337

UUUAUAAGAUUAAUGCAAA 221 UUUAUAAGAUUAAUGCAAATT 548 UUUGCAUUAAUCUUAUAAATT 875 ND-10338

GAAUCCAUAUUUGAGACUC 222 GAAUCCAUAUUUGAGACUCTT 549 GAGUCUCAAAUAUGGAUUCTT 876 ND-10339

UUAUUGGAGUAUGAAGGGA 223 UUAUUGGAGUAUGAAGGGATT 550 UCCCUUCAUACUCCAAUAATT 877 ND-10340

AGCUUCCGGAAAGUUAAAC 224 AGCUUCCGGAAAGUUAAACTT 551 GUUUAACUUUCCGGAAGCUTT 878 ND-10341

UACUCUGAUUGGCAUAGUA 225 UACUCUGAUUGGCAUAGUATT 552 UACUAUGCCAAUCAGAGUATT 879 ND-10342

AGUUUUUUCUAGUGCUGAG 226 AGUUUUUUCUAGUGCUGAGTT 553 CUCAGCACUAGAAAAAACUTT 880 ND-10343

UGUUCACAUACGAUGAAUC 227 UGUUCACAUACGAUGAAUCTT 554 GAUUCAUCGUAUGUGAACATT 881 ND-10344

AAUCUCCCUUAAAGUACUU 228 AAUCUCCCUUAAAGUACUUTT 555 AAGUACUUUAAGGGAGAUUTT 882 ND-10345

GACUCAAAGUUAGACGUGA 229 GACUCAAAGUUAGACGUGATT 556 UCACGUCUAACUUUGAGUCTT 883 ND-10346

AAUUUAACAGUCGAAAUCU 230 AAUUUAACAGUCGAAAUCUTT 557 AGAUUUCGACUGUUAAAUUTT 884 ND-10347

AUGUGCUUUUACUUCCGGA 231 AUGUGCUUUUACUUCCGGATT 558 UCCGGAAGUAAAAGCACAUTT 885 ND-10348

UUGUCACCUAACGUGGAAU 232 UUGUCACCUAACGUGGAAUTT 559 AUUCCACGUUAGGUGACAATT 886 ND-10349

AUGUGACUUGACGUAUCAC 233 AUGUGACUUGACGUAUCACTT 560 GUGAUACGUCAAGUCACAUTT 887 ND-10350

GUGAACGAAGAAUCACUGU 234 GUGAACGAAGAAUCACUGUTT 561 ACAGUGAUUCUUCGUUCACTT 888 ND-10351

GAAUCUAGAUUUCCAAGCA 235 GAAUCUAGAUUUCCAAGCATT 562 UGCUUGGAAAUCUAGAUUCTT ^■ 889 ND-10352

GUCAAUCUUUAUUCUGACU 236 GUCAAUCUUUAUUCUGACUTT 563 AGUCAGAAUAAAGAUUGACTT 890 ND-10353

ACACAAAUCACAAUGAAGA 237 ACACAAAUCACAAUGAAGATT 564 UCUUCAUUGUGAUUUGUGUTT 891 ND-10354

GUGACCAAUGAAUCUCCCU 238 GUGACCAAUGAAUCUCCCUTT 565 AGGGAGAUUCAUUGGUCACTT 892 ND-10355

UGAAUCUCCCUUAAAGUAC 239 UGAAUCUCCCUUAAAGUACTT 566 GUACUUUAAGGGAGAUUCATT 893 ND-10356

AUGAACCACUGAAUGAGGU 240 AUGAACCACUGAAUGAGGUTT 567 ACCUCAUUCAGUGGUUCAUTT 894 ND-10357

GCUUCCGGAAAGUUAAACA 241 GCUUCCGGAAAGUUAAACATT 568 UGUUUAACUUUCCGGAAGCTT 895 ND-10358

GAGGGUCAGUUUACUCUGA 242 GAGGGUCAGUUUACUCUGATT 569 UCAGAGUAAACUGACCCUCTT 896 ND-10359

CAUGUACAGUGAACGAAGA 243 CAUGUACAGUGAACGAAGATT 570 UCUUCGUUCACUGUACAUGTT 897 ND-10360

AAGAGUUUUUUCUAGUGCU 244 AAGAGUUUUUUCUAGUGCUTT 571 AGCACUAGAAAAAACUCUUTT 898 ND-10361

CAAUGCAGACCAGAUUCGG 245 CAAUGCAGACCAGAUUCGGTT 572 CCGAAUCUGGUCUσCAUUGTT 899 ND-10362

AUUUCAGCAACUUAUUACU 246 AUUUCAGCAACUUAUUACUTT 573 AGUAAUAAGUUGCUGAAAUTT 900 ND-10363

UAUAUUUGUCACCUAACGU 247 UAUAUUUGUCACCUAACGUTT 574 ACGUUAGGUGACAAAUAUATT 901 ND-10364

CUGAGAUAAAAAUGAACAA 248 CUGAGAUAAAAAUGAACAATT 575 UUGUUCAUUUUUAUCUCAGTT 902 ND-10365

CCUUAAAGUACUUAUUCAG 249 CCUUAAAGUACUUAUUCAGTT 576 CUGAAUAAGUACUUUAAGGTT 903 ND-10366

GUGACCAUAUCAUAGAUGA 250 GUGACCAUAUCAUAGAUGATT 577 UCAUCUAUGAUAUGGUCACTT 904 ND-10367

GCUUUAAUGUGCUUUUACU 251 GCUUUAAUGUGCUUUUACUTT 578 AGUAAAAGCACAUUAAAGCTT 905 ND-10368

GAAUCUACAAAAUUGUUUU 252 GAAUCUACAAAAUUGUUUUTT 579 AAAACAAUUUUGUAGAUUCTT 906 ND-10369

UUUUCUAGUGCUGAGGCAU 253 UUUUCUAGUGCUGAGGCAUTT 580 AUGCCUCAGCACUAGAAAATT 907 ND-10370

UGACCAAUGAAUCUCCCUU 254 UGACCAAUGAAUCUCCCUUTT 581 AAGGGAGAUUCAUUGGUCATT 908 ND-10371

GGGAAAUCGUUCAUUCAUU 255 GGGAAAUCGUUCAUUCAUUTT 582 AAUGAAUGAACGAUUUCCCTT 909 ND-10372

AUGGAUAAUAAUGCAGCAG 256 AUGGAUAAUAAUGCAGCAGTT 583 CUGCUGCAUUAUUAUCCAUTT 910 ND-10373

GAAGGGUCUACACCAGAUU 257 GAAGGGUCUACACCAGAUUTT 584 AAUCUGGUGUAGACCCUUCTT 911 ND-10374

CAAUGUAUACUCUCGAGAU 258 CAAUGUAUACUCUCGAGAUTT 585 AUCUCGAGAGUAUACAUUGTT 912 ND-10375

AGUGCUGAGGCAUUGGUAC 259 AGUGCUGAGGCAUUGGUACTT 586 GUACCAAUGCCUCAGCACUTT 913 ND-10376

UGUAUACUCUCGAGAUCCU 260 UGUAUACUCUCGAGAUCCUTT 587 AGGAUCUCGAGAGUAUACATT 914 ND-10377

CGAGCUUUAUAAGAUUAAU 261 CGAGCUUUAUAAGAUUAAUTT 588 AUUAAUCUUAUAAAGCUCGTT 915 ND-10378

UAUCACAAUGUAUACUCUC 262 UAUCACAAUGUAUACUCUCTT 589 GAGAGUAUACAUUGUGAUATT 916 ND-10379

CGAAGUGCUUGAAAAUGGU 263 CGAAGUGCUUGAAAAUGGUTT 590 ACCAUUUUCAAGCACUUCGTT 917 ND-10380

GCUGUCACAAAGAAUUUGG 264 GCUGUCACAAAGAAUUUGGTT 591 CCAAAUUCUUUGUGACAGCTT 918 ND-10381

AUCACAAUGUAUACUCUCG 265 AUCACAAUGUAUACUCUCGTT 592 CGAGAGUAUACAUUGUGAUTT 919 ND-10382

AUGUACAGUGAACGAAGAA 266 AUGUACAGUGAACGAAGAATT 593 UUCUUCGUUCACUGUACAUTT 920 ND-10383

CUACAGAAUAUGACGGUGG 267 CUACAGAAUAUGACGGUGGTT 594 CCACCGUCAUAUUCUGUAGTT 921 ND-10384

UUCAGCAACUUAUUACUUA 268 UUCAGCAACUUAUUACUUATT 595 UAAGUAAUAAGUUGCUGAATT 922 ND-10385

AAAGAUGUGACUUACUUAA 269 AAAGAUGUGACUUACUUAATT 596 UUAAGUAAGUCACAUCUUUTT 923 ND-10386

GUGCUGAGGCAUUGGUACA 270 GUGCUGAGGCAUUGGUACATT 597 UGUACCAAUGCCUCAGCACTT 924 ND-10387

AUAUUUGUCACCUAACGUG 271 AUAUUUGUCACCUAACGUGTT 598 CACGUUAGGUGACAAAUAUTT 925 ND-10388

UGGAAGCCGGAAUCUAGAU 272 UGGAAGCCGGAAUCUAGAUTT 599 AUCUAGAUUCCGGCUUCCATT 926 ND-10389

AGAGAUUGUUGAAGGCCAU 273 AGAGAUUGUUGAAGGCCAUTT 600 AUGGCCUUCAACAAUCUCUTT 927 ND-10390

UACAGAAUAUGACGGUGGC 274 UACAGAAUAUGACGGUGGCTT 601 GCCACCGUCAUAUUCUGUATT 928 ND-10391

GGGAGACAACAAUUUGCAA 275 GGGAGACAACAAUUUGCAATT 602 UUGCAAAUUGUUGUCUCCCTT 929 ND-10392

AAUUUGUUCAUUAUCGUAA 276 AAUUUGUUCAUUAUCGUAATT 603 UUACGAUAAUGAACAAAUUTT 930 ND-10393

UGAACAGAAAAGACUCUUC 277 UGAACAGAAAAGACUCUUCTT 604 GAAGAGUCUUUUCUGUUCATT 931 ND-10394

AAUCAGUAGAAAAACAGUU 278 AAUCAGUAGAAAAACAGUUTT 605 AACUGUUUUUCUACUGAUUTT 932 ND-10395

GACUCUUCUUGCAGUUUAC 279 GACUCUUCUUσCAGUUUACTT 606 GUAAACUGCAAGAAGAGUCTT 933 ND-10396

UAAUGUGCUUUUACUUCCG 280 UAAUGUGCUUUUACUUCCGTT 607 CGGAAGUAAAAGCACAUUATT 934 ND-10397

UUUCAGCAACUUAUUACUU 281 UUUCAGCAACUUAUUACUUTT 608 AAGUAAUAAGUUGCUGAAATT 935 ND-10398

GGAGACAACAAUUUGCAAA 282 GGAGACAACAAUUUGCAAATT 609 UUUGCAAAUUGUUGUCUCCTT 936 ND-10399

CAUAUUUGAGACUCAAAGU 283 CAUAUUUGAGACUCAAAGUTT 610 ACUUUGAGUCUCAAAUAUGTT 937 ND-10400

UCAUAGAUGAUGCACUUGU 284 UCAUAGAUGAUGCACUUGUTT 611 ACAAGUGCAUCAUCUAUGATT 938 ND-10401

CAUAGAUGAUGCACUUGUC 285 CAUAGAUGAUGCACUUGUCTT 612 GACAAGUGCAUCAUCUAUGTT 939 ND-10402

AAACUACAGAAUAUGACGG 286 AAACUACAGAAUAUGACGGTT _, 613 CCGUCAUAUUCUGUAGUUUTT 940 ND-10403

UGAACCACUGAAUGAGGUU 287 UGAACCACUGAAUGAGGUUTT 614 AACCUCAUUCAGUGGUUCATT 941 ND-10404

GAAAUCUAGUGAAUGAUGA 288 GAAAUCUAGUGAAUGAUGATT 615 UCAUCAUUCACUAGAUUUCTT 942 ND-10405

GAAGCGAGCAGCUGCAAAG 289 GAAGCGAGCAGCUGCAAAGTT 616 CUUUGCAGCUGCUCGCUUCTT 943 ND-10406

AUAUUGAAUGCUGUCACAA 290 AUAUUGAAUGCUGUCACAATT 617 UUGUGACAGCAUUCAAUAUTT 944 ND-10407

UUAAUCCAUCUUCUUUUGA 291 UUAAUCCAUCUUCUUUUGATT 618 UCAAAAGAAGAUGGAUUAATT 945 ND-10408

UGGUUUAAUCCAUCUUCUU 292 UGGUUUAAUCCAUCUUCUUTT 619 AAGAAGAUGGAUUAAACCATT 946 ND-10409

CGAAAUCUAGUGAAUGAUG 293 CGAAAUCUAGUGAAUGAUGTT 620 CAUCAUUCACUAGAUUUCGTT 947 ND-10410

UAUUGAAUGCUGUCACAAA 294 UAUUGAAUGCUGUCACAAATT 621 UUUGUGACAGCAUUCAAUATT 948 ND-10411

UUUGGUUUAAUCCAUCUUC 295 UUUGGUUUAAUCCAUCUUCTT 622 GAAGAUGGAUUAAACCAAATT 949 ND-10412

CCCUUUAUAUUGAAUGCUG 296 CCCUUUAUAUUGAAUGCUGTT 623 CAGCAUUCAAUAUAAAGGGTT 950 ND-10413

UUUUGGUUUAAUCCAUCUU 297 UUUUGGUUUAAUCCAUCUUTT 624 AAGAUGGAUUAAACCAAAATT 951 ND-10414

AGCCGAAUGAAGCGAGCAG 298 AGCCGAAUGAAGCGAGCAGTT 625 CUGCUCGCUUCAUUCGGCUTT 952 ND-10415

UGCGUGAAAGUGUUACAUA 299 UGCGUGAAAGUGUUACAUATT 626 UAUGUAACACUUUCACGCATT 953 ND-10416

GUUACAUAUUCUUUCACUU 300 GUUACAUAUUCUUUCACUUTT 627 AAGUGAAAGAAUAUσUAACTT 954 ND-10417

UUACUGCUUGAGGUUGAGC 301 UUACUGCUUGAGGUUGAGCTT 628 GCUCAACCUCAAGCAGUAATT 955 ND-10418

UGAAGCUAGCCGAAUGAAG 302 UGAAGCUAGCCGAAUGAAGTT 629 CUUCAUUCGGCUAGCUUCATT 956 ND-10419

GGGUUAAAUCACUUUUGCU 303 GGGUUAAAUCACUUUUGCUTT 630 AGCAAAAGUGAUUUAACCCTT 957 ND-10420

AGGGUUAAAUCACUUUUGC 304 AGGGUUAAAUCACUUUUGCTT 631 GCAAAAGUGAUUUAACCCUTT 958 ND-10421

UUAGUAACAGCACAACAAA 305 UUAGUAACAGCACAACAAATT 632 UUUGUUGUGCUGUUACUAATT 959 ND-10422

UAUUACUσCUUGAGGUUGA 306 UAUUACUGCUUGAGGUUGATT 633 UCAACCUCAAGCAGUAAUATT 960 ND-10423

UGUUUCAGUAGCCAAUCCU 307 UGUUUCAGUAGCCAAUCCUTT 634 AGGAUUGGCUACUGAAACATT 961 ND-10424

CAUUGAAGCUAGCCGAAUG 308 CAUUGAAGCUAGCCGAAUGTT 635 CAUUCGGCUAGCUUCAAUGTT 962 ND-10425

GACAUUGAAGCUAGCCGAA 309 GACAUUGAAGCUAGCCGAATT 636 UUCGGCUAGCUUCAAUGUCTT 963 ND-10426

ACAUUGAAGCUAGCCGAAU 310 ACAUUGAAGCUAGCCGAAUTT 637 AUUCGGCUAGCUUCAAUGUTT 964 ND-10427

AAUGAAGCGAGCAGCUGCA 311 AAUGAAGCGAGCAGCUGCATT 638 UGCAGCUGCUCGCUUCAUUTT 965 ND-10428

AGUGUUACAUAUUCUUUCA 312 AGUGUUACAUAUUCUUUCATT 639 UGAAAGAAUAUGUAACACUTT 966 ND-10429

GCUUGAGGUUGAGCCUUUU 313 GCUUGAGGUUGAGCCUUUUTT 640 AAAAGGCUCAACCUCAAGCTT 967 ND-10430

GUCUUGCAAUGAACUGUUU 314 GUCUUGCAAUGAACUGUUUTT 641 AAACAGUUCAUUGCAAGACTT 968 ND-10431

GCCGAAUGAAGCGAGCAGC 315 GCCGAAUGAAGCGAGCAGCTT 642 GCUGCUCGCUUCAUUCGGCTT 969 ND-10432

UUGCGUGAAAGUGUUACAU 316 UUGCGUGAAAGUGUUACAUTT 643 AUGUAACACUUUCACGCAATT 970 ND-10433

UCUUGCAAUGAACUGUUUC 317 UCUUGCAAUGAACUGUUUCTT 644 GAAACAGUUCAUUGCAAGATT 971 ND-10434

AGUAUAUGAAAGGACAGGG 318 AGUAUAUGAAAGGACAGGGTT 645 CCCUGUCCUUUCAUAUACUTT 972 ND-10435

UUGAAGCUAGCCGAAUGAA 319 UUGAAGCUAGCCGAAUGAATT 646 UUCAUUCGGCUAGCUUCAATT 973 ND-10436

AGAACUUUAGUAACAGCAC 320 AGAACUUUAGUAACAGCACTT 647 GUGCUGUUACUAAAGUUCUTT 974 ND-10437

UUAAGGGUUAAAUCACUUU 321 UUAAGGGUUAAAUCACUUUTT 648 AAAGUGAUUUAACCCUUAATT 975 ND-10438

UUGUAUGUACAGAGAGGUU 322 UUGUAUGUACAGAGAGGUUTT 649 AACCUCUCUGUACAUACAATT 976 ND-10439

AAUCACUUUUGCUUGUGUU 323 AAUCACUUUUGCUUGUGUUTT 650 AACACAAGCAAAAGUGAUUTT 977 ND-10440

UACAUAUUCUUUCACUUGU 324 UACAUAUUCUUUCACUUGUTT 651 ACAAGUGAAAGAAUAUGUATT 978 ND-10441

CAGAGAGGUUUUUCUGAAU 325 CAGAGAGGUUUUUCUGAAUTT 652 AUUCAGAAAAACCUCUCUGTT 979 ND-10442

UUUGCUUGUGUUUAUUACU 326 UUUGCUUGUGUUUAUUACUTT 653 AGUAAUAAACACAAGCAAATT 980 ND-10443

GCAAUGAACUGUUUCAGUA 327 GCAAUGAACUGUUUCAGUATT 654 UACUGAAACAGUUCAUUGCTT 981 ND-10444

dsRNA synthesis

Source of reagents

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, 50θA, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2 -O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and T-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, Unterschleifiheim, 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 - 90°C for 3 minutes and cooled to room temperature over a period of 3 - 4 hours. The annealed RNA solution was stored at -20 ⁰ C 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- 1 ,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-ylmethoxycarbony l-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 O ⁰ C. 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)-ethoxycarbonylmethyl-amino] -propionic acid ethyl ester AC

AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylam ino)-hexanoyl]- amino} -propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at O ⁰ C. 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 l, 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 0 ⁰ C 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-2,3,4,7,8,9, 10,l 1,12,13, 14,15,16,17- tetradecahydro- lH-cyclopenta[a] phenanthren-3-yloxycarbonylamino]-hexanoyl} -4-oxo- pyrrolidine-3-carboxylic acid ethyl ester AF,

AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to O ⁰ C 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 0 ⁰ C and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH ₂ PO ₄ -H ₂ O 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 0 ⁰ C 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-pyrroIidin- 1 -yl)-6-oxo-hexyl]-carbamic acid 17-( 1,5- dimethyl-hexyl)- 10 ₅ 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/CHCls) (89%).

(6- {3 -[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyr rolidin- 1 -yl } -6- oxo-hexyl)-carbamic acid 17-( 1 ,5-dimethyl-hexyl)- 10, 13 -dimethyl-

2,3,4,7,8,9,10,1 l,l2,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]ρhenanth ren-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% MeOH/CHCl ₃ ) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-l-{6-[ 17-(l,5- dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-lH cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl} -pyrrolidin-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 40 ⁰ C 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; Skillera, 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. ScL USA (1995) 92: 1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al.,

1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al.,

1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640- 19 ; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. NatLAcad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; 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 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-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-116 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. Fl 015) 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% CO ₂ in a humidified incubator.

For transfection with siRNA, HCT-116 cells were seeded at a density of 2.0 x 10 ⁴ cells/well in 96- well 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, CA)(formerly 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 % of mean % of transcript Standard transcript Standard duplex remaining deviation duplex remaining deviation name at 3OnM at 3 OnM name at 3 OnM at 3 OnM

ND-10118 17.66 1.99 ND-10283 17.85 2.99

ND-10119 22.63 2.07 ND-10284 27.81 3.86

ND-10120 25.30 2.17 ND-10285 14.01 1.66

ND-10121 22.16 2.12 ND-10286 25.23 2.10

ND-10122 21.29 3.19 ND-10287 6.37 0.50

ND-10123 14.25 2.24 ND-10288 14.58 1.39

ND-10124 29.18 2.04 ND-10289 16.54 2.25

ND-10125 21.76 2.58 ND-10290 12.05 2.53

ND-10126 34.49 2.87 ND-10291 22.07 3.30

ND-10127 20.06 1.59 ND-10292 70.58 9.44

ND-10128 43.10 4.23 ND-10293 14.72 2.05

ND-10129 21.23 3.33 ND-10294 11.42 0.42

ND-10130 26.72 2.38 ND-10295 19.95 1.07

ND-10131 14.42 1.85 ND-10296 14.97 0.38

ND-10132 22.26 4.11 ND-10297 15.64 1.55

ND-10133 25.69 1.98 ND-10298 16.99 0.80

ND-10134 28.12 0.88 ND-10299 18.26 2.01

ND-10135 38.42 3.24 ND-10300 20.01 1.31

ND-10136 15.54 2.55 ND-10301 14.44 0.64

ND-10137 36.32 4.42 ND-10302 41.22 3.73

ND-10138 36.77 5.69 ND-10303 37.74 6.03

ND-10139 19.09 2.75 ND-10304 10.90 0.75

ND-10140 10.30 1.79 ND-10305 14.71 3.13

ND-10141 48.13 12.88 ND-10306 52.68 4.91

ND-10142 35.73 2.65 ND-10307 24.24 0.33

ND-10143 21.24 2.26 ND-10308 54.90 3.97

ND-10144 17.64 3.47 ND-10309 54.76 10.28

ND-10145 19.02 3.28 ND-10310 22.17 1.68

ND-10146 20.39 3.57 ND-10311 24.79 2.11

ND-10147 20.99 1.21 ND-10312 17.33 2.88

ND-10148 76.62 11.31 ND-10313 17.55 4.00

ND-10149 39.27 5.88 ND-10314 23.02 4.28

ND-10150 26.90 1.86 ND-10315 13.33 1.88

ND-10151 18.88 1.08 ND-10316 19.61 3.16

ND-10152 21.77 2.79 ND-10317 17.29 1.13

ND-10153 15.93 2.07 ND-10318 18.78 1.73

ND-10154 45.64 3.75 ND-10319 17.46 4.72

ND-10155 15.85 1.61 ND-10320 19.37 2.32

ND-10156 17.86 2.88 ND-10321 64.52 3.68

ND-10157 25.83 2.64 ND-10322 17.02 3.52

ND-10158 46.13 3.63 ND-10323 47.08 4.36

ND-10159 12.76 1.03 ND-10324 20.06 1.65

ND-10160 17.45 1.40 ND-10325 93.65 11.68

ND-10161 11.21 0.96 ND-10326 18.09 3.77

ND-10162 42.00 5.65 ND-10327 23.52 4.09

ND-10163 38.02 4.20 ND-10328 19.58 4.04

ND-10164 58.42 8.32 ND-10329 71.26 4.08

ND-10165 18.58 2.63 ND-10330 22.76 1.92

ND-10166 26.03 3.03 ND-10331 23.96 1.40

ND-10167 16.87 5.53 ND-10332 31.98 2.53

ND-10168 18.86 1.96 ND-10333 19.35 2.76

ND-10169 32.31 3.92 ND-10334 22.21 2.20

ND-10170 22.71 3.45 ND-10335 58.81 6.69

ND-10171 12.39 3.02 ND-10336 25.38 3.14

ND-10172 13.53 1.72 ND-10337 14.70 1.37

ND-10173 67.35 4.47 ND-10338 30.50 3.75

ND-10174 13.44 1.15 ND-10339 36.81 5.22

ND-10175 19.24 1.89 ND-10340 65.85 2.35

ND-10176 34.75 2.35 ND-10341 57.67 2.06

ND-10177 27.58 3.78 ND-10342 16.25 1.61

ND-10178 10.76 1.32 ND-10343 22.54 1.78

ND-10179 45.45 7.56 ND-10344 17.76 1.90

ND-10180 88.36 11.08 ND-10345 31.65 2.34

ND-10181 33.41 5.36 ND-10346 15.63 1.69

ND-10182 16.39 1.93 ND-10347 29.52 2.48

ND-10183 14.21 1.26 ND-10348 15.28 1.01

ND-10184 17.63 1.94 ND-10349 20.37 2.26

ND-10185 31.21 2.96 ND-10350 36.74 4.34

ND-10186 15.85 1.59 ND-10351 42.98 2.06

ND-10187 20.72 3.28 ND-10352 10.78 1.82

ND-10188 43.08 3.80 ND-10353 18.53 1.48

ND-10189 18.50 2.95 ND-10354 14.95 0.66

ND-10190 43.59 2.94 ND-10355 34.81 3.09

ND-10191 13.33 1.46 ND-10356 86.73 4.10

ND-10192 14.69 2.18 ND-10357 28.86 3.35

ND-10193 9.86 0.91 ND-10358 10.04 1.16

ND-10194 19.71 3.49 ND-10359 9.50 0.88

ND-10195 11.75 1.75 ND-10360 14.63 0.18

ND-10196 16.32 3.59 ND-10361 26.72 1.84

ND-10197 21.00 3.62 ND-10362 24.12 1.32

ND-10198 24.06 3.53 ND-10363 17.32 1.54

ND-10199 57.92 11.64 ND-10364 25.85 2.53

ND-10200 14.31 1.47 ND-10365 15.99 2.50

ND-10201 42.05 2.20 ND-10366 20.32 3.27

ND-10202 58.23 7.77 ND-10367 18.23 3.67

ND-10203 13.85 1.41 ND-10368 11.45 1.91

ND-10204 16.18 1.26 ND-10369 15.69 1.49

ND-10205 15.76 1.71 ND-10370 53.37 4.67

ND-10206 14.78 2.55 ND-10371 24.71 2.42

ND-10207 17.67 2.81 ND-10372 14.08 1.42

ND-10208 7.50 2.37 ND-10373 85.92 6.67

ND-10209 91.36 15.11 ND-10374 17.84 1.52

ND-10210 27.56 2.65 ND-10375 15.51 2.28

ND-10211 41.43 5.64 ND-10376 17.49 1.75

ND-10212 30.82 4.81 ND-10377 15.98 2.79

ND-10213 12.97 1.52 ND-10378 16.99 2.71

ND-10214 19.53 2.31 ND-10379 65.20 6.45

ND-10215 47.38 9.79 ND-10380 12.26 1.75

ND-10216 18.12 2.03 ND-10381 15.82 1.59

ND-10217 12.51 1.97 ND-10382 36.31 2.72

ND-10218 25.56 3.83 ND-10383 15.15 1.72

ND-10219 81.47 13.73 ND-10384 18.34 1.31

ND-10220 42.93 3.74 ND-10385 17.14 1.51

ND-10221 9.22 2.09 ND-10386 14.83 1.67

ND-10222 13.38 1.89 ND-10387 13.41 2.42

ND-10223 14.84 0.96 ND-10388 25.62 3.19

ND-10224 15.56 1.67 ND-10389 21.84 1.17

ND-10225 35.58 3.00 ND-10390 12.71 2.10

ND-10226 48.38 3.47 ND-10391 48.10 4.21

ND-10227 8.24 1.03 ND-10392 14.42 1.77

ND-10228 48.31 3.86 ND-10393 24.34 2.27

ND-10229 16.51 1.79 ND-10394 21.08 2.07

ND-10230 43.19 4.51 ND-10395 16.74 3.30

ND-10231 41.29 3.49 ND-10396 12.06 1.13

ND-10232 14.21 1.34 ND-10397 44.74 4.77

ND-10233 54.04 4.03 ND-10398 60.44 7.22

ND-10234 19.76 1.64 ND-10399 9.71 1.59

ND-10235 16.66 1.24 ND-10400 23.92 3.78

ND-10236 93.09 4.20 ND-10401 22.11 2.14

ND-10237 12.28 0.91 ND-10402 20.63 1.38

ND-10238 13.66 1.20 ND-10403 65.43 5.95

ND-10239 21.61 1.86 ND-10404 27.26 2.88

ND-10240 55.26 3.89 ND-10405 14.97 1.44

ND-10241 18.24 1.87 ND-10406 13.69 0.97

ND-10242 16.05 2.18 ND-10407 27.65 1.98

ND-10243 33.65 4.73 ND-10408 25.92 1.30

ND-10244 42.58 6.33 ND-10409 6.55 0.76

ND- 10245 11.90 0.94 ND-10410 17.62 2.42

ND-10246 19.15 2.37 ND-10411 14.78 2.72

ND-10247 73.91 9.75 ND-10412 92.60 9.76

ND-10248 37.44 5.96 ND-10413 13.62 0.69

ND-10249 37.62 4.47 ND-10414 34.45 1.77

ND-10250 68.05 8.23 ND-10415 29.44 4.14

ND-10251 10.47 1.51 ND-10416 14.70 1.87

ND-10252 89.61 8.08 ND-10417 21.80 2.69

ND-10253 21.85 3.32 ND-10418 107.83 9.24

ND-10254 19.81 2.12 ND-10419 36.97 5.30

ND-10255 7.20 0.84 ND-10420 15.00 2.31

ND-10256 45.03 3.65 ND-10421 19.17 3.65

ND-10257 20.71 2.14 ND-10422 23.86 2.63

ND-10258 30.07 2.59 ND-10423 21.55 0.35

ND-10259 24.85 2.94 ND-10424 31.81 3.81

ND-10260 14.58 1.79 ND-10425 27.34 2.29

ND-10261 32.58 3.24 ND-10426 42.72 2.80

ND-10262 26.63 3.78 ND-10427 24.49 2.04

ND-10263 72.06 6.75 ND-10428 24.97 1.00

ND-10264 93.19 12.63 ND-10429 15.47 2.36

ND-10265 19.70 1.96 ND-10430 17.65 2.25

ND-10266 108.26 10.65 ND-10431 20.23 3.72

ND-10267 14.69 1.22 ND-10432 30.69 5.52

ND-10268 28.16 3.50 ND-10433 16.89 1.30

ND-10269 43.98 7.03 ND-10434 44.68 4.63

ND-10270 13.09 1.36 ND-10435 50.13 4.22

ND-10271 13.92 0.99 ND-10436 35.13 1.63

ND-10272 14.43 1.25 ND-10437 48.34 3.87

ND-10273 17.91 1.03 ND-10438 35.36 2.60

ND-10274 17.81 2.72 ND-10439 17.92 1.54

ND-10275 19.72 3.09 ND-10440 21.45 2.96

ND-10276 8.92 1.34 ND-10441 25.77 4.43

ND-10277 49.34 5.11 ND-10442 16.20 2.39

ND-10278 48.61 6.12 ND-10443 27.37 1.63

ND-10279 16.49 1.61 ND-10444 20.49 1.49

ND-10280 14.14 0.91

ND-10281 13.55 1.86

ND-10282 27.29 4.14

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.