# Patent

## Title
dsRNA compositions and methods for treating HPV infection

## Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for treating human papilloma virus (HPV) infection. The dsRNA comprises an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of an HPV Target gene selected from among HPV E1, HPV E6 and the human E6AP gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by HPV infection and the expression of the E6AP gene using the pharmaceutical composition; and methods for inhibiting the expression of the HPV Target genes in a cell.

## Background
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-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human more than 100 types of papillomavirus (HPV) have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincott—Raven Pub., Philadelphia, N.Y.; Bernard, H-U., 2005. J. Clin. Virol. 328: S1-S6).
Human papillomavirus (HPV) is one of the most prevalent sexually transmitted infections in the world. The majority of HPV infections are harmless. Some types of HPV cause genital warts, which appear as single or multiple bumps in the genital areas of men and women including the vagina, cervix, vulva (area outside of the vagina), penis, and rectum. Many people infected with HPV have no symptoms.
While most HPV subtypes result in benign lesions, certain subtypes are considered high-risk and can lead to more serious lesions, such as cervical and anal dysplasia. Fifteen HPV types were recently classified as high-risk types (Munoz, N. et al. 2003. N. Engl. J. Med. 348(6):518-27.) These high-risk subtypes are genetically diverse, demonstrating >10% sequence divergence at the L1 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, Md., 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-1 designated cells (“cervical intraepithelial neoplasia-1”)); or HSIL (High-grade squamous intraepithelial lesions), including CIN-2 and CIN-3 designated cells, having a higher likelihood of progressing to cancer.
About 85% of low-grade lesions spontaneously regress, and the remainder either stay unchanged, or progress to high-grade lesions. About 10% of high-grade lesions, if left untreated, are expected to transform into cancerous tissues. HPV-16 and HPV-18 are most often associated with dysplasias, although several other transforming HPV subtypes are also associated with dysplasias.
Recent studies indicate that up to 89% of HIV positive homosexual males may be infected with these high-risk subtypes of HPV. 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 HIV Positive Patients will include treatment guidelines for patients diagnosed with anal dysplasia.
There is no known cure for HPV infection. There are treatments for genital warts, although they often disappear even without treatment. The method of treatment depends on factors such as the size and location of the genital warts. Among the treatments used are Imiquimod cream, 20 percent podophyllin antimitotic solution, 0.5 percent podofilox solution, 5 percent 5-fluorouracil cream, and Trichloroacetic acid. The use of podophyllin or podofilox is not recommended for pregnant women because they are absorbed by the skin and may cause birth defects. The use of 5-fluorouracil cream is also not recommended for pregnant women. Small genital warts can be physically removed by freezing (cryosurgery), burning (electrocautery) or laser treatment. Large warts that do not responded to other treatment may have to be removed by surgery. Genital warts have been known to return following physical removal; in these instances α-interferon has been directly injected into these warts. However, α-interferon is expensive, and its use does not reduce the rate of return of the genital warts.
As such there exists an unmet need for effective HPV treatment. Surprisingly, compounds have been discovered that meet this need, and provide other benefits as well.
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
PCT Publication WO 03/008573 discloses a previous effort to develop a nucleic acid based medicament for the treatment of disease caused by HPV infection. This publication reports the use of two siRNAs directed to HPV mRNA to inhibit HPV replication in a cell based system; a related publication is found at Jiang, M. et al. 2005. N. A. R. 33(18): e151.
Despite significant advances in the field of RNAi and advances in the treatment of pathological processes mediated by HPV infection, there remains a need for agents that can inhibit the progression of HPV infection and that can treat diseases associated with HPV infection. The challenge is exacerbated because such agents must be designed to inhibit all the high-risk HPV subtypes, which together display a wide degree of genotypic diversity.
SUMMARY OF THE INVENTION
The invention provides a solution to the problem of treating diseases associated with HPV infection, by using double-stranded ribonucleic acid (dsRNA) to silence gene expression essential for HPV propagation. E6AP is a conserved gene of the human host species required by HPV for proliferation.
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the E6AP gene in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by the expression of the E6AP gene in connection with HPV infection, such as in cervical cancer and genital 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-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such modified sequence will be based on a first sequence of said dsRNA selected from the group consisting of the sense sequences of 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 genital warts, comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.
In another embodiment, the invention provides vectors for inhibiting the expression of the E6AP gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of the E6AP gene in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
BRIEF DESCRIPTION OF THE FIGURES
No Figures are presented


DETAILED DESCRIPTION OF THE INVENTION
The invention provides a solution to the problem of treating diseases associated with HPV infection, by using double-stranded ribonucleic acid (dsRNA) to silence expression of genes essential for HPV proliferation. In particular, the dsRNA of the invention silence the HPV genes E1 or E6 or human E6AP, a conserved gene of the human host species required by HPV for proliferation. Herein, these genes are sometimes collectively called the HPV Target genes.
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods, for inhibiting the expression of the E1, E6 or E6AP gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of the E1, E6 or E6AP gene in association with HPV infection using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of the HPV Target mRNA transcript. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and/or maintenance of an HPV in mammals. Using cell-based and animal-based assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the E1, E6 or E6AP gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by HPV infection by targeting a host factor gene involved in the HPV life cycle.
Description of the HPV Targets: HPV E1 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).
E1 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 E10RF encodes a 68-76 kD protein essential for plasmid DNA replication. The full-length E1 product is a phosphorylated nuclear protein that binds to the origin of replication in the LCR of BPV1. E1 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 E1 for the origin of DNA replication. In HPV-16, E1 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 may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “E6AP” refers to the ubiquitin protein ligase E3A (ube3A, also referred to as E6-associated protein or E6AP) gene or protein. Human mRNA sequences to E6AP representing different isoforms are provided as GenBank Accession numbers NM—130838.1, NM—130839.1, and NM—000462.2.
As used herein, “E1” refers to the human papillomavirus type 16 (HPV16) E1 gene (GenBank accession number NC—001526, nucleotides 865 to 2813). As used herein, “E6” refers to the human papillomavirus type 16 (HPV16) E6 gene (GenBank accession number NC—001526, nucleotides 65 to 559). Many variants of the E1 and E6 genes have also been publicly disclosed. These and future published E1 and E6 gene variants are intended to be covered herein by the use of “E1” 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 oligo nucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding 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




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to the HPV Target gene transcription, e.g. the amount of protein encoded by the HPV Target gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, HPV Target gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of the HPV Target gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of the E6AP gene is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the E6AP gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the E6AP gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. Table 2 provides a wide range of values for inhibition of transcription obtained in an in vitro assay using various E6AP dsRNA molecules at various concentrations. Likewise, Table 6 provides a wide range of values for the inhibition of transcription of E1; and Table 8 provides a wide range of values for the inhibition of transcription of E6.
As used herein in the context of HPV infection, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by HPV infection. Such description includes use of the therapeutic agents of the invention for prophylaxis or prevention of HPV infection, and relief from symptoms or pathologies caused by HPV infection. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by HPV infection), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by HPV infection or an overt symptom of pathological processes mediated by HPV infection. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by HPV infection, the patient's history and age, the stage of pathological processes mediated by HPV infection, and the administration of other anti-pathological agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of a dsRNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the HPV Target gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the HPV Target gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said HPV Target gene, inhibits the expression of said HPV Target gene by at least 10%, 25%, or 40%.
The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the HPV Target gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand; such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the HPV Target gene is the human E6AP gene. In specific embodiments, the antisense strand of the dsRNA comprises a strand selected from the sense sequences of Table 1 and a second sequence selected from the group consisting of the antisense sequences of Table 1. Alternative antisense agents that target elsewhere in the target sequence provided in Table 1 can readily be determined using the target sequence and the flanking E6AP sequence.
In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Table 1. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the E6AP gene. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Table 1 and the second oligonucleotide is described as the antisense strand in Table 1. Table 1 provides a duplex name and sequence ID number for each preferred dsRNA.
In further embodiments, the dsRNA comprises at least one named duplex dsRNA selected from the groups of sequences provided in Table 5 (E1 dsRNA) or Table 7 (E6 dsRNA).
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 1, Table 5 or Table 7, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably-expected that shorter dsRNAs comprising one of the sequences of Table 1, Table 5 or Table 7, minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 1, Table 5 or Table 7, and differing in their ability to inhibit the expression of the HPV Target gene in a FACS assay or other assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Table 1, Table 5 or Table 7 can readily be made using the reference sequence and the target sequence provided.
In addition, the RNAi agents provided in Table 1, Table 5 and Table 7 identify a site in the respective HPV Target mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 1, Table 5 or Table 7 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the HPV Target gene. For example, the last 15 nucleotides of SEQ ID NO: 1 (minus the added AA sequences) combined with the next 6 nucleotides from the target E6AP gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Table 1.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the HPV Target gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the HPV Target gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the HPV Target gene is important, especially if the particular region of complementarity in the HPV Target gene is known to have polymorphic sequence variation in the virus (if E1 or E6) or within the human population (for E6AP).
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, modifications at other sites of the sugar or base of an oligonucleotide, introduction of non-natural bases into the oligonucleotide chain, covalent attachment to a ligand or chemical moiety, and replacement of internucleotide phosphate linkages with alternate linkages such as thiophosphates. More than one such modification may be employed.
Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Generally, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2-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 hexaethylene 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-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.
In certain instances, conjugation of a cationic ligand to oligonucleotides results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide. In certain instances, an dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.
The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems. (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.
In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
The incorporation of a 2′-O-methyl, 2′-O-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′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT Publication WO 200370918.
In some embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-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 CH2 component parts.
Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
In certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., 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 9 sets out patterns of chemical modifications preferred for use with the duplex dsRNA set out in Table 1, Table 5 and Table 7 of the invention. Some of these modifications are also illustrated in Table 3.









TABLE 9





Chemical




Modification
Changes made to sense
Changes made


Series
strand (5′-3′)
to antisense stand (5′-3′)







1 (single
dTsdT
dTsdT


phosphorothioate


at the ends of


both strands)


2 (single
dTsdT, 2′OMe@all Py
dTsdT, 2′OMe@uA, cA


phosphorothioate


at the ends of


both strands plus,


2′OMe sense


strand


modification of


all pyrimidines


and 2′Ome


modification of


all U's followed


by and A and all


C's followed by


A)


3 (single
dTsdT, 2′OMe@all Py
dTsdT, 2′OMe@uA, cA, uG,


phosphorothioate

uU


at the ends of


both strands plus,


2′OMe sense


strand


modification of


all pyrimidines


and, 2′Ome of


indicted bases all


U's followed by


an A, all C's


followed by an


A, all U's


followed by a G


and all U's


followed by a U


on the antisense


strand)


4 (same as 1
Chol (“exo”)
dTsdT (“exo”)


except addition


of cholesterol


conjugated to the


sense strand)


5 (same as 2
Chol (“endo”)
dTsdT, 2′OMe@uA, cA


except


cholesterol


conjugated to the


sense strand)


6 (same as 3
Chol (“endo”)
dTsdT, 2′OMe@uA, cA, uG,


except

uU


cholesterol


conjugated to the


sense strand)









Vector Encoded RNAi Agents
The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.
dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
III. Pharmaceutical Compositions Comprising dsRNA
In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of 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 desirable 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 a biologic means as described in, for example, U.S. Pat. No. 6,271,359. A biologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DOTMA (N-[1,2(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOSPA (2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-deimethyl-1-propanaminium), DOGS (dioctadecyl amido glycil spermine), and DC-chol (3,[N—N1,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 liposomes) such as lipoplexes and nanoemulsions. Additionally, condensing polymeric delivery systems (i.e., DNA-polymer complexes, or “polyplexes”) may be used, including but not limited to chitosans, poly(L-lysine) (PLL), polyethylenimine (PEI), dendrimers (e.g., polyamidoamine (PANAM) dendrimers), and poloxamines. Additionally, noncondensing polymeric delivery systems may be used, including but not limited to poloxamers, gelatin, PLGA (polylactic-co-glycolic acid), PVP (polyvinylpyrrolidone), and PVA (polyvinyl alcohol).
Procedures for the above-mentioned delivery or administration techniques are well known in the art. For instance, condensing polymeric delivery systems work by easily complexing with anionic DNA molecules; for example, poly(L-lysine)(PLL) works by forming a positively charged complex that interacts with negatively charged cell surface and subsequently undergoing rapid internalization.
Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to skin cells and cervical cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.
dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
In addition, dsRNA that target the HPV Target gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the E6AP gene can contain other therapeutic agents such as anti-inflammatory drugs (e.g., nonsteroidal anti-inflammatory drugs and corticosteroids) and antiviral drugs (e.g., ribivirin, vidarabine, acyclovir, and ganciclovir). In some embodiments, a composition can contain one or more dsRNAs having a sequence complementary to the HPV Target gene in combination with a keratolytic agent. Keratolytic agents are agents that separate or loosen the horny layer of the epidermis. An example of a keratolytic agent includes, without limitation, salicylic acid. Other examples are provided in U.S. Pat. No. 5,543,417. Keratolytic agents can be used in an amount effective to enhance the penetration of dsRNAs, for example, into tissues such as skin. For example, a keratolytic agent can be used in an amount that allows a dsRNA applied to a genital wart to penetrate throughout the wart.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by HPV infection. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Combinations of dsRNA can be tested in vitro and in vivo using the same methods employed for identification of preferred single dsRNA. Such combinations may be selected based on a purely bioinformatics basis, wherein the minimum number of siRNA are selected which provide coverage over the widest range of genotypes. Alternatively, such combinations may be selected based on in vitro or in vivo evaluations along the lines of those described herein for single dsRNA agents. A preferred assay for testing combinations of dsRNA is to evaluate the phenotypic consequences of siRNA mediated HPV target knockdown in HPV16 positive cancer cell lines (e.g. SiHa or Caski, as described in, e.g., Hengstermann et al. (2005) Journal Vir. 79(14): 9296; and Butz et al. (2003) Oncogene 22: 5938), or in organotypic culture systems, as described in, e.g., Jeon et al. (1995) Journal Vir. 69(5):2989.
The inventors have identified certain preferred combinations of dsRNA which may be used to treat HPV infection. In the most general terms, the combination of dsRNA comprises more than one dsRNA selected from among Table 1, Table 3, Table 5 and Table 7. Thus the invention contemplates the use of 2, 3, 4, 5 or more dsRNA duplexes selected from among Table 1, Table 3, Table 5 and Table 7 in a combination therapy. In principle, the smallest number of dsRNA is preferred for simplicity of the therapeutic product. This forces the selection of dsRNA which will cover the greatest number of deleterious or potentially deleterious HPV genotypes, and indeed may justify selection of a combination that does not necessarily cover all such HPV genotypes.
The following dsRNA are particularly amenable to combination:
From E1: ND-9072; ND-9142; ND-9092; ND-9162; ND-9097; ND-9167; ND-9066; ND-9123; AL-DP-8082; AL-DP-8095;
From E6: ND-8903; ND-8991; ND-8914; ND-9002; ND-8906; ND-8994; ND-8943; ND-9031; ND-9032; ND-8920; ND-8952; ND-8951; ND-9008; ND-9040; ND-9039; AL-DP-7783; AL-DP-7784;
From E6AP: AL-DP-7365; AL-DP-7371; AL-DP-7499; AL-DP-7545; AL-DP-7492; AL-DP-7473; AL-DP-7478; AL-DP-7554; AL-DP-7514; AL-DP-7397, ND-9300.
Methods for Treating Diseases Caused by HPV Infection
The methods and compositions described herein can be used to treat diseases and conditions caused by human papillomavirus, which can be the result of clinical or sub-clinical papillomavirus infections. Such diseases and conditions, herein sometimes called “HPV associated disorders” or “pathological processes mediated by HPV infection”, include, e.g., epithelial malignancies, skin cancer (non-melanoma or melanoma), anogenital malignancies such as cervical cancer, HPV associated precancerous lesions (including LSIL or HSIL cervical tissue), anal carcinoma, malignant lesions, benign lesions, papillomacarcinomas, 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 gonadotropindependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.
In a further alternative, the dsRNA targeting E6AP may be employed to treat neurological and behavioural disorders. E6AP has been implicated in neurological and behavioural disorders through the identification of E6AP mutations in patients having Angelman syndrome. Angelman syndrome (AS) is an imprinted neurobehavioral disorder characterized by mental retardation, absent speech, excessive laughter, seizures, ataxia, and a characteristic EEG pattern. (Hitchins, M. P. et al. 2004. Am J Med Genet A. 125(2):167-72.) It would not, presumably, be the intent of treatment to induce such conditions; rather, as observed in many hereditary defects, this evidence that E6AP has a critical role in neurological and behavioural conditions also indicates that this target may have a variety of roles in human pathologies and is likely a suitable target for other diseases in this class where silencing of E6AP will compensate for other biochemical defects or diseases. As used herein “E6AP associated disorders” include the HPV associated disorders noted above and other neurological and behavioural disorders.
Methods for Inhibiting Expression of the E6AP Gene
In yet another aspect, the invention provides a method for inhibiting the expression of the E6AP gene in a mammal. The method comprises administering a composition of Table 1 of the invention to the mammal such that expression of the target E6AP gene is silenced. Because of their high specificity, such dsRNAs of the invention specifically target RNAs (primary or processed) of the target E6AP gene. Compositions and methods for inhibiting the expression of these E6AP genes using such dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the E6AP gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by topical/vaginal administration or by intravenous infusion or injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Gene Walking of the E6AP gene sIRNA design was carried out to identify siRNAs targeting human ubiquitin protein ligase E3A (ube3A, also referred to as E6AP). Human mRNA sequences to E6AP representing different isoforms (NM—130838.1, NM—130839.1, 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—130838.1 were identified, resulting in 4473 19mer candidate sequences. Combined, these candidate target sequences cover the 5′UTR, coding and 3′UTR domains of the E6AP mRNA, and the junction sites of these domains.
In order to rank and select siRNAs out of the pool of candidates, the predicted potential for interacting with irrelevant targets (off-target potential) was taken as a ranking parameter. siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.
For predicting siRNA-specific off-target potential, the following assumptions were made:

    
    
        1) complementarity to a target gene in positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) may be sufficient for interaction of that strand with the mRNA transcribed from the target gene and subsequent downregulation (Jackson A L, et al. Nat. Biotechnol. 2003 June; 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 according descending to the off-target score and sequences containing stretches with more than 3 Gs in a row were excluded from selection.
The 156 siRNAs with an off-target score of >=3 were selected and synthesized (Table 1).







TABLE 1







dsRNA targeting E6AP














Target sequence of mRNA









from human reference

Sense strand

antisense strand


sequence NM_130838

(target sequence)

(guide sequence)


(human iso3)
SEQ
having double
SEQ
having
SEQ


sequence of total 19mer
ID.
overhang
ID.
double overhang
ID.
duplex


target site + AA at ends
NO.
sequence (5′-3′)
NO.
sequence (5′-3′)
NO.
name

















AAAUACGAUGAAUCUACAAAAAA
1
AUACGAUGAAUCUACAAAATT
157
UUUUGUAGAUUCAUCGUAUTT
313
AL-DP-7545



 


AAUGACUACAUUCUCAAUAAAAA
2
UGACUACAUUCUCAAUAAATT
158
UUUAUUGAGAAUGUAGUCATT
314
AL-DP-7558


 


AAAGCCUGCACGAAUGAGUUUAA
3
AGCCUGCACGAAUGAGUUUTT
159
AAACUCAUUCGUGCAGGCUTT
315
AL-DP-7548


 


AAGGAUUGUCGAAAACCACUUAA
4
GGAUUGUCGAAAACCACUUTT
160
AAGUGGUUUUCGACAAUCCTT
316
AL-DP-7509


 


AACUCUCGAGAUCCUAAUUAUAA
5
CUCUCGAGAUCCUAAUUAUTT
161
AUAAUUAGGAUCUCGAGAGTT
317
AL-DP-7492


 


AAAUGUGACUUACUUAACAGAAA
6
AUGUGACUUACUUAACAGATT
162
UCUGUUAAGUAAGUCACAUTT
318
AL-DP-7554


 


AAGUAUACUCUCGAGAUCCUAAA
7
GUAUACUCUCGAGAUCCUATT
163
UAGGAUCUCGAGAGUAUACTT
319
AL-DP-7557


 


AAAGGUUACCUACAUCUCAUAAA
8
AGGUUACCUACAUCUCAUATT
164
UAUGAGAUGUAGGUAACCUTT
329
AL-DP-7476


 


AAAGUACUUAUUCAGACCAGAAA
9
AGUACUUAUUCAGACCAGATT
165
UCUGGUCUGAAUAAGUACUTT
321
AL-DP-7514


 


AAAUCCUAAUUAUCUGAAUUUAA
10
AUCCUAAUUAUCUGAAUUUTT
166
AAAUUCAGAUAAUUAGGAUTT
322
AL-DP-7540


 


AAAAGGAUAGGUGAUAGCUCAAA
11
AACGAUAGGUGAUAGCUCATT
167
UCAGCUAUCACCUAUCCUUTT
323
AL-DP-7397


 


AAGGAAGCCGGAAUCUAGAUUAA
12
GGAAGCCGGAAUCUAGAUUTT
168
AAUCUAGAUUCCGGCUUCCTT
324
AL-DP-7526


 


AAUGCUUCGAAGUGCUUGAAAAA
13
UGCUUCGAAGUGCUUGAAATT
169
UUUCAAGCACUUCGAAGCATT
325
AL-DP-7473


 


AAUGGAUUGUCGAAAACCACUAA
14
UGGAUUGUCGAAAACCACUTT
170
AGUGGUUUUCGACAAUCCATT
326
AL-DP-7478


 


AACGGCUAGAGAUGAUCGCUAAA
15
CGGCUAGAGAUGAUCGCUATT
171
UAGCGAUCAUCUCUAGCCGTT
327
AL-DP-7553


 


AAACAGUCGAAAUCUAGUGAAAA
16
ACAGUCGAAAUCUAGUGAATT
172
UUCACUAGAUUUCGACUGUTT
328
AL-DP-7395


 


AAGAUCAGACUGUGGUCUAAAAA
17
GAUCAGACUGUGGUCUAAATT
173
UUUAGACCACAGUCUGAUCTT
329
AL-DP-7522


 


AACUCGAGAUCCUAAUUAUCUAA
18
CUCGAGAUCCUAAUUAUCUTT
174
AGAUAAUUAGGAUCUCGAGTT
330
AL-DP-7499


 


AAUAUCGUAAUGGAGAAUAGAAA
19
UAUCGUAAUGGAGAAUAGATT
175
UCUAUUCUCCAUUACGAUATT
331
AL-DP-7527


 


AACUCAAAGUUAGACGUGACCAA
20
CUCAAAGUUAGACGUGACCTT
176
GGUCACGUCUAACUUUGAGTT
332
AL-DP-7544


 


AAAGGAUAGGUGAUAGCUCACAA
21
AGGAUAGGUGAUAGCUCACTT
177
GUGAGCUAUCACCUAUCGUTT
333
AL-DP-7489


 


AACACCUAACGUGGAAUGUGAAA
22
CACCUAACGUGGAAUGUGATT
178
UCACAUUCCACGUUAGGUGTT
334
AL-DP-7365


 


AAAAUCGUUCAUUCAUUUACAAA
23
AAUCGUUCAUUCAUUUACATT
179
UGUAAAUGAAUGAACGAUUTT
335
AL-DP-7390


 


AACUUGACGUAUCACAAUGUAAA
24
CUUGACGUAUCACAAUGUATT
180
UACAUUGUGAUACGUCAAGTT
336
AL-DP-7458


 


AAUGGUAUGUUCACAUACGAUAA
25
UGGUAUGUUCACAUACGAUTT
181
AUCGUAUGUGAACAUACCATT
337
AL-DP-7532


 


AAGAUAGGUGAUAGCUCACAGAA
26
GAUAGGUGAUAGCUCACAGTT
182
CUGUGAGCUAUCACCUAUCTT
338
AL-DP-7546


 


AACCGGCUAGAGAUGAUCGCUAA
27
CCGGCUAGAGAUGAUCGCUTT
183
AGCGAUCAUCUCUAGCCGGTT
339
AL-DP-7512


 


AACAUAGUACUGGGUCUGGCUAA
28
CAUAGUACUGGGUCUGGCUTT
184
AGCCAGACCCAGUACUAUGTT
340
AL-DP-7470


 


AAAAUGUAUACUCUCGAGAUCAA
29
AAUGUAUACUCUCGAGAUCTT
185
GAUCUCGAGAGUAUACAUUTT
341
AL-DP-7406


 


AAAACUUUUCGUGACUUGGGAAA
30
AACUUUUCGUGACUUGGGATT
186
UCCCAAGUCACGAAAAGUUTT
342
AL-DP-7382


 


AAAAAGUUAGACGUGACCAUAAA
31
AAAGUUAGACGUGACCAUATT
187
UAUGGUCACGUCUAACUUUTT
343
AL-DP-7547


 


AAUGAUUACGGAGUUCUGGGAAA
32
UGAUUAGGGAGUUCUGGGATT
188
UCCCAGAACUCCCUAAUCATT
344
AL-DP-7490


 


AAUACGAUCAAUCUACAAAAUAA
33
UACGAUGAAUCUACAAAAUTT
189
AUUUUGUAGAUUCAUCGUATT
345
AL-DP-7493


 


AACUUGUCCGGCUAGAGAUGAAA
34
CUUGUCCGGCUAGAGAUGATT
190
UCAUCUCUAGCCGGACAAGTT
346
AL-DP-7529


 


AAUAUACUCUCGAGAUCCUAAAA
35
UAUACUCUCGAGAUCCUAATT
191
UUAGGAUCUCGAGAGUAUATT
347
AL-DP-7400


 


AAACUUGACGUAUCACAAUGUAA
36
ACUUGACGUAUCACAAUGUTT
192
ACAUUGUGAUACGUCAAGUTT
348
AL-DP-7391


 


AAAACAGUCGAAAUCUAGUGAAA
37
AACAGUCGAAAUCUAGUCATT
193
UCACUAGAUUUCGACUGUUTT
349
AL-DP-7393


 


AAUCAUUAUCGUAAUGGAGAAAA
38
UCAUUAUCGUAAUGGAGAATT
194
UUCUCCAUUACGAUAAUGATT
350
AL-DP-7511


 


AAAUAGUACUGGGUCUGGCUAAA
39
AUAGUACUGGGUCUGGCUATT
195
UAGCCAGACCCAGUACUAUTT
351
AL-DP-7454


 


AACCUAACGUGGAAUGUGACUAA
40
CCUAACGUGGAAUGUGACUTT
196
AGUCACAUUCCACGUUAGGTT
352
AL-DP-7450


 


AAUUGUCCGGCUAGAGAUGAUAA
41
UUGUCCGGCUAGAGAUGAUTT
197
AUCAUCUCUAGCCGGACAATT
353
AL-DP-7533


 


AAACCUAACGUGGAAUGUGACAA
42
ACCUAACGUGGAAUGUGACTT
198
GUCACAUUCCACGUUAGGUTT
354
AL-DP-7485


 


AAUUAACAGUCGAAAUCUAGUAA
43
UUAACAGUCGAAAUCUAGUTT
199
ACUAGAUUUCGACUGUUAATT
355
AL-DP-7495


 


AAUUGGCAUAGUACUGGGUCUAA
44
UUGGCAUAGUACUGGGUCUTT
200
AGACCCAGUACUAUGCCAATT
356
AL-DP-7456


 


AAGAACUUUUCGUGACUUGGGAA
45
GAACUUUUCGUGACUUGGGTT
201
CCCAAGUCACGAAAAGUUCTT
357
AL-DP7538


 


AAGUCCGGCUAGAGAUGAUCGAA
46
GUCCGGCUAGAGAUGAUCGTT
202
CGAUCAUCUCUAGCCGGACTT
358
AL-DP-7377


 


AAGCCCUCGAGCUUUAUAAGAAA
47
GCCCUCGAGCUUUAUAAGATT
203
UCUUAUAAAGCUCGAGGGCTT
359
AL-DP-7405


 


AACUCGAGCUUUAUAAGAUUAAA
48
CUCGAGCUUUAUAAGAUUATT
204
UAAUCUUAUAAAGCUCGAGTT
360
AL-DP-7392


 


AAUGGCAUAGUACUGGGUCUGAA
49
UGGCAUAGUACUGGGUCUGTT
205
CAGACCCAGUACUAUGCCATT
361
AL-DP-7453


 


AAACGAAUGAGUUUUGUGCUUAA
50
ACGAAUGAGUUUUGUGCUUTT
206
AAGCACAAAACUCAUUCGUTT
362
AL-DP-7366


 


AAUUUCUUCGUAUGGAUAAUAAA
51
UUUCUUCGUAUGGAUAAUATT
207
UAUUAUCCAUACCAAGAAATT
363
AL-DP-7534


 


AAAGACGUGACCAUAUCAUAGAA
52
AGACGUGACCAUAUCAUAGTT
208
CUAUGAUAUGGUCACGUCUTT
364
AL-DP-7401


 


AAUAGUACUGGGUCUGGCUAUAA
53
UAGUACUGGGUCUGGCUAUTT
209
AUACCCAGACCCAGUACUATT
365
AL-DP-7523


 


AACCUAUGGAUAAUAAUGCAGAA
54
CGUAUGGAUAAUAAUGCACTT
210
CUGCAUUAUUAUCCAUACGTT
366
AL-DP-7555


 


AAUGGCUAUUUACAAUAACUGAA
55
UGGCUAUUUACAAUAACUGTT
211
CAGUUAUUGUAAAUAGCCATT
367
AL-DP-7536


 


AAAAUUCGCAUGUACAGUGAAAA
56
AAUUCGCAUGUACAGUGAATT
212
UUCACUGUACAUGCGAAUUTT
368
AL-DP-7371


 


AAAAUAGAAUUCGCAUGUACAAA
57
AAUAGAAUUCGCAUGUACATT
213
UGUACAUGCGAAUUCUAUUTT
369
AL-DP-7372


 


AAUGGUAACCCAAUGAUGUAUAA
58
UGGUAACCCAAUGAUGUAUTT
214
AUACAUCAUUGGGUUACCATT
370
AL-DP-7370


 


AAAGCCGGAAUCUAGAUUUCCAA
59
AGCCGGAAUCUAGAUUUCCTT
215
GGAAAUCUAGAUUCCGGCUTT
371
AL-DP-7474


 


AAACUUUUCGUGACUUGGGAGAA
60
ACUUUUCGUGACUUGGGAGTT
216
CUCCCAAGUCACGAAAAGUTT
372
AL-DP-7452


 


AAAGCCCUCGAGCUUUAUAAGAA
61
AGCGCUCGAGCUUUAUAAGTT
217
CUUAUAAAGCUCGAGGGCUTT
373
AL-DP-7498


 


AAGAACGAAGAAUCACUGUUCAA
62
GAACGAAGAAUCACUGUUCTT
218
GAACAGUGAUUCUUCGUUCTT
374
AL-DP-7551


 


AAUAUUCUGACUACAUUCUCAAA
63
UAUUCUGACUACAUUCUCATT
219
UGAGAAUGUAGUCAGAAUATT
375
AL-DP-7552


 


AAGCAUCUAAUAGAACGCUACAA
64
GCAUCUAAUAGAACGCUACTT
220
GUAGCGUUCUAUUAGAUGCTT
376
AL-DP-7504


 


AAUCGAAAUCUAGUGAAUGAUAA
65
UCGAAAUCUAGUGAAUGAUTT
221
AUCAUUCACUAGAUUUCGATT
377
AL-DP-7467


 


AAAGUCGAAAUCUAGUGAAUGAA
66
AGUCGAAAUCUAGUGAAUGTT
222
CAUUCACUAGAUUUCGACUTT
378
AL-DP-7463


 


AAGAAAGGCGCUAGAAUUCAUAA
67
GAAAGGCGCUAGAAUUGAUTT
223
AUCAAUUCUAGCGCCUUUCTT
379
AL-DP-7399


 


AAAAAGGCGCUAGAAUUGAUUAA
68
AAAGGCGCUAGAAUUGAUUTT
224
AAUCAAUUCUAGCGCCUUUTT
380
AL-DP-7501


 


AAAGGCGCUAGAAUUGAUUUUAA
69
AGGCCCUAGAAUUGAUUUUTT
225
AAAAUCAAUUCUAGCGCCUTT
381
AL-DP-7385


 


AAAGCAUCUAAUAGAACGCUAAA
70
AGCAUCUAAUAGAACGCUATT
226
UAGCGUUCUAUUAGAUGCUTT
382
AL-DP-7480


 


AACAAAGCGAUGAGCAAGCUAAA
71
CAAAGCGAUGAGCAAGCUATT
227
UAGCUUGCUCAUCGCUUUGTT
383
AL-DP-7528


 


AACCAUGGUUGUCUACAGGAAAA
72
CCAUGGUUGUCUACAGGAATT
228
UUCCUGUAGACAACCAUGGTT
384
AL-DP-7535


 


AAAGAAAGGCGCUAGAAUUGAAA
73
AGAAAGGCGCUAGAAUUGATT
229
UCAAUUCUAGCGCCUUUCUTT
385
AL-DP-7403


 


AAAACGCUACUACUACCAGUUAA
74
AACGCUACUACCACCAGUUTT
230
AACUGGUGGUAGUAGCGUUTT
386
AL-DP-7380


 


AAGCGCUAGAAUUGAUUUUAAAA
75
GCGCUAGAAUUGAUUUUAATT
231
UUAAAAUCAAUUCUAGCGCTT
387
AL-DP-7364


 


AAGCACGUGAUCAGUGUUGCAAA
76
GCACGUGAUCAGUGUUGCATT
232
UGCAACACUGAUCACGUGCTT
388
AL-DP-7469


 


AAGAUAGUGUCCCAGUACAAAAA
77
GAUAGUGUCCCAGUACAAATT
233
UUUGUACUGGGACACUAUCTT
389
AL-DP-7518


 


AAUUUGCGUGAAAGUGUUACAAA
78
UUUGCGUGAAAGUGUUACATT
234
UGUAACACUUUCACGCAAATT
390
AL-DP-7464


 


AAAGUAUGUGCUACUUUUUUGAA
79
AGUAUGUGCUACUUUUUUGTT
235
CAAAAAAGUAGCACAUACUTT
391
AL-DP-7560


 


AAGUAUGUCGUCUUCAUGUGUAA
80
GUAUGUCGUCUUCAUGUGUTT
236
ACACAUGAAGACGACAUACTT
392
AL-DP-7461


 


AAGGUAGUCAAGCCUAUUGCAAA
81
GCUACUCAAGCCUAUUGCATT
237
UGCAAUAGGCUUGACUACCTT
393
AL-DP-7472


 


AAACGUAACCUUCAAGUAUGUAA
82
ACGUAACCUUCAAGUAUGUTT
238
ACAUACUUGAAGGUUACGUTT
394
AL-DP-7459


 


AAACCACGUAACCUUCAAGUAAA
83
ACCACGUAACCUUCAAGUATT
239
UACUUGAAGGUUACGUGGUTT
395
AL-DP-7381


 


AACAGUAAGCUGACCUGGAAAAA
84
CAGUAAGCUGACCUGGAAATT
240
UUUCCAGGUCAGCUUACUGTT
396
AL-DP-7515


 


AAAGUAGGUUUACAUUACUGAAA
85
AGUAGGUUUACAUUACUGATT
241
UCAGUAAUGUAAACCUACUTT
397
AL-DP-7517


 


AAAAUGGUAGUCAAGCCUAUUAA
86
AAUGGUAGUCAAGCCUAUUTT
242
AAUAGGCUUGACUACCAUUTT
398
AL-DP-7521


 


AAGCCUAUUGCAACAAAGUUAAA
87
GCCUAUUGCAACAAAGUUATT
243
UAACUUUGUUGCAAUAGGCTT
399
AL-DP-7530


 


AAGACCACGUAACCUUCAAGUAA
88
GACCACGUAACCUUCAAGUTT
244
ACUUGAAGGUUACGUGGUCTT
400
AL-DP-7388


 


AAUGUUAAACGUUACUUUCAUAA
89
UGUUAAACGUUACUUUCAUTT
245
AUGAAAGUAACGUUUAACATT
401
AL-DP-7451


 


AAAUUGAAGCUAGCCGAAUGAAA
90
AUUGAAGCUAGCCGAAUGATT
246
UCAUUCGGCUAGCUUCAAUTT
402
AL-DP-7484


 


AAAUAUAACGAGGGAUAAAUUAA
91
AUAUAACGAGGGAUAAAUUTT
247
AAUUUAUCCCUCGUUAUAUTT
403
AL-DP-7376


 


AAACUUACUUAUUACCUAGAUAA
92
ACUUACUUAUUACCUAGAUTT
248
AUCUAGGUAAUAAGUAAGUTT
404
AL-DP-7500


 


AAUGUUCUCGUUGUUGUUUUAAA
93
UGUUCUCGUUGUUGUUUUATT
249
UAAAACAACAACGAGAACATT
405
AL-DP-7488


 


AAUUUUAAGGGUUAAAUCACUAA
94
UUUUAAGGGUUAAAUCACUTT
250
AGUGAUUUAACCCUUAAAATT
406
AL-DP-7541


 


AAAGUAACAGCACAACAAAUUAA
95
AGUAACAGCACAACAAAUUTT
251
AAUUUGUUGUGCUGUUACUTT
407
AL-DP-7550


 


AACAACUCCUGCUCUGAGAUAAA
96
CAACUCCUGCUCUGAGAUATT
252
UAUCUCAGAGCAGGAGUUGTT
4O8
AL-DP-7776


 


AAGAUGUGACUUACUUAACAGAA
97
GAUGUGACUUACUUAACAGTT
253
CUGUUAAGUAAGUCACAUCTT
409
AL-DP-7777


 


AACAUUAUCGUAAUGGAGAAUAA
98
CAUUAUCGUAAUGGAGAAUTT
254
AUUCUCCAUUACGAUAAUGTT
410
AL-DP-7510


 


AACCAUUUUAUCGAGGCACGUAA
99
CCAUUUUAUCGAGGCACGUTT
255
ACGUGCCUCGAUAAAAUGGTT
411
AL-DP-7507


 


AAAGUAGCCAAUCCUCUUUCUAA
100
AGUAGCCAAUCCUCUUUCUTT
256
AGAAAGAGGAUUGGCUACUTT
412
AL-DP-7479


 


AAUAAUAGAACGCUACUACCAAA
101
UAAUAGAACGCUACUACCATT
257
UGGUAGUAGCGUUCUAUUATT
413
AL-DP-7542


 


AACUUCGUGCAACUGUAGUCAAA
102
CUUCGUGCAACUGUAGUCATT
258
UGACUACAGUUGCACGAAGTT
414
AL-DP-7494


 


AAUCAUAUGGUGACCAAUGAAAA
103
UCAUAUGGUGACCAAUGAATT
259
UUCAUUGGUCACCAUAUGATT
415
AL-DP-7531


 


AAUUAAUCCGUGUUAUUGGAAAA
104
UUAAUCCGUGUUAUUGGAATT
260
UUCCAAUAACACGGAUUAATT
416
AL-DP-7373


 


AAAUACGCUACCUUGAUGAAAAA
105
AUACGCUACCUUGAUGAAATT
261
UUUCAUCAAGGUAGCGUAUTT
417
AL-DP-7508


 


AAAGCUAGCCGAAUGAAGCGAAA
106
AGCUAGCCGAAUGAAGCGATT
262
UCGCUUCAUUCGGCUAGCUTT
418
AL-DP-7487


 


AAUACAUACGCUACCUUGAUGAA
107
UACAUACGCUACCUUGAUGTT
263
CAUCAAGGUAGCGUAUGUATT
419
AL-DP-7375


 


AAAAUAUAACGAGGGAUAAAUAA
108
AAUAUAACGAGGGAUAAAUTT
264
AUUUAUCCCUCGUUAUAUUTT
420
AL-DP-7462


 


AAGUUCUCGUUGUUGUUUUAAAA
109
GUUCUCGUUGUUGUUUUAATT
265
UUAAAACAACAACGAGAACTT
421
AL-DP-7513


 


AAUGAUUGACUGAUUGUUUUAAA
110
UGAUUGACUGAUUGUUUUATT
266
UAAAACAAUCAGUCAAUCATT
422
AL-DP-7455


 


AAUUUAAUCCGUGUUAUUGGAAA
111
UUUAAUCCGUGUUAUUGGATT
267
UCCAAUAACACCGAUUAAATT
423
AL-DP-7374


 


AAUGUCCGGCUAGAGAUGAUCAA
112
UGUCCGGCUAGAGAUGAUCTT
268
GAUCAUCUCUAGCCGGACATT
424
AL-DP-7475


 


AAGCUAGCCGAAUGAAGCGAGAA
113
GCUAGCCGAAUGAAGCGAGTT
269
CUCGCUUCAUUCGGCUAGCTT
425
AL-DP-7369


 


AAGUACAUACGCUACCUUGAUAA
114
GUACAUACGCUACCUUGAUTT
270
AUCAAGGUAGCGUAUGUACTT
426
AL-DP-7466


 


AAUCGAGCUUUAUAAGAUUAAAA
115
UCGAGCUUUAUAAGAUUAATT
271
UUAAUCUUAUAAAGCUCGATT
427
AL-DP-7491


 


AACUACCACCAGUUAACUGAGAA
116
CUACCACCAGUUAACUGAGTT
272
CUCAGUUAACUGGUGGUAGTT
426
AL-DP7482


 


AAUUUUAUCGAGGCACGUGAUAA
117
UUUUAUCGAGGCACGUGAUTT
273
AUCACGUGCCUCGAUAAAATT
429
AL-DP-7398


 


AAAUUUUAUCGAGGCACGUGAAA
118
AUUUUAUCGAGGCACGUGATT
274
UCACGUGCCUCGAUAAAAUTT
430
AL-DP-7471


 


AAAACGUUACUUUCAUGUACUAA
119
AACGUUACUUUCAUGUACUTT
275
AGUACAUGAAAGUAACGUUTT
431
AL-DP-7383


 


AAAGAAUUCGCAUGUACAGUGAA
120
AGAAUUCGGAUGUACAGUGTT
276
CACUGUACAUGCGAAUUCUTT
432
AL-DP-7367


 


AAUCACGUAUGCCAAAGGAUUAA
121
UCACGUAUGCCAAAGGAUUTT
277
AAUCCUUUGGCAUACGUGATT
433
AL-DP-7386


 


AACUUUAAUCCGUGUUAUUGGAA
122
CUUUAAUCCGUGUUAUUGGTT
278
CCAAUAACACGGAUUAAAGTT
434
AL-DP-7525


 


AAACAUACGCUACCUUGAUGAAA
123
ACAUACGCUACCUUGAUGATT
279
UCAUCAAGGUAGCGUAUGUTT
435
AL-DP-7486


 


AAUCCUAGUCUUCUGUGUAUGAA
124
UCCUAGUCUUCUGUGUAUGTT
280
CAUACACAGAAGACUAGGATT
436
AL-DP-7539


 


AACAUUUUAUCGAGGCACGUGAA
125
CAUUUUAUCGAGGCACGUGTT
281
CACGUGCCUCGAUAAAAUGTT
437
AL-DP-7483


 


AAUUAACUGAUUACUGUAGAUAA
126
UUAACUGAUUACUGUAGAUTT
282
AUCUACAGUAAUCAGUUAATT
438
AL-DP-7503


 


AACCUUCGUGCAACUGUAGUCAA
127
CCUUCGUGCAACUGUAGUCTT
283
GACUACAGUUGCACGAAGGTT
439
AL-DP-7537


 


AAUGUUAGGGUACAUACGCUAAA
128
UGUUAGGGUACAUACGCUATT
284
UAGCGUAUGUACCCUAACATT
440
AL-DP-7396


 


AACACUGUUAGGGUACAUACGAA
129
CACUGUUAGGGUACAUACGTT
265
CGUAUGUACCCUAACACUCTT
441
AL-DP-7404


 


AAUGUGGCACUUUUCACCAUAAA
130
UGUGGCACUUUUCACCAUATT
286
UAUGGUGAAAAGUGCCACATT
442
AL-DP-7543


 


AAAUCACGUAUGCCAAAGGAUAA
131
AUCACGUAUGCCAAAGGAUTT
287
AUCCUUUGGCAUACGUGAUTT
443
AL-DP-7379


 


AAAGGGUACAUACGCUACCUUAA
132
AGGGUACAUACGCUACCUUTT
288
AAGGUAGCGUAUGUACCCUTT
444
AL-DP-7502


 


AAACUAAGGUGAGACAUUGAUAA
133
ACUAAGGUGAGACAUUGAUTT
289
AUCAAUGUCUCACCUUAGUTT
445
AL-DP-7519


 


AAUGUCACCUAACGUGGAAUGAA
134
UGUCACCUAACGUGGAAUGTT
290
CAUUCCACGUUAGGUGACATT
446
AL-DP-7506


 


AAAUAGAAUUCGCAUGUACAGAA
135
AUAGAAUUCGCAUGUACAGTT
291
CUGUACAUGCGAAUUCUAUTT
447
AL-DP-7457


 


AAUUCGUGACUUGGGAGACUCAA
136
UUCGUGACUUGGGAGACUCTT
292
GAGUCUCCCAAGUCACGAATT
448
AL-DP-7468


 


AAUAGAAUUCGCAUGUACAGUAA
137
UAGAAUUCGCAUGUACAGUTT
293
ACUGUACAUGCGAAUUCUATT
449
AL-DP-7368


 


AAUGAGGGCGUUUUAUAUAAUAA
138
UGAGGGCGUUUUAUAUAAUTT
294
AUUAUAUAAAACGCCCUCATT
450
AL-DP-7402


 


AAACACUGUUAGGGUACAUACAA
139
ACACUGUUAGGGUACAUACTT
295
GUAUGUACCCUAACAGUGUTT
451
AL-DP-7481


 


AAUUCUCGUUGUUGUUUUAAGAA
140
UUCUCGUUGUUGUUUUAAGTT
296
CUUAAAACAACAACGAGAATT
452
AL-DP-7465


 


AACUGUUAGGGUACAUACGCUAA
141
CUGUUAGGGUACAUACGCUTT
297
AGCGUAUGUACCCUAACAGTT
453
AL-DP-7496


 


AAUUUAACUAAGGUGAGACAUAA
142
UUUAACUAAGGUGAGACAUTT
298
AUGUCUCACCUUAGUUAAATT
454
AL-DP-7549


 


AAUAACAGUCGAAAUCUAGUGAA
143
UAACAGUCGAAAUCUAGUGTT
299
CACUAGAUUUCGACUGUUATT
455
AL-DP-7394


 


AAUGUGGUCUAAAUACAAUGCAA
144
UGUGGUCUAAAUACAAUGCTT
300
GCAUUGUAUUUAGACCACATT
456
AL-DP-7477


 


AAUCACCUAACGUGGAAUGUGAA
145
UCACCUAACGUGGAAUGUGTT
301
CACAUUCCACGUUAGGUGATT
457
AL-DP-7516


 


AAUUCUUAGUAUAUGAAAGGAAA
146
UUCUUAGUAUAUGAAAGGATT
302
UCCUUUCAUAUACUAAGAATT
458
AL-DP-7556


 


AAAAUAGAACGCUACUACCACAA
147
AAUAGAACGCUACUACCACTT
303
GUGGUAGUAGCGUUCUAUUTT
459
AL-DP-7387


 


AACUGAGGGCGUUUUAUAUAAAA
148
CUGAGGGCGUUUUAUAUAATT
304
UUAUAUAAAACGCCCUCAGTT
460
AL-DP-7524


 


AAAAAUCGUUCAUUCAUUUACAA
149
AAAUCGUUCAUUCAUUUACTT
305
GUAAAUGAAUGAACGAUUUTT
461
AL-DP-7378


 


AAUUUUCGUGACUUGGGAGACAA
150
UUUUCGUGACUUGGGAGACTT
306
GUCUCCCAAGUCACGAAAATT
462
AL-DP-7389


 


AAUCGUGCAACUGUAGUCAUCAA
151
UCGUGCAACUGUACUCAUCTT
307
GAUGACUACAGUUdCACCATT
463
AL-DP-7384


 


AAUCAUACAGUAAGCUGACCUAA
152
UCAUACAGUAAGCUGACCUTT
308
AGGUCAGCUUACUGUAUGATT
464
AL-DP-7497


 


AACGUGCAACUGUAGUCAUCUAA
153
CGUdCAACUGUAGUCAUCUTT
309
AGAUGACUACAGUUGCACGTT
465
AL-DP-7559


 


AAUGCCAUUAGAAGGGUCUACAA
154
UGCCAUUAGAACGGUCUACTT
310
GUAGACCCUUCUAAUGGCATT
466
AL-DP-7520


 


AAUUACAAUAACUGUAUACUGAA
155
UUACAAUAACUGUAUACUGTT
311
CAGUAUACAGUUAUUGUAATT
467
AL-DP-7505


 


AAUUCGCAUGUACAGUGAACGAA
156
UUCGCAUGUACAGUGAACGTT
312
CGUUCACUGUACAUGCGAATT
468
AL-DP-7460









dsRNA Synthesis
Source of 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, 500 Å, 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 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., 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, Unterschleiβheim, 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° 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 (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB




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 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-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




3-{(Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 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-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD




The hydrochloride salt of 3-[(6-Amino-hexanoyl)-thoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 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%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE




Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 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° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 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-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester 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 (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 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 1M 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/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester 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° 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 (2×20 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




Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
dsRNA Expression Vectors
In another aspect of the invention, E6AP specific dsRNA molecules that modulate E6AP gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3614-3018; 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. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA-polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:25111-2515)).
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-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single 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. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
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. FI 015) supplemented to contain 10% fetal calf serum (FCS), Penicillin 100 U/ml, Streptomycin 100 μg/ml and 2 mM L-Glutamin at 37° C. in an atmosphere with 5% CO2 in a humidified incubator.
For transfection with siRNA, HCT-116 cells were seeded at a density of 2.0×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30 nM and 3 nM 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, Calif.) (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
Standard
mean
Standard



duplex
activity
deviation
activity
deviation



name
at 30 nM
at 30 nM
at 3 nM
at 3 nM

















AL-DP-7545
9.35
3.36
14.04
3.82



AL-DP-7558
12.36
3.07
18.49
4.36



AL-DP-7548
12.55
5.85
18.92
4.72



AL-DP-7509
14.42
3.99
19.39
2.71



AL-DP-7492
11.25
2.53
19.61
7.89



AL-DP-7554
14.16
4.56
19.83
5.15



AL-DP-7557
16.00
6.50
19.97
7.04



AL-DP-7476
14.15
7.05
20.21
6.19



AL-DP-7514
24.01
12.46
20.54
6.13



AL-DP-7540
15.61
5.14
21.78
3.95



AL-DP-7397
13.05
5.68
22.03
11.42



AL-DP-7526
15.87
5.65
22.28
5.61



AL-DP-7473
17.22
6.09
22.65
6.64



AL-DP-7478
16.76
9.85
22.69
6.84



AL-DP-7553
23.50
5.15
23.19
3.34



AL-DP-7395
17.30
7.48
23.22
8.88



AL-DP-7522
26.16
10.71
23.51
8.18



AL-DP-7499
14.21
6.15
23.81
12.13



AL-DP-7527
24.11
5.05
23.98
8.89



AL-DP-7544
17.23
5.90
24.03
2.56



AL-DP-7489
23.56
10.21
24.54
7.57



AL-DP-7365
14.54
7.13
24.56
8.90



AL-DP-7390
16.44
6.37
24.74
6.73



AL-DP-7458
14.25
5.11
25.28
6.56



AL-DP-7532
21.47
4.18
25.48
6.40



AL-DP-7546
17.66
4.28
25.91
7.73



AL-DP-7512
27.88
6.58
26.22
5.07



AL-DP-7470
28.22
6.50
26.31
8.12



AL-DP-7406
20.23
6.01
26.62
6.35



AL-DP-7382
17.82
7.24
26.93
9.30



AL-DP-7547
24.63
6.66
28.80
10.23



AL-DP-7490
25.94
9.32
28.95
10.29



AL-DP-7493
12.53
5.36
29.56
12.54



AL-DP-7529
17.61
8.36
29.59
10.53



AL-DP-7400
21.03
14.86
30.04
12.58



AL-DP-7391
26.74
12.00
30.06
8.07



AL-DP-7393
22.40
9.77
30.69
8.51



AL-DP-7511
26.50
6.02
30.88
6.43



AL-DP-7454
25.16
14.85
31.09
8.75



AL-DP-7450
20.09
8.43
32.10
8.57



AL-DP-7533
26.93
5.86
33.91
5.52



AL-DP-7485
27.45
4.36
34.12
10.28



AL-DP-7495
28.51
13.42
34.45
11.20



AL-DP-7456
16.82
5.62
34.54
10.30



AL-DP-7538
29.04
5.12
34.71
6.42



AL-DP-7377
22.98
8.19
35.31
12.53



AL-DP-7405
21.93
10.30
35.66
15.22



AL-DP-7392
23.83
8.93
36.14
6.31



AL-DP-7453
25.78
12.10
36.98
5.22



AL-DP-7366
19.60
7.30
37.20
13.88



AL-DP-7534
26.35
5.24
37.69
8.49



AL-DP-7401
28.74
9.10
37.75
7.70



AL-DP-7523
33.88
6.85
39.81
9.45



AL-DP-7555
29.13
8.87
40.35
6.23



AL-DP-7536
32.33
3.49
41.08
8.00



AL-DP-7371
25.49
9.83
42.19
16.08



AL-DP-7372
21.83
12.03
42.87
17.78



AL-DP-7370
24.51
12.64
43.75
14.09



AL-DP-7474
32.57
13.13
44.40
7.78



AL-DP-7452
30.12
12.02
46.66
9.19



AL-DP-7498
32.38
11.81
54.11
12.74



AL-DP-7504
15.04
6.39
19.69
5.67



AL-DP-7467
19.81
6.42
21.66
8.12



AL-DP-7463
26.63
8.84
21.73
8.80



AL-DP-7399
15.62
8.32
22.98
7.65



AL-DP-7501
17.32
5.24
23.45
7.44



AL-DP-7385
17.60
5.11
28.00
11.84



AL-DP-7480
21.89
8.21
29.42
8.64



AL-DP-7528
26.47
2.94
30.76
10.87



AL-DP-7535
26.65
3.13
31.77
4.34



AL-DP-7403
24.10
6.21
38.79
14.41



AL-DP-7380
29.84
7.65
40.42
5.72



AL-DP-7469
17.18
7.41
21.13
6.29



AL-DP-7518
15.71
6.00
21.89
6.68



AL-DP-7464
29.18
12.30
22.13
8.99



AL-DP-7560
17.33
4.85
24.84
5.80



AL-DP-7461
30.55
8.26
25.62
9.48



AL-DP-7472
25.17
11.50
26.31
8.61



AL-DP-7459
29.60
7.71
27.27
9.68



AL-DP-7381
17.29
6.63
27.31
7.42



AL-DP-7515
32.18
10.22
29.76
6.01



AL-DP-7517
29.75
6.99
29.87
5.69



AL-DP-7521
28.60
8.06
31.68
5.72



AL-DP-7530
31.09
8.09
31.94
3.36



AL-DP-7388
22.81
3.80
32.28
6.23



AL-DP-7451
22.66
8.92
32.45
8.26



AL-DP-7484
26.77
13.00
32.84
6.95



AL-DP-7376
34.18
14.11
39.93
8.41



AL-DP-7500
32.69
8.47
41.55
13.32



AL-DP-7776
17.91
4.95
21.77
5.04



AL-DP-7777
21.10
7.60



AL-DP-7510
34.70
5.83



AL-DP-7507
35.11
6.78



AL-DP-7479
35.29
13.76



AL-DP-7542
36.32
5.00



AL-DP-7494
38.34
12.68



AL-DP-7531
38.58
14.26



AL-DP-7373
39.04
16.08



AL-DP-7508
39.95
12.87



AL-DP-7487
40.48
15.20



AL-DP-7375
41.19
15.06



AL-DP-7462
41.61
17.23



AL-DP-7513
41.69
9.15



AL-DP-7455
43.35
12.72



AL-DP-7374
43.37
12.26



AL-DP-7475
43.68
11.45



AL-DP-7369
43.99
15.44



AL-DP-7466
44.27
15.53



AL-DP-7491
45.06
10.32



AL-DP-7482
45.06
12.37



AL-DP-7398
45.79
9.50



AL-DP-7471
46.11
13.53



AL-DP-7383
46.87
20.08



AL-DP-7367
46.96
16.88



AL-DP-7386
47.46
10.01



AL-DP-7525
49.60
14.11



AL-DP-7486
49.64
8.95



AL-DP-7539
49.97
12.73



AL-DP-7483
49.97
12.50



AL-DP-7503
51.28
7.08



AL-DP-7537
53.19
7.75



AL-DP-7396
54.11
13.02



AL-DP-7404
54.96
17.72



AL-DP-7543
55.48
9.23



AL-DP-7379
55.82
18.47



AL-DP-7502
56.15
16.52



AL-DP-7519
56.15
13.30



AL-DP-7506
57.24
21.04



AL-DP-7457
57.30
15.19



AL-DP-7468
57.83
15.40



AL-DP-7368
59.38
22.50



AL-DP-7402
59.57
13.42



AL-DP-7481
60.17
14.54



AL-DP-7465
61.44
28.49



AL-DP-7496
61.65
17.78



AL-DP-7549
61.90
12.36



AL-DP-7394
61.94
17.08



AL-DP-7477
63.20
14.74



AL-DP-7516
67.72
19.24



AL-DP-7556
69.49
13.89



AL-DP-7387
72.14
16.20



AL-DP-7524
72.52
19.76



AL-DP-7378
73.44
19.20



AL-DP-7389
73.74
23.83



AL-DP-7384
76.45
21.99



AL-DP-7497
77.66
22.60



AL-DP-7559
78.86
16.61



AL-DP-7520
85.45
14.83



AL-DP-7505
86.86
39.07



AL-DP-7460
100.95
22.69










Testing of Chemically Modified dsRNA Targeting E6AP
Chemically modified dsRNA were tested to identify their relative abilities to reduce the expression level of mRNA encoding E6AP in a cell. The assay conditions described above for HCT-116 cells were employed. The activity of a given E6AP specific siRNA duplex was expressed as percent E6AP mRNA concentration in treated cells relative to E6AP mRNA concentration in cells treated with the control siRNA duplex.
1. Chemically Modified dsRNA
Table 3 sets forth dsRNA compositions of the invention. In this table the unmodified sequence is followed by the same sequence containing one or more nucleotide modifications.












TABLE 3






SEQ

SEQ




sense strand
ID
antisense strand
ID
duplex


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







AUACGAUGAAUCUACAAAATT
469
UUUUGUAGAUUCAUCGUAUTT
644
AL-DP-7545



 


AUACGAUGAAUCUACAAAATsT
470
UUUUGUAGAUUCAUCGUAUTsT
645
ND-8763


 


AuAcGAuGAAucuAcAAAATsT
471
UUUUGuAGAUUcAUCGuAUTsT
646
ND-8782


 


AuAcGAuGAAucuAcAAAATsT
472
uuuuGuAGAuUcAUCGuAUTsT
647
ND-8801


 


AUACGAUGAAUCUACAAAATTChol
473
UUUUGUAGAUUCAUCGUAUTsT
648
ND-8820


 


AuAcGAuGAAucuAcAAAATTchol
474
UUUUGuAGAUUcAUCGUAUTsT
649
ND-8845


 


AuAcGAuGAAucuAcAAAATTchol
475
uuuuGuAGAuUcAUCGuAUTsT
650
ND-8870


 


UGACUACAUUCUCAAUAAATT
476
UUUAUUGAGAAUGUAGUCATT
651
AL-DP-7558


 


UGACUACAUUCUCAAUAAATST
477
UUUAUUGAGAAUGUAGUCATsT
652
ND-8764


 


uGAcuAcAuucucAAuAAATsT
478
UUuAUUGAGAAUGuAGUcATsT
653
ND-8783


 


uGAcuAcAuucucAAuAAATsT
479
uuuAuuGAGAAuGuAGUcATsT
654
ND-8802


 


UGACUACAUUCUCAAUAAATTChol
480
UUUAUUOAGAAUGUAGUCATsT
655
ND-8821


 


uGAcuAcAuucucAAuAAATTchol
481
UUuAUUGAGAAUGuAGUcATsT
656
ND-8846


 


uGAcuAcAuucucAAuAAATTchol
482
uuuAuuGAGAAuGuAGUcATsT
657
ND-8871


 


AGCCUGCACGAAUGAGUUUTT
483
AAACUCAUUCGUGCAGGCUTT
658
AL-DP-7548


 


AGCCUGCACGAAUGAGUUUTsT
484
AAACUCAUUCGUGCAGGCUTsT
659
ND-8765


 


AGccuGcAcGAAuGAGuuuTsT
485
AAACUcAUUCGUGcAGGCUTsT
660
ND-8784


 


AGccuGcAcGAAuGAGuuuTST
486
AAACUcAuUCGuGcAGGCUTsT
661
ND-8803


 


AGCCUGCACGAAUGAGUUUTTChol
487
AAACUCAUUCGUGCAGGCUTsT
662
ND-8822


 


AGccuGcAcGAAuGAGuuuTTchol
488
AAACUcAUUCGUGcAGGCUTsT
663
ND-8847


 


AGccuGcAcGAAuGAGuuuTTchol
489
AAACUcAuUCGuGcAGGCUTsT
664
ND-8872


 


GGAUUGUCGAAAACCACUUTT
490
AAGUGGUUUUCGACAAUCCTT
665
AL-DP-7509


 


GGAUUGUCGAAAACCACUUTsT
491
AAGUGGUUUUCGACAAUCCTsT
666
ND-8766


 


GGAuuGuCGAAAAccAcuuTsT
492
AAGUGGUUUUCGAcAAUCCTST
667
ND-8785


 


GGAuuGucGAAAAccAcuuTsT
493
AAGuGGuUuUCGAcAAUCCTsT
668
ND-8804


 


GGAUUGUCGAAAACCACUUTTChol
494
AAGUGGUUUUCGACAAUCCTsT
669
ND-8823


 


GGAuuGucGAAAAccAcuuTTchol
495
AAGUGGUUUUCGAcAAUCCTsT
670
ND-8848


 


GGAuuGucGAAAAccAcuuTTchol
496
AAGuGGuUuUCGAcAAUCCTsT
671
ND-8873


 


CUCUCGAGAUCCUAAUUAUTT
497
AUAAUUAGGAUCUCGAGAGTT
672
AL-DP-7492


 


CUCUCGAGAUCCUAAUUAUTsT
498
AUAAUUAGGAUCUCGAGAGTsT
673
ND-8767


 


cucucGAGAuccuAAuuAuTsT
499
AuAAUuAGGAUCUCGAGAGTsT
674
ND-8786


 


cucucGAGAuccuAAuuAuTsT
500
AuAAuUAGGAUCUCGAGAGTsT
675
ND-8805


 


CUCUCGAGAUCCUAAUUAUTTChol
501
AUAAUUAGGAUCUCGAGAGTsT
676
ND-8824


 


cucucGAGAuccuAAuuAuTTchol
502
AuAAUuAGGAUCUCGAGAGTsT
677
ND-8849


 


cucucGAGAuccuAAuuAuTTchol
503
AuAAuUAGGAUCUCGAGAGTsT
678
ND-8874


 


AUGUGACUUACUUAACAGATT
504
UCUGUUAAGUAAGUCACAUTT
679
AL-DP-7554


 


AUGUGACUUACUUAACAGATsT
505
UCUGUUAAGUAAGUCACAUTsT
680
ND-8768


 


AuGuGAcuuAcuuAAcAGATsT
506
UCUGUuAAGuAAGUcACAUTsT
681
ND-8787


 


AuGuGAcuuAcuuAAcAGATsT
507
UCuGuuAAGUAAGUcAcAUTsT
682
ND-8806


 


AUGUGACUUACUUAACAGATTChol
508
UCUGUUAAGUAAGUCACAUTsT
683
ND-8825


 


AuGuGAcuuAcuuAAcAGATTchol
509
UCUGUuAAGUAAGUcAcAUTsT
684
ND-8850


 


AuGuGAcuuAcuuAAcAGATTchol
510
UCuGuuAAGuAAGUcAcAUTsT
685
ND-8875


 


GUAUACUCUCGAGAUCCUATT
511
UAGGAUCUCGAGAGUAUACTT
686
AL-DP-7557


 


GUAUACUCUCGAGAUCCUATsT
512
UAGGAUCUCGAGAGUAUACTsT
687
ND-8769


 


GuAuAcucucGAGAuccuATsT
513
uAGGAUCUCGAGAGUAUACTsT
688
ND-8788


 


GuAuAcucucGAGAuccuATsT
514
uAGGAUCUCGAGAGuAuACTsT
689
ND-8788


 


GUAUACUCUCGAGAUCCUATTChol
515
UAGGAUCUCGAGAGUAUACTsT
690
ND-8826


 


GuAuAcucucGAGAuccuATTchol
516
uAGGAUCUCGAGAGuAuACTsT
691
ND-8851


 


GuAuAcucucGAGAuccuATTchol
517
uAGGAUCUCGAGAGuAuACTsT
692
ND-8851


 


AGGUUACCUACAUCUCAUATT
518
UAUGAGAUGUAGGUAACCUTT
693
AL-DP-7476


 


AGGUUACCUACAUCUCAUATsT
519
UAUGAGAUGUAGGUAACCUTsT
694
ND-8770


 


AGGuuAccuAcAucucAuATsT
520
uAUGAGAUGuAGGuAACCUTsT
695
ND-8789


 


AGGuuAccuAcAucucAuATsT
521
uAuGAGAuGuAGGuAACCUTST
696
ND-8808


 


AGGUUACCUACAUCUCAUATTChol
522
UAUGAGAUGUAGGUAACCUTsT
697
ND-8827


 


AGGuuAccuAcAucucAuATTchol
523
uAUGAGAUGuAGGUAACCUTsT
698
ND-8852


 


AGGuuAccuAcAucucAuATTchol
524
uAuGAGAuGuAGGuAACCUTsT
699
ND-8877


 


AGUACUUAUUCAGACCAGATT
525
UCUGGUCUGAAUAAGUACUTT
700
AL-DP-7514


 


AGUACUUAUUCAGACCAGATST
526
UCUGGUCUGAAUAAGUACUTsT
701
ND-8771


 


AGuAcuuAuucAGAccAGATsT
527
UCUGGUCUGAAUAAGuACUTsT
702
ND-8790


 


AGuAcuuAuucAGAccAGATsT
528
UCuGGUCuGAAuAAGuACUTsT
703
ND-8809


 


AGUACUUAUUCAGACCAGATTChol
529
UCUGGUCUGAAUAAGUACUTsT
704
ND-8828


 


AGuAcuuAuucAGAccAGATTchol
530
UCUGGUCUGAAuAAGuACUTsT
705
ND-8853


 


AGuAcuuAuucAGAccAGATTchol
531
UCuGGUCuGAAuAAGuACUTsT
706
ND-8878


 


AUCCUAAUUAUCUGAAUUUTT
532
AAAUUCAGAUAAUUAGGAUTT
707
AL-DP-7540


 


AUCCUAAUUAUCUcAAUUUTsT
533
AAAUUCAGAUAAUUAGGAUTST
708
ND-8772


 


AuccuAAuuAucuGAAuuuTsT
534
AAAUUcAGAuAAUuAGGAUTsT
709
ND-8791


 


AuccuAAuuAucuGAAuuuTsT
535
AAAuUcAGAuAAuUAGGAUTsT
710
ND-8810


 


AUCCUAAUUAUCUGAAUUUTTChol
536
AAAUUCAGAUAAUUAGGAUTsT
711
ND-8829


 


AuccuAAuuAucuGAAuuuTTchol
537
AAAUUcAGAuAAUuAGGAUTsT
712
ND-8854


 


AuccuAAuuAucuGAAuuuTTchol
538
AAAuUcAGAuAAuUAGGAUTsT
713
ND-8879


 


AAGGAUAGGUGAUAGCUCATT
539
UGAGCUAUCACCUAUCCUUTT
714
AL-DP-7397


 


AAGGAUAGGUGAUAGCUCATsT
540
UGAGCUAUCACCUAUCCUUTsT
715
ND-8731


 


AAGGAuAGGuGAuAGcucATsT
541
UGAGCuAUcACCuAUCCUUTsT
716
ND-8743


 


AAGGAuAGGuGAUAGcucATsT
542
uGAGCuAUcACCUAUCCuuTsT
717
ND-8754


 


AAGGAUAGGUGAUAGCUCATTChol
543
UGAGCUAUCACCUAUCCUUTsT
718
ND-8839


 


AAGGAuAGGuGAuAGcucATTchol
544
UGAGCuAUCACCuAUCCUUTsT
719
ND-8864


 


AAGGAuAGGuGAUAGcucATTchol
545
uGAGCuAUcACCuAUCCuuTsT
720
ND-8889


 


GGAAGCCGGAAUCUAGAUUTT
546
AAUCUAGAUUCCGGCUUCCTT
721
AL-DP-7526


 


GGAAGCCGGAAUCUAGAUUTsT
547
AAUCUAGAUUCCGGCUUCCTsT
722
ND-8773


 


GGAAGccGGAAucuAGAuuTsT
548
AAUCuAGAUUCCGGCUUCCTsT
723
ND-8792


 


GGAAGccGGAAucuAGAuuTsT
549
AAUCuAGAuUCCGGCuUCCTsT
724
ND-8811


 


GGAAGCCGGAAUCUAGAUUTTChol
550
AAUCUAGAUUCCGGCUUCCTsT
725
ND-8830


 


GGAAGccGGAAucuAGAuuTTChol
551
AAUCuAGAUUCCGGCUUCCTsT
726
ND-8855


 


GGAAGccGGAAucuAGAuuTTchol
552
AAUCuAGAuUCCGGCuUCCTsT
727
ND-8880


 


UGCUUCGAAGUGCUUGAAATT
553
UUUCAAGCACUUCGAAGCATT
728
AL-DP-7473


 


UGCUUCGAAGUGCUUGAAATsT
554
UUUCAAGCACUUCGAAGCATsT
729
ND-8774


 


uGcuucGAAGuGcUuGAAATsT
555
UUUcAAGcACUUCGAAGcATsT
730
ND-8793


 


uGcuucGAAGuGcuuGAAATsT
556
uuUcAAGcACuUCGAAGcATsT
731
ND-8812


 


UGCUUCGAAGUGCUUGAAATTChol
557
UUUCAAGCACUUCGAAGCATsT
732
ND-8831


 


uGCuuCGAAGuGCUUGAAATTChol
558
UUUcAAGcACUUCGAAGcATsT
733
ND-8856


 


uGcuucGAAGuGcuuGAAATTchol
559
uuUcAAGcACuUCGAAGcATsT
734
ND-8881


 


UGGAUUGUCGAAAACCACUTT
560
AGUGGUUUUCGACAAUCCATT
735
AL-DP-7478


 


UGGAUUGUCGAAAACCACUTsT
561
AGUGGUUUUCGACAAUCCATST
736
ND-8775


 


uGGAuuGucGAAAACCAcuTsT
562
AGUGGUUUUCGAcAAUCcATsT
737
ND-8794


 


uGGAuuGucGAAAAccAcuTsT
563
AGuGGuuuUCGAcAAUCcATsT
738
ND-8813


 


UGGAUUGUCGAAAACCACUTTChol
564
AGUGGUUUUCGACAAUCCATsT
739
ND-8832


 


uGGAuuGucGAAAAccAcuTTchol
565
AGUGGUUUUCGAcAAUCcATsT
746
ND-8857


 


uGGAuuGucGAAAAccAcuTTchol
566
AGuGGuuuUCGAcAAUCCATST
741
ND-8882


 


CGGCUAGAGAUGAUCGCUATT
567
UAGCGAUCAUCUCUAGCCGTT
742
AL-DP-7553


 


CGGCUAGAGAUGAUCGCUATsT
568
UAGCGAUCAUCUCUAGCCGTsT
743
ND-8776


 


cGGcuAGAGAuGAucGCUATsT
569
uAGCGAUcAUCUCuAGCCGTsT
744
ND-8795


 


cGGcuAGAGAuGAucGcuATsT
570
uAGCGAUcAUCUCuAGCCGTsT
745
ND-8795


 


CGGCUAGAGAUGAUCGCUATTChol
571
UAGCGAUCAUCUCUAGCCGTsT
746
ND-8833


 


cGGcuAGAGAuGAucGcuATTchol
572
uAGCGAUcAUCUCuAGCCGTsT
747
ND-8858


 


cGGcuAGAGAuGAucGcuATTChol
573
uAGCGAUcAUCUCuAGCCGTsT
748
ND-8858


 


ACAGUCGAAAUCUAGUGAATT
574
UUCACUAGAUUUCGACUGUTT
749
AL-DP-7395


 


ACAGUCGAAAUCUAGUGAATsT
575
UUCACUAGAUUUCGACUGUTsT
750
ND-8730


 


AcAGucGAAAucuAGuGAATsT
576
UUcACuAGAUUUCGACUGUTsT
751
ND-8742


 


AcAGuCGAAAucuAGuGAATST
577
uucACuAGAuuUCGACuGUTsT
752
ND-8753


 


ACAGUCGAAAUCUAGUGAATTChol
578
UUCACUAGAUUUCGACUGUTsT
753
ND-8840


 


AcAGucGAAAucuAGuGAATTChol
579
UUcACuAGAUUUCGACUGUTsT
754
ND-8865


 


AcAGucGAAAucuAGuGAATTchol
580
uucACuAGAuuUCGACuGUTsT
755
ND-8890


 


CUCGAGAUCCUAAUUAUCUTT
581
AGAUAAUUAGGAUCUCGAGTT
756
AL-DP-7499


 


CUCGAGAUCCUAAUUAUCUTsT
582
AGAUAAUUAGGAUCUCGAGTsT
757
ND-8777


 


cucCAGAuccuAAuuAucuTsT
583
AGAuAAUuAGGAUCUCGAGTsT
758
ND-8796


 


cucGAGAuccuAAuuAucuTsT
584
AGAuAAuUAGGAUCUCGAGTsT
759
ND-8815


 


CUCGAGAUCCUAAUUAUCUTTChol
585
AGAUAAUUAGGAUCUCGAGTST
760
ND-8834


 


cucGAGAuccuAAuuAucuTTchol
586
AGAuAAUuAGGAUCUCGAGTsT
761
ND-8859


 


cucGAGAuccuAAuuAucuTTchol
587
AGAuAAuUAGGAUCUCGAGTsT
762
ND-8884


 


CACCUAACGUGGAAUGUGATT
588
UCACAUUCCACGUUAGGUGTT
763
AL-DP-7365


 


CACCUAACGUGGAAUGUGATsT
589
UCACAUUCCACGUUAGGUGTsT
764
ND-8724


 


cAccuAAcGuGGAAuGuGATsT
590
UcAcAUUCcACGUuAGGUGTsT
765
ND-8736


 


CAccuAAcGuGGAAUGuGATsT
591
UcAcAuuCcACGuUAGGuGTsT
766
ND-8748


 


CACCUAACGUGGAAUGUGATTChol
592
UCACAUUCCACGUUAGGUGTsT
767
ND-8841


 


cAccuAAcGuGGAAuGuGATTchol
593
UcAcAUUCcACGUuAGGUGTsT
768
ND-8866


 


cAccuAACGuGGAAuGuGATTchol
594
UcAcAuuCcACGuuAGGuGTsT
769
ND-8891


 


AAUCGUUCAUUCAUUUACATT
595
UGUAAAUGAAUGAACGAUUTT
770
AL-DP-7390


 


AAUCGUUCAUUCAUUUACATsT
596
UGUAAAUGAAUGAACGAUUTsT
771
ND-8727


 


AAucGuucAuucAuuuAcATsT
597
UGuAAAUGAAUGAACGAUUTsT
772
ND-8739


 


AAucGuucAuucAuuuAcATsT
598
uGuAAAuGAAuGAACGAuuTsT
773
ND-8750


 


AAUCGUUCAUUCAUUUACATTChol
599
UGUAAAUGAAUGAACGAUUTsT
774
ND-8842


 


AAucGuucAuucAuuuAcATTchol
600
UGuAAAUGAAUGAACGAUUTsT
775
ND-8867


 


AAucGuucAuucAuuuAcATTchol
601
uGuAAAuGAAuGAACGAuuTsT
776
ND-8892


 


AACUUUUCGUGACUUGGGATT
602
UCCCAAGUCACGAAAAGUUTT
777
AL-DP-7382


 


AACUUUUCGUGACUUGGGATsT
603
UCCCAAGUCACGAAAAGUUTsT
778
ND-8726


 


AAcuuuucGuGAcuuGGGATsT
604
UCCcAAGUcACGAAAAGUUTsT
779
ND-8738


 


AAcuuuucGuGAcuuGGGATsT
605
UCCcAAGUcACGAAAAGuuTsT
780
ND-8749


 


AACUUEUUCGUGACUUGGGATTChol
606
UCCCAAGUCACGAAAAGUUTsT
781
ND-8843


 


AAcuuuucGuGAcuuGGGATTchol
607
UCCcAAGUcACGAAAAGUUTsT
782
ND-8868


 


AAcuuuucGuGAcuuGGGATTchol
608
UCCcAAGUCACGAAAAGuuTsT
783
ND-8893


 


AACAGUCGAAAUCUAGUGATT
609
UCACUAGAUUUCGACUGUUTT
784
AL-DP-7393


 


AACAGUCGAAAUCUAGUGATsT
610
UcACUAGAUUUCGACUGUUTsT
785
ND-8729


 


AAcAGucGAAAucuAGuGATsT
611
UcACuAGAUUUCGACUGUUTsT
786
ND-8741


 


AAcAGucGAAAucuAGuGATsT
612
UcACuAGAuuUCGACuGuuTsT
787
ND-8752


 


AACAGUCGAAAUCUAGUGATTChol
613
UCACUAGAUUUCGACUGUUTsT
788
ND-8844


 


AAcAGucGAAAucuAGuGATTchol
614
UcACuAGAUUUCGACUGUUTsT
789
ND-8869


 


AAcAGucGAAAucuAGuGATTchol
615
UcACuAGAuuUCGACuGuuTsT
790
ND-8894


 


ACGAAUGAGUUUUGUGCUUTT
616
AAGCACAAAACUCAUUCGUTT
791
AL-DP-7366


 


ACGAAUGAGUUUUGUGCUUTsT
617
AAGCACAAAACUCAUUCGUTST
792
ND-8778


 


AcGAAuGAGuuuuGuGcuuTsT
618
AAGcAcAAAACUcAUUCGUTsT
793
ND-8797


 


AcGAAuGAGuuuuGuGcuuTsT
619
AAGcAcAAAACUcAuUCGUTsT
794
ND-8816


 


ACGAAUGAGUUUUGUGCUUTTChol
620
AAGCACAAAACUCAUUCGUTsT
795
ND-8835


 


AcGAAuGAGuuuuGuGcuuTTchol
621
AAGcAcAAAACUcAUUCGUTsT
796
ND-8860


 


AcGAAuGAGuuuuGuGcuuTTchol
622
AAGcAcAAAACUcAuUCGUTsT
797
ND-8885


 


AAUUCGCAUGUACAGUGAATT
623
UUCACUGUACAUGCGAAUUTT
798
AL-DP-7371


 


AAUUCGCAUGUACAGUGAATsT
624
UUCACUGUACAUGCGAAUUTsT
799
ND-8779


 


AAuucGcAuGuAcAGuGAATsT
625
UUcACUGuAcAUGCGAAUUTsT
800
ND-8798


 


AAuuCGcAuGuAcAGuGAATsT
626
uUcACuGuAcAuGCGAAuUTsT
801
ND-8817


 


AAUUCGCAUGUACAGUGAATTChol
627
UUCACUGUACAUGCGAAUUTsT
802
ND-8836


 


AAuucGcAuGuAcAGuGAATTChol
628
UUcACUGuAcAUGCGAAUUTsT
803
ND-8861


 


AAuucGcAUGUACAGuGAATTChol
629
uUCACuGuAcAUGCGAAUUTsT
804
ND-8886


 


AAUAGAAUUCGCAUGUACATT
630
UGUACAUGCGAAUUCUAUUTT
805
AL-DP-7372


 


AAUAGAAUUCGCAUGUACATsT
631
UGUACAUGCGAAUUCUAUUTsT
806
ND87 80


 


AAuAGAAuucGcAuGuAcATsT
632
UGuAcAUGCGAAUUCuAUUTsT
807
ND-8799


 


AAuAGAAuuCGcAuGUAcATsT
633
uGuAcAuGCGAAuUCuAuUTsT
808
ND-8818


 


AAUAGAAUUCGCAUGUACATTChol
634
UGUACAUGCGAAUUCUAUUTsT
809
ND-8837


 


AAuAGAAuucGcAuGuAcATTChol
635
UGuAcAUGCGAAUUCuAUUTsT
810
ND-8862


 


AAuAGAAuucGcAuGuACATTchol
636
uGuAcAuGCGAAuUCuAuUTsT
811
ND-8887


 


UGGUAACCCAAUGAUGUAUTT
637
AUACAUCAUUGGGUUACCATT
812
AL-DP-7370


 


UGGUAACCCAAUGAUGUAUTsT
638
AUACAUCAUUGGGUUACCATsT
813
ND-8781


 


uGGuAAcccAAuGAUGUAuTsT
639
AuAcAUcAUUGGGUuACcATsT
814
ND-8800


 


uGGuAAcccAAuGAuGuAuTsT
640
AuAcAUcAuUGGGuUACcATsT
815
ND-8819


 


UGGUAACCCAAUGAUGUAUTTChol
641
AUACAUCAUUGGGUUACCATsT
816
ND-8838


 


uGGuAAcccAAuGAuGuAuTTchol
642
AuAcAUcAUUGGGUuACcATsT
817
ND-8863


 


uGGuAAcCcAAuGAuGuAUTTchol
643
AuAcAUcAuUGGGuUACcATsT
818
ND-8888


 


cucGAGAuccuAAuuAucuTsT
1748
AGAuAAuuAGGAUCUCGAGTst
1749
ND-9300





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


Lower case letters: ribonucloetide bearing 2′-O-methyl substituent on ribose moiety


s: Indicates position of phosphorothioate intemucleoside linkage


chol: cholesterol moiety conjugated to 3′ ribonucleotide.


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






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











TABLE 4






Mean

Mean




activity

activity



remaining

remaining



after

after


Duplex
30 nM
Standard
100 pM
Standard


name
treatment
deviation
treatment
deviation



















AL-DP-7545
6.74
1.80
18.41
4.14


ND-8763
6.28
1.79
21.38
5.93


ND-8782
7.21
1.59
23.76
7.49


ND-8801
18.52
2.58
51.10
12.01


ND-8820
9.26
1.88
58.34
10.61


ND-8845
34.08
7.03
69.28
14.97


ND-8870
30.96
5.97
77.58
12.41


AL-DP-7558
11.93
1.66
25.71
3.57


ND-8764
8.97
1.53
25.81
7.79


ND-8783
27.33
3.10
51.35
6.52


ND-8802
28.82
4.39
91.90
14.32


ND-8821
8.96
2.36
71.92
10.68


ND-8846
75.94
17.07
88.87
7.90


ND-8871
58.02
9.96
91.79
13.92


AL-DP-7548
11.23
1.92
35.58
6.27


ND-8765
8.24
1.01
45.42
10.63


ND-8784
25.07
4.28
68.74
7.10


ND-8803
45.89
10.22
97.46
12.87


ND-8822
11.59
2.94
75.22
17.11


ND-8847
64.96
9.30
100.47
16.50


ND-8872
78.50
14.11
86.77
5.96


AL-DP-7509
17.62
2.26
21.58
2.98


ND-8766
15.26
1.22
25.45
2.92


ND-8785
19.66
3.35
43.13
4.50


ND-8804
21.66
2.34
50.36
8.66


ND-8823
15.66
2.09
48.14
4.88


ND-8848
27.84
3.58
95.42
20.53


ND-8873
29.97
3.32
91.79
13.36


AL-DP-7492
11.09
1.19
19.22
3.29


ND-8767
11.90
1.73
20.65
2.66


ND-8786
11.69
1.72
19.78
2.74


ND-8805
14.97
1.46
26.41
6.08


ND-8824
11.53
1.51
43.76
5.00


ND-8849
25.37
11.97
43.95
10.44


ND-8874
16.84
2.99
53.87
6.12


AL-DP-7554
15.01
1.22
23.48
4.39


ND-8768
14.46
1.30
26.79
4.77


ND-8787
15.20
2.47
24.76
4.44


ND-8806
15.01
2.02
33.77
10.43


ND-8825
17.00
3.82
72.33
14.34


ND-8850
29.25
7.49
93.94
19.23


ND-8875
23.33
3.94
79.79
9.03


AL-DP-7557
13.10
1.34
22.30
8.07


ND-8769
11.17
1.10
24.91
4.44


ND-8788
21.84
2.02
60.20
10.58


ND-8788
23.53
1.55
69.43
13.87


ND-8826
12.81
1.35
50.68
10.86


ND-8851
36.41
3.49
116.14
48.06


ND-8851
36.42
5.05
100.91
26.50


AL-DP-7476
17.11
2.75
25.33
7.43


ND-8770
13.36
1.65
30.58
8.25


ND-8789
46.06
6.35
76.12
14.80


ND-8808
43.15
5.55
98.81
21.90


ND-8827
14.76
2.03
56.08
13.96


ND-8852
70.35
13.51
107.70
22.62


ND-8877
58.73
8.08
90.83
10.87


AL-DP-7514
15.63
2.76
18.89
0.67


ND-8771
14.96
1.69
23.31
10.62


ND-8790
15.91
1.57
31.71
2.88


ND-8809
16.79
2.80
36.42
5.40


ND-8828
14.61
2.09
53.50
8.13


ND-8853
34.20
4.88
81.95
16.33


ND-8878
26.63
2.95
87.21
33.73


AL-DP-7540
18.18
3.06
32.59
5.25


ND-8772
19.31
2.99
36.01
5.41


ND-8791
35.43
4.60
55.34
7.39


ND-8810
17.83
2.64
25.48
7.36


ND-8829
18.93
3.20
68.53
14.55


ND-8854
50.71
6.95
89.19
9.26


ND-8879
21.76
5.10
62.43
16.86


AL-DP-7397
17.10
2.37
22.44
4.36


ND-8731
17.09
2.86
31.25
8.34


ND-8743
15.89
2.29
27.33
4.67


ND-8754
19.53
2.97
41.57
9.22


ND-8839
18.18
2.95
66.39
13.77


ND-8864
19.51
3.79
59.13
5.60


ND-8889
19.91
2.30
92.91
14.85


AL-DP-7526
17.67
2.32
41.09
7.63


ND-8773
15.59
1.57
42.07
6.55


ND-8792
19.42
2.08
46.87
6.99


ND-8811
34.56
7.82
72.57
9.85


ND-8830
19.49
3.09
69.87
7.25


ND-8855
27.49
4.52
85.38
13.45


ND-8880
38.04
6.41
87.78
13.97


AL-DP-7473
15.81
2.07
31.86
6.43


ND-8774
15.81
2.61
30.89
8.60


ND-8793
14.04
1.44
25.98
2.91


ND-8812
21.16
3.28
49.59
8.12


ND-8831
19.07
3.60
75.06
16.79


ND-8856
17.86
5.51
65.08
11.14


ND-8881
28.56
6.18
83.97
12.49


AL-DP-7478
16.41
2.58
33.38
6.20


ND-8775
16.74
1.63
31.52
2.92


ND-8794
19.05
3.19
24.88
3.34


ND-8813
17.04
2.34
26.53
3.90


ND-8832
16.40
2.16
66.67
15.18


ND-8857
26.53
6.20
69.13
9.30


ND-8882
20.04
2.43
68.67
8.67


AL-DP-7553
20.83
2.66
28.97
4.93


ND-8776
21.10
2.76
29.95
5.15


ND-8795
26.00
3.54
79.53
11.78


ND-8795
25.14
3.95
80.83
12.02


ND-8833
21.76
3.23
52.28
7.24


ND-8858
33.25
7.09
92.18
20.40


ND-8858
31.50
5.36
84.22
13.01


AL-DP-7395
18.01
2.33
25.01
4.17


ND-8730
18.63
2.22
35.55
6.30


ND-8742
18.04
2.92
29.24
6.48


ND-8753
19.03
3.21
50.35
10.66


ND-8840
24.81
3.87
81.78
17.12


ND-8865
27.65
3.29
72.55
12.44


ND-8890
22.03
1.60
105.32
26.89


AL-DP-7499
12.40
1.94
25.24
3.83


ND-8777
12.78
2.14
25.07
6.35


ND-8796
11.28
0.83
21.19
2.68


ND-8815
10.85
1.12
27.56
7.33


ND-8834
9.88
1.77
48.81
8.56


ND-8859
38.05
5.09
56.68
8.15


ND-8884
38.13
7.42
75.98
15.04


AL-DP-7365
15.72
2.57
23.60
5.58


ND-8724
14.88
2.37
27.95
11.09


ND-8736
71.51
11.99
81.07
19.08


ND-8748
71.98
14.80
82.12
16.76


ND-8841
18.39
3.01
66.82
19.67


ND-8866
79.40
15.36
80.86
15.81


ND-8891
73.79
17.04
86.53
21.21


AL-DP-7390
17.45
3.14
30.46
4.87


ND-8727
17.98
3.47
44.60
4.60


ND-8739
23.47
4.83
53.99
8.89


ND-8750
25.98
3.55
83.20
10.09


ND-8842
21.10
2.77
109.29
34.23


ND-8867
44.74
4.83
91.06
22.68


ND-8892
57.70
9.50
96.07
23.52


AL-DP-7382
16.90
3.54
30.39
3.91


ND-8726
17.17
3.84
38.93
6.26


ND-8738
19.51
2.77
41.20
3.80


ND-8749
17.03
3.66
34.11
8.30


ND-8843
26.36
4.99
83.57
8.12


ND-8868
26.78
3.25
88.44
7.96


ND-8893
22.70
2.05
86.71
12.41


AL-DP-7393
24.38
3.02
38.04
7.48


ND-8729
29.07
4.34
59.65
11.35


ND-8741
68.38
7.91
87.12
8.74


ND-8752
50.68
7.27
86.26
11.15


ND-8844
36.14
5.29
102.26
16.83


ND-8869
71.02
12.42
97.57
17.41


ND-8894
52.86
8.43
106.24
17.77


AL-DP-7366
18.69
2.05
44.08
7.35


ND-8778
18.46
2.08
41.29
5.42


ND-8797
15.49
2.21
36.71
5.29


ND-8816
13.61
1.76
33.13
6.21


ND-8835
22.00
3.84
76.17
11.70


ND-8860
17.81
4.03
68.48
8.32


ND-8885
15.76
2.33
70.03
10.95


AL-DP-7371
18.77
2.20
52.94
11.86


ND-8779
19.88
2.86
56.27
9.68


ND-8798
24.79
4.89
59.87
8.65


ND-8817
26.06
2.89
85.76
15.79


ND-8836
33.17
7.60
87.15
20.65


ND-8861
78.78
18.21
88.22
14.97


ND-8886
70.66
10.62
96.55
14.35


AL-DP-7372
19.23
3.22
46.80
10.62


ND-8780
19.94
0.97
62.29
12.87


ND-8799
42.73
4.67
81.61
10.68


ND-8818
88.99
6.53
104.90
8.74


ND-8837
25.18
3.70
99.41
12.93


ND-8862
79.80
15.30
92.36
10.28


ND-8887
78.27
16.96
92.55
14.48


AL-DP-7370
20.04
1.52
46.57
9.68


ND-8781
16.68
1.83
62.43
12.82


ND-8800
41.69
5.44
77.39
12.71


ND-8819
35.98
3.15
78.56
17.49


ND-8838
21.49
3.65
84.71
30.30


ND-8863
59.44
19.74
91.91
17.80


ND-8888
49.74
16.09
97.57
15.46









Design of siRNA Targeting HPV E1 Gene Expression
Table 5 sets forth dsRNA compositions of the invention.














TABLE 5





Target sequence of mRNA



antisense strand





from HPV E1 reference

Sense strand

(guide sequence)


sequence (sequence of
SEQ
(target sequence)
SEQ
having double TT
SEQ


total 19mer target
ID.
having double TT
ID.
overhang
ID.
duplex


site + AA at both ends)
NO.
overhang (5′-3′)
NO.
(5′-3′)
NO.
name






















AAAAAUCAACGUGUUGCGAUUAA
819
AAAUCAACGUGUUGCGAUUTT
945
AAUCGCAACACGUUGAUUUTT
1141
ND-9061



 


AAGAGCCUCCAAAAUUGCGUAAA
820
GAGCCUCCAAAAUUGCGUATT
946
UACGCAAUUUUGGAGGCUCTT
1142
ND-9062


 


AAUCAACGUGUUGCGAUUGGUAA
821
UCAACGUGUUGCGAUUGGUTT
947
ACCAAUCGCAACACGUUGATT
1143
ND-9063


 


AAUCCAAAAUUGCGUAGUACAAA
822
UCCAAAAUUGCGUAGUACATT
948
UGUACUACGCAAUUUUGGATT
1144
ND-9064


 


AAAAUCAACGUGUUGCGAUUGAA
823
AAUCAACGUGUUGCGAUUGTT
949
CAAUCGCAAGACGUUGAUUTT
1145
ND-9065


 


AACCUCCAAAAUUGCGUAGUAAA
824
CCUCCAAAAUUGCGUAGUATT
950
UACUACGCAAUUUUGGAGGTT
1146
ND-9066


 


AAAGAGCCUCCAAAAUUGCGUAA
825
AGAGCCUCCAAAAUUGCGUTT
951
ACGCAAUUUUGGAGGCUCUTT
1147
ND-9067


 


AACAACGUGUUGCGAUUGGUGAA
826
CAACGUGUUGCGAUUGGUGTT
952
CACCAAUCGCAACACGUUGTT
1148
ND-9068


 


AAAUAGAUGUGAUAGGGUAGAAA
827
AUAGAUGUGAUAGGGUAGATT
953
UCUACCCUAUCACAUCUAUTT
1149
ND-9069


 


AAGGGAAGAGGGUACGGGAUGAA
828
GGGAAGAGGGUACGGGAUGTT
954
CAUCCCGUACCCUCUUCCCTT
1150
ND-9070


 


AAAGAUUAAGUUUGCACGAGGAA
829
AGAUUAAGUUUGCACGAGGTT
955
CCUCGUGCAAACUUAAUCUTT
1151
ND-9071


 


AAGGUAUCAAGGUGUAGAGUUAA
830
GGUAUCAAGGUGUAGAGUUTT
956
AACUCUACACCUUGAUACCTT
1152
ND-9072


 


AAACUUAGUGAUAUUAGUGGAAA
831
ACUUAGUGAUAUUAGUGGATT
957
UCCACUAAUAUCACUAAGUTT
1153
ND-9073


 


AAGAGAUUAUUUGAAAGCGAAAA
832
GAGAUUAUUUGAAAGCGAATT
958
UUCGCUUUCAAAUAAUCUCTT
1154
ND-9074


 


AAAACACCAUGUAGUCAGUAUAA
833
AACACCAUGUAGUCAGUAUTT
959
AUACUGACUACAUGGUGUUTT
1155
ND-9075


 


AAAGCCUCCAAAAUUGCGUAGAA
834
AGCCUCCAAAAUUGCGUACTT
960
CUACGCAAUUUUGGAGGCUTT
1156
ND-9076


 


AAGCCUCCAAAAUUGCGUAGUAA
835
GCCUCCAAAAUUGCGUAGUTT
961
ACUACGCAAUUUUGGAGGCTT
1157
ND-9077


 


AAGUGUAUGGAGACACGCCAGAA
836
GUGUAUGGAGACACGCCAGTT
962
CUGGCGUGUCUCCAUACACTT
1158
ND-9078


 


AAGUACAAUGGGCCUACGAUAAA
837
GUACAAUGGGCCUACGAUATT
963
UAUCGUAGGCCCAUUGUACTT
1159
ND-9079


 


AAUACAAUGGGCCUACGAUAAAA
838
UACAAUGGGCCUACGAUAATT
964
UUAUCGUAGGCCCAUUGUATT
1160
ND-9080


 


AAUGACAUAGUAGACGAUAGUAA
839
UGACAUAGUAGACGAUAGUTT
965
ACUAUCGUCUACUAUGUCATT
1161
ND-9081


 


AAGACAUAGUAGACGAUAGUGAA
840
GACAUAGUAGACGAUAGUGTT
966
CACUAUCGUCUACUAUGUCTT
1162
ND-9082


 


AAACUCUUUGCCAACGUUUAAAA
841
ACUCUUUGCCAACGUUUAATT
967
UUAAACGUUGGCAAAGAGUTT
1163
ND-9083


 


AAAUAAUGACAUAGUAGACGAAA
842
AUAAUGACAUAGUAGACGATT
968
UCGUCUACUAUGUCAUUAUTT
1164
ND-9084


 


AAAAGUAUUUGGGUAGUCCACAA
843
AAGUAUUUGGGUAGUCCACTT
969
GUGGACUACCCAAAUACUUTT
1165
ND-9085


 


AAACGUGUUGCGAUUGGUGUAAA
844
ACGUGUUGCGAUUGGUGUATT
970
UACACCAAUCGCAACACGUTT
1166
ND-9086


 


AACGAAAGUAUUUGGGUAGUCAA
845
CGAAAGUAUUUGGGUAGUCTT
971
GACUACCCAAAUACUUUCGTT
1167
ND-9087


 


AACUCCAAAAUUGCGUAGUACAA
846
CUCCAAAAUUGCGUAGUACTT
972
GUACUACGCAAUUUUGGAGTT
1168
ND-9088


 


AAUGGUACAAUGGGCCUACGAAA
847
UGGUACAAUGGGCCUACGATT
973
UCGUAGGCCCAUUGUACCATT
1169
ND-9089


 


AAUAAUGACAUAGUAGACGAUAA
848
UAAUCACAUAGUAGACGAUTT
974
AUCGUCUACUAUGUCAUUATT
1170
ND-9090


 


AAACAUAGUAGACGAUAGUGAAA
849
ACAUAGUAGACGAUAGUGATT
975
UCACUAUCGUCUACUAUGUTT
1171
ND-9091


 


AAGUGUAGACAUUAUAAACGAAA
850
GUGUAGACAUUAUAAACGATT
976
UCGUUUAUAAUGUCUACACTT
1172
ND-9092


 


AAUAGACAUUAUAAACGAGCAAA
851
UAGACAUUAUAAACGAGCATT
977
UGCUCGUUUAUAAUGUCUATT
1173
ND-9093


 


AAUUGCGAUUGGUGUAUUGCUAA
852
UUGCGAUUGGUGUAUUGCUTT
978
AGCAAUACACCAAUCGCAATT
1174
ND-9094


 


AAUUGGCAGACACUAAUAGUAAA
853
UUGGCAGACACUAAUAGUATT
979
UACUAUUAGUGUCUGCCAATT
1175
ND-9095


 


AAUUCAGAAUUAGUAAGACCAAA
854
UUCAGAAUUAGUAAGACCATT
980
UGGUCUUACUAAUUCUGAATT
1176
ND-9096


 


AAGGAGAUUAUUUGAAAGCGAAA
855
GGAGAUUAUUUGAAAGCGATT
981
UCGCUUUCAAAUAAUCUCCTT
1177
ND-9097


 


AAACCAUGUAGUCAGUAUAGUAA
856
ACCAUGUAGUCAGUAUAGUTT
982
ACUAUACUGACUACAUGGUTT
1178
ND-9098


 


AAGAAGAGGGUACGGGAUGUAAA
857
GAAGAGGGUACGGGAUGUATT
983
UACAUCCCGUACCCUCUUCTT
1179
ND-9099


 


AAAUAAAUCAACGUGUUGCGAAA
858
AUAAAUCAACGUGUUGCGATT
984
UCGCAACACGUUGAUUUAUTT
1180
ND-9100


 


AACGUGUUGCGAUUGGUGUAUAA
859
CGUGUUGCGAUUGGUGUAUTT
985
AUACACCAAUCGCAACACGTT
1181
ND-9101


 


AAUGGGCCUACGAUAAUGACAAA
860
UGGGCCUACGAUAAUGACATT
986
UGUCAUUAUCGUAGGCCCATT
1182
ND-9102


 


AAUGCAUAUUACUAUAUGGUGAA
861
UGCAUAUUACUAUAUGGUGTT
987
CACCAUAUAGUAAUAUGCATT
1183
ND-9103


 


AACCAGAUUAAGUUUGCACGAAA
862
CCAGAUUAAGUUUGCACGATT
988
UCGUGCAAACUUAAUCUGGTT
1184
ND-9104


 


AAAGAUUAUUUGAAAGCGAAGAA
863
AGAUUAUUUGAAAGCGAAGTT
989
CUUCGCUUUCAAAUAAUCUTT
1185
ND-9105


 


AAGUUACAGGUAGAAGGGCGCAA
864
GUUACAGGUAGAAGGGCGCTT
990
GCGCCCUUCUACCUGUAACTT
1186
ND-9106


 


AAGCCAAAAUAGGUAUGUUAGAA
865
GCCAAAAUAGGUAUGUUAGTT
991
CUAACAUACCUAUUUUGGCTT
1187
ND-9107


 


AAGCAUAGACCAUUGGUACAAAA
866
GCAUAGACCAUUGGUACAATT
992
UUGUACCAAUGGUCUAUGCTT
1188
ND-9108


 


AAAAAUUGCGUAGUACAGCAGAA
867
AAAUUGCGUAGUACAGCAGTT
993
GUGCUGUACUACGCAAUUUTT
1189
ND-9109


 


AAAGUAUUUGGGUAGUCCACUAA
868
AGUAUUUGGGUAGUCCACUTT
994
AGUGGACUACCCAAAUACUTT
1190
ND-9110


 


AAAUGACAUAGUAGACGAUAGAA
869
AUGACAUAGUAGACGAUAGTT
995
CUAUCGUCUACUAUGUCAUTT
1191
ND-9111


 


AAGGUAGUCCACUUAGUGAUAAA
870
GGUAGUCCACUUAGUGAUATT
996
UAUCACUAAGUGGACUACCTT
1192
ND-9112


 


AAAGGAUAGACCAUUGGUACAAA
871
AGCAUAGACCAUUGGUACATT
997
UGUACCAAUGGUCUAUGCUTT
1193
ND-9113


 


AAUUUCAGAAUUAGUAAGACCAA
872
UUUCAGAAUUAGUAAGACCTT
998
GGUCUUACUAAUUCUGAAATT
1194
ND-9114


 


AAUGCGAUUGGUGUAUUGCUGAA
873
UGCGAUUGGUGUAUUGCUGTT
999
CAGCAAUACACCAAUCGCATT
1195
ND-9115


 


AACCAAAAUUGCGUAGUACAGAA
874
CCAAAAUUGCGUAGUACAGTT
1000
CUGUACUACGCAAUUUUGGTT
1196
ND-9116


 


AAGACAGCACAUGCGUUGUUUAA
875
GACAGCACAUGCGUUGUUUTT
1001
AAACAACGCAUGUGCUGUCTT
1197
ND-9117


 


AAUGUUACAGGUAGAAGGGCGAA
876
UGUUACAGGUAGAAGGGCGTT
1002
CGCCCUUCUACCUGUAACATT
1198
ND-9118


 


AAAGACAAUAAUAUUAGUCCUAA
877
AGACAAUAAUAUUAGUCCUTT
1003
AGGACUAAUAUUAUUGUCUTT
1199
ND-9119


 


AAAUGAUUAUUUAACACAGGCAA
878
AUGAUUAUUUAACACAGGCTT
1004
GCCUGUGUUAAAUAAUCAUTT
1200
ND-9120


 


AAAGAUGUGAUAGGGUAGAUGAA
879
AGAUGUGAUAGGGUAGAUGTT
1005
CAUCUACCCUAUCACAUCUTT
1201
ND-9121


 


AACUGCAAGGGUCUGUAAUAUAA
880
CUGCAAGGGUCUGUAAUAUTT
1006
AUAUUACAGACCCUUGCAGTT
1202
ND-9122


 


AAUCAGAUGACGAGAACGAAAAA
881
UCAGAUGACGAGAACGAAATT
1007
UUUCGUUCUCGUCAUCUGATT
1203
ND-9123


 


AACACCAUGUAGUCAGUAUAGAA
882
CACCAUGUAGUCAGUAUAGTT
1008
CUAUACUGACUACAUGGUGTT
1204
ND-9124


 


AAAGACAGCACAUGCGUUGUUAA
883
AGACAGCACAUGCGUUGUUTT
1009
AACAACGCAUGUGCUGUCUTT
1205
ND-9125


 


AAAGAGGGUACGGGAUGUAAUAA
884
AGAGGGUACGGGAUGUAAUTT
1010
AUUACAUCCCGUACCCUCUTT
1206
ND-9126


 


AAAAAAGUAAUAAAUCAACGUAA
885
AAAAGUAAUAAAUCAACGUTT
1011
ACGUUGAUUUAUUACUUUUTT
1207
ND-9127


 


AAAAAGCAUAGACCAUUGGUAAA
886
AAAGCAUAGACCAUUGGUATT
1012
UACCAAUGGUCUAUGCUUUTT
1208
ND-9128


 


AAUUGUACAUUUGAAUUAUCAAA
887
UUGUACAUUUGAAUUAUCATT
1013
UGAUAAUUCAAAUCUACAATT
1209
ND-9129


 


AAGUAAAGCAUAGACCAUUGGAA
888
GUAAAGCAUAGACCAUUGGTT
1014
CCAAUGGUCUAUGCUUUACTT
1210
ND-9130


 




AAAucAAcGuGuuGcGAuuTsT
1015
AAUCGcAAcACGUUGAUUUTsT
1211
ND-9131


 




GAGccuccAAAAuuGcGuATsT
1016
uACGcAAUUUUGGAGGCUCTsT
1212
ND-9132


 




ucAAcGuGuuGcGAuuGGuTsT
1017
ACcAAUCGcAAcACGUUGATsT
1213
ND-9133


 




uccAAAAuuGcGuAGuAcATsT
1018
UGuACuACGcAAUUUUGGATsT
1214
ND-9134


 




AAucAAcGuGuuGcGAuuGTsT
1019
cAAUCGcAAcACGUUGAUUTsT
1215
ND-9135


 




ccuccAAAAuuGcGuAGuATsT
1020
uACuACGcAAUUUUGGAGGTsT
1216
ND-9136


 




AGAGccuccAAAAuuGcGuTsT
1021
ACGcAAUUUUGGAGGCUCUTST
1217
ND-9137


 




cAAcGuGuuGcGAuuGGuGTsT
1022
cACcAAUCGcAAcACGUUGTsT
1218
ND-9138


 




AuAGAuGuGAuAGGGuAGATsT
1023
UCuACCCuAUcAcAUCuAUTsT
1219
ND-9139


 




GGGAAGAGGGuAcGGGAuGTsT
1024
cAUCCCGuACCCUCUUCCCTsT
1220
ND-9140


 




AGAuuAAGuuuGcAcGAGGTsT
1025
CCUCGUGcAAACUuAAUCUTsT
1221
ND-9141


 




GGuAucAAGGuGuAGAGuuTsT
1026
AACUCuAcACCUUGAuACCTsT
1222
ND-9142


 




AcuuAGuGAuAuuAGuGGATsT
1027
UCcACuAAuAUcACuAAGUTsT
1223
ND-9143


 




GAGAuuAuuuGAAAGcGAATsT
1028
UUCGCUUUcAAAuAAUCUCTsT
1224
ND-9144


 




AAcAccAuGuAGucAGuAuTsT
1029
AuACUGACuACAUGGUGUUTsT
1225
ND-9145


 




AGccuccAAAAuuGcGuAGTsT
1030
CuACGcAAUUUUGGAGGCUTsT
1226
ND-9146


 




GccuccAAAAuuGcGuAGuTsT
1031
ACuACGcAAUUUUGGAGGCTsT
1227
ND-9147


 




GuGuAuGGAGAcAcGccAGTsT
1032
CUGGCGUGUCUCcAuACACTST
1228
ND-9148


 




GuAcAAuGGGccuAcGAuATsT
1033
uAUCGuAGGCCcAUUGuACTsT
1229
ND-9149


 




uAcAAuGGGccuAcGAuAATsT
1034
UuAUCGuAGGCCcAUUGuATsT
1230
ND-9150


 




uGAcAuAGuAGAcGAuAGuTsT
1035
ACuAUCGUCuACuAUGUcATsT
1231
ND-9151


 




GAcAuAGuAGAcGAuAGuGTsT
1036
cACuAUCGUCuACuAUGUCTsT
1232
ND-9152


 




AcucuuuGccAAcGuuuAATsT
1037
UuAAACGUUGGcAAAGAGUTsT
1233
ND-9153


 




AuAAuGAcAuAGuAGAcGATsT
1038
UCGUCuACuAUGUcAUuAUTsT
1234
ND-9154


 




AAGuAuuuGGGuAGuccAcTsT
1039
GUGGACuACCCAAAUACUUTST
1235
ND-9155


 




AcGuGuuGcGAuuGGuGuATsT
1040
uAcACcAAUCGcAAcACGUTsT
1236
ND-9156


 




cGAAAGuAuuuGGGuAGucTsT
1041
GACuACCcAAAuACUUUCGTsT
1237
ND-9157


 




cuccAAAAuuGcGuAGuAcTsT
1042
GuACuACGcAAUUUUGGAGTsT
1238
ND-9158


 




uGGuAcAAuGGGccuAcGATsT
1043
UCGuAGGCCcAUUGuACcATsT
1239
ND-9159


 




uAAuGAcAuACuAGAcGAuTsT
1044
AUCGUCuACuAUGUCAUuATsT
1240
ND-9160


 




AcAuAGuAGAcGAuAGuGATsT
1045
UcACuAUCGUCuACuAUGUTsT
1241
ND-9161


 




GuGuAGAcAuuAuAAAcGATsT
1046
UCGUUuAuAAUGUCuAcACTsT
1242
ND-9162


 




uAGAcAuuAuAAAcGAGcATsT
1047
UGCUCGUUuAuAAUGUCuATsT
1243
ND-9163


 




uuGcGAuuGGuGuAuuGcuTsT
1048
AGcAAuAcACcAAUCGcAATsT
1244
ND-9164


 




uuGGcAGAcAcuAAuAGuATsT
1049
uACuAUuAGUGUCUGCcAATsT
1245
ND-9165


 




uucAGAAuuAGuAAGAccATsT
1050
UGGUCUuACuAAUUCUGAATsT
1246
ND-9166


 




GGAGAuuAuuuGAAAGcGATsT
1051
UCGCUUUcAAAuAAUCUCCTsT
1247
ND-9167


 




AccAuGuAGucAGuAuAGuTST
1052
ACuAuACUGACuAcAUGGUTsT
1248
ND-9168


 




GAAGAGGGuAcGGGAuGuATsT
1053
uAcAUCCCGuACCCUCUUCTsT
1249
ND-9169


 




AuAAAucAAcGuGuuGcGATsT
1054
UCGcAAcACGUUGAUUuAUTsT
1250
ND-9170


 




cGuGuuGcGAuuGGuGuAuTsT
1055
AuAcACcAAUCGcAAcACGTsT
1251
ND-9171


 




uGGGccuAcGAuAAuGAcATsT
1056
UGUcAUuAUCGuAGGCCcATsT
1252
ND-9172


 




uGcAuAuuAcuAuAuGGuGTsT
1057
cACcAuAuAGuAAuAUGcATsT
1253
ND-9173


 




ccAGAuuAAGuuuGcAcGATsT
1058
UCGUGcAAACUuAAUCUGGTsT
1254
ND-9174


 




AGAuuAuuuGAAAGcGAAGTsT
1059
CUUCGCUUUcAAAuAAUCUTsT
1255
ND-9175


 




GuuAcAGGuAGAAGGGcGcTsT
1060
GCGCCCUUCuACCUGuAACTsT
1256
ND-9176


 




GccAAAAuAGGuAuGuuAGTsT
1061
CuAAcAuACCuAUUUUGGCTsT
1257
ND-9177


 




GcAuAGAccAuuGGuAcAATsT
1062
UUGuACcAAUGGUCuAUGCTsT
1258
ND-9178


 




AAAuuGcGuAGuAcAGcAGTsT
1063
CUGCUGuACuACGcAAUUUTsT
1259
ND-9179


 




AGuAuuuGGGuAGuccAcuTsT
1064
AGUGGACuACCcAAAuACUTsT
1260
ND-9180


 




AuGAcAuAGuAGAcGAuAGTsT
1065
CuAUCGUCuACuAUGUcAUTsT
1261
ND-9181


 




GGuAGuccAcuuAGuGAuATsT
1066
uAUcACuAAGUGGACuACCTsT
1262
ND-9182


 




AGcAuAGAccAuuGGuAcATsT
1067
UGuACcAAUGGUCuAUGCUTsT
1263
ND-9183


 




uuucAGAAuuAGuAAGAccTsT
1068
GGUCUuACuAAUUCUGAAATsT
1264
ND-9184


 




uCcGAuuGGuGuAuuGcuGTsT
1069
cAGcAAuAcACcAAUCGcATsT
1265
ND-9185


 




ccAAAAuuGcGuAGuAcAGTsT
1070
CUGuACuACGcAAUUUUGGTsT
1266
ND-9186


 




GAcAGcAcAuGcGuuGuuuTsT
1071
AAAcAACGcAUGUGCUGUCTsT
1267
ND-9187


 




uGuuAcAGGuAGAAGGGcGTsT
1072
CGCCCUUCuACCUGuAAcATsT
1268
ND-9188


 




AGAcAAuAAuAuuAGuccuTsT
1073
AGGACuAAuAUuAUUGUCUTsT
1269
ND-9189


 




AuGAuuAuuuAAcAcAGGcTsT
1074
GCCUGUGUuAAAuAAUcAUTsT
1270
ND-9190


 




AGAuGuGAuAGGGUAGAuGTsT
1075
cAUCuACCCuAUcAcAUCUTsT
1271
ND-9191


 




cuGcAAGGGucuGuAAuAuTsT
1076
AuAUuAcAGACCCUUGcAGTsT
1272
ND-9192


 




ucAGAuGAcGAGAACGAAATsT
1077
UUUCGUUCUCGUcAUCUGATsT
1273
ND-9193


 




cAccAuGuAGucAGuAuAGTsT
1078
CuAuACUGACuAcAUGGUGTsT
1274
ND-9194


 




AGAcAGcAcAuGcGuuGuuTsT
1079
AAcAACGcAUGUGCUGUCUTsT
1275
ND-9195


 




AGAGGGuAcGGGAuGuAAuTsT
1080
AUuAcAUCCCGuACCCUCUTsT
1276
ND-9196


 




AAAAGuAAuAAAucAAcGuTsT
1081
ACGUUGAUUuAUuACUUUUTsT
1277
ND-9197


 




AAAGcAuAGAccAuuGGuATsT
1082
uACcAAUGGUCuAUGCUUUTsT
1278
ND-9198


 




uuGuAcAuuuGAAuuAucATsT
1083
UGAuAAUUcAAAUGuAcAATsT
1279
ND-9199


 




GuAAAGcAuAGAccAuuGGTsT
1084
CcAAUGGUCuAUGCUUuACTsT
1280
ND-9200


 


AAAUAUCAAAUAUUAGUGAAGAA
889
AUAUCAAAUAUUAGUGAAGTT
1085
CUUCACUAAUAUUUGAUAUTT
1281
AL-DP-8042


 


AAUAUCAAAUAUUAGUGAAGUAA
890
UAUCAAAUAUUAGUGAAGUTT
1086
ACUUCACUAAUAUUUGAUATT
1282
AL-DP-8043


 


AAGGGUAUGGCAAUACUGAAGAA
891
GGGUAUGGCAAUACUGAAGTT
1087
CUUCAGUAUUGCCAUACCCTT
1283
AL-DP-8044


 


AACAACGUUUAAAUGUGUGUCAA
892
CAACGUUUAAAUGUGUGUCTT
1088
GACACACAUUUAAACGUUGTT
1284
AL-DP-8045


 


AAAACGUUUAAAUGUGUGUCAAA
893
AACGUUUAAAUGUGUGUCATT
1089
UGACACACAUUUAAACGUUTT
1285
AL-DP-8046


 


AAGAAAACGAUGGAGACUCUUAA
894
GAAAACGAUGGAGACUCUUTT
1090
AAGAGUCUCCAUCGUUUUCTT
1286
AL-DP-8047


 


AAGCGGGUAUGGCAAUACUGAAA
895
GCGGGUAUGGCAAUACUGATT
1091
UCAGUAUUGCCAUACCCGCTT
1287
AL-DP-8048


 


AACGGGUAUGGCAAUACUGAAAA
896
CGGGUAUGGCAAUACUGAATT
1092
UUCAGUAUUGCCAUACCCGTT
1288
AL-DP-8049


 


AAUUAUAAACGAGCAGAAAAAAA
897
UUAUAAACGAGCAGAAAAATT
1093
UUUUOCUGCUCGUUUAUAATT
1289
AL-DP-8050


 


AAACAAUGUGUAGACAUUAUAAA
898
ACAAUGUGUAGACAUUAUATT
1094
UAUAAUGUCUACACAUUGUTT
1290
AL-DP-8051


 


AAAUUAUAAACGAGCAGAAAAAA
899
AUUAUAAACGAGCAGAAAATT
1095
UUUUCUGCUCGUUUAUAAUTT
1291
AL-DP-8052


 


AAUGUGUAGACAUUAUAAACGAA
900
UGUGUAGACAUUAUAAACGTT
1096
CGUUUAUAAUGUCUACACATT
1292
AL-DP-8053


 


AAUGUGUGUCAGGACAAAAUAAA
901
UGUGUGUCAGGACAAAAUATT
1097
UAUUUUGUCCUGACACACATT
1293
AL-DP-8054


 


AAACAUUAUAAACGAGCAGAAAA
902
ACAUUAUAAACGAGCAGAATT
1098
UUCUGCUCGUUUAUAAUGUTT
1294
AL-DP-8055


 


AAAGACAGCGGGUAUGGCAAUAA
903
AGACAGCGGGUAUGGCAAUTT
1099
AUUGCCAUACCCGCUGUCUTT
1295
AL-DP-8056


 


AAGACAGCGGGUAUGGCAAUAAA
904
GACAGCGGGUAUGGCAAUATT
1100
UAUUGCCAUACCCGCUGUCTT
1296
AL-DP-8057


 


AAACAGCGGGUAUGGCAAUACAA
905
ACAGCGGGUAUGGCAAUACTT
1101
GUAUUGCCAUACCCGCUGUTT
1297
AL-DP-8058


 


AAAGCGGGUAUGGCAAUACUGAA
906
AGCGGGUAUGGCAAUACUGTT
1102
CAGUAUUGCCAUACCCGCUTT
1298
AL-DP-8059


 


AAAACAAUGUGUAGACAUUAUAA
907
AACAAUGUGUAGACAUUAUTT
1103
AUAAUGUCUACACAUUGUUTT
1299
AL-DP-8060


 


AACAUUAUAAACGAGCAGAAAAA
908
CAUUAUAAACGAGCAGAAATT
1104
UUUCUGCUCGUUUAUAAUGTT
1300
AL-DP-8061


 


AAACGUUUAAAUGUGUGUCAGAA
909
ACGUUUAAAUGUGUGUCAGTT
1105
CUGACACACAUUUAAACGUTT
1301
AL-DP-8062


 


AAAAAUGUGUGUCAGGACAAAAA
910
AAAUGUGUGUCAGGACAAATT
1106
UUUGUCCUGACACACAUUUTT
1302
AL-DP-8063


 


AAGGUUCUAAAACGAAAGUAUAA
911
GGUUCUAAAACGAAAGUAUTT
1107
AUACUUUCGUUUUAGAACCTT
1303
AL-DP-8064


 


AAAUGUGUAGACAUUAUAAACAA
912
AUGUGUAGACAUUAUAAACTT
1108
GUUUAUAAUGUCUACACAUTT
1304
AL-DP-8065


 


AAAGUAAGACCAUUUAAAAGUAA
913
AGUAAGACCAUUUAAAAGUTT
1109
ACUUUUAAAUGGUCUUACUTT
1305
AL-DP-8066


 


AAUAGUAAGACCAUUUAAAAGAA
914
UAGUAAGACCAUUUAAAAGTT
1110
CUUUUAAAUGGUCUUACUATT
1306
AL-DP-8067


 


AAGAAUUAGUAAGACCAUUUAAA
915
GAAUUAGUAACACCAUUUATT
1111
UAAAUGGUCUUACUAAUUCTT
1307
AL-DP-8068


 


AAAAUUAGUAAGACCAUUUAAAA
916
AAUUAGUAAGACCAUUUAATT
1112
UUAAAUGGUCUUACUAAUUTT
1308
AL-DP-8069


 


AAAUUAGUAAGACCAUUUAAAAA
917
AUUAGUAAGACCAUUUAAATT
1113
UUUAAAUGGUCUUACUAAUTT
1309
AL-DP-8070


 


AAUUAGUAAGACCAUUUAAAAAA
918
UUAGUAAGACCAUUUAAAATT
1114
UUUUAAAUGGUCUUACUAATT
1310
AL-DP-8071


 


AAAAUACUGAAGUGGAAACUCAA
919
AAUACUGAAGUGGAAACUCTT
1115
GAGUUUCCACUUCAGUAUUTT
1311
AL-DP-8072


 


AAAUACUGAAGUGGAAACUCAAA
920
AUACUGAAGUGGAAACUCATT
1116
UGAGUUUCCACUUCAGUAUTT
1312
AL-DP-8073


 


AACAAUACUGAAGUGGAAACUAA
921
CAAUACUGAAGUGGAAACUTT
1117
AGUUUCCACUUCAGUAUUGTT
1313
AL-DP-8074


 


AAUACUGAAGUGGAAACUCAGAA
922
UACUGAAGUGGAAACUCAGTT
1118
CUGAGUUUCCACUUCAGUATT
1314
AL-DP-8075


 


AAACUGAAGUGGAAACUCAGCAA
923
ACUGAAGUGGAAACUCAGCTT
1119
GCUGAGUUUCCACUUCAGUTT
1315
AL-DP-8076


 


AACUGAAGUGGAAACUCAGCAAA
924
CUGAAGUGGAAACUCAGCATT
1120
UGCUGAGUUUCCACUUCAGTT
1316
AL-DP-8077


 


AAUGAAGUGGAAACUCAGCAGAA
925
UGAAGUGGAAACUCAGCAGTT
1121
CUGCUGAGUUUCCACUUCATT
1317
AL-DP-8078


 


AAGAAGUGGAAACUCAGCAGAAA
926
GAAGUGGAAACUCAGCAGATT
1122
UCUGCUGAGUUUCCACUUCTT
1318
AL-DP-8079


 


AAAAGUGGAAACUCAGCAGAUAA
927
AAGUGGAAACUCAGCAGAUTT
1123
AUCUGCUGAGUUUCCACUUTT
1319
AL-DP-8080


 


AAAGUGGAAACUCAGCAGAUGAA
928
AGUGGAAACUCAGCAGAUGTT
1124
CAUCUGCUGAGUUUCCACUTT
1320
AL-DP-8081


 


AAAUGGCAAUACUGAAGUGGAAA
929
AUGGCAAUACUGAAGUGGATT
1125
UCCACUUCAGUAUUGCCAUTT
1321
AL-DP-8082


 


AAAAAUCCUUUUUCUCAAGGAAA
930
AAAUCCUUUUUCUCAAGGATT
1126
UCCUUGAGAAAAAGGAUUUTT
1322
AL-DP-8083


 


AAUCCUUUUUCUCAAGGACGUAA
931
UCCUUUUUCUCAAGGACGUTT
1127
ACGUCCUUGAGAAAAAGGATT
1323
AL-DP-8084


 


AAAUCCUUUUUCUCAAGGACGAA
932
AUCCUUUUUCUCAAGGACGTT
1128
CGUCCUUGAGAAAAAGGAUTT
1324
AL-DP-8085


 


AAGGCAAUACUGAAGUGGAAAAA
933
GGCAAUACUGAAGUGGAAATT
1129
UUUCCACUUCAGUAUUGCCTT
1325
AL-DP-8086


 


AACUUUUUCUCAAGGACGUGGAA
934
CUUUUUCUCAAGGACGUGGTT
1130
CCACGUCCUUGAGAAAAAGTT
1326
AL-DP-8087


 


AACCUUUUUCUCAAGGACGUGAA
935
CCUUUUUCUCAAGGACGUGTT
1131
CACGUCCUUGAGAAAAAGGTT
1327
AL-DP-8088


 


AAUUUUUCUCAAGGACGUGGUAA
936
UUUUUCUCAAGGACGUGGUTT
1132
ACCACGUCCUUGAGAAAAATT
1328
AL-DP-8089


 


AAUGGAAAUCCUUUUUCUCAAAA
937
UGGAAAUCCUUUUUCUCAATT
1133
UUGAGAAAAAGGAUUUCCATT
1329
AL-DP-8090


 


AAGGAAAUCCUUUUUCUCAAGAA
938
GGAAAUCCUUUUUCUCAAGTT
1134
CUUGAGAAAAAGGAUUUCCTT
1330
AL-DP-8091


 


AAGAAAUCCUUUUUCUCAAGGAA
939
GAAAUCCUUUUUCUCAAGGTT
1135
CCUUGAGAAAAAGGAUUUCTT
1331
AL-DP-8092


 


AAAAUCCUUUUUCUCAAGGACAA
940
AAUCCUUUUUCUCAAGGACTT
1136
GUCCUUGAGAAAAAGGAUUTT
1332
AL-DP-8093


 


AAUAUGGCAAUACUCAAGUGGAA
941
UAUGCCAAUACUGAAGUGGTT
1137
CCACUUCAGUAUUGCCAUATT
1333
AL-DP-8094


 


AAUGGCAAUACUGAAGUGGAAAA
942
UGGCAAUACUGAAGUGGAATT
1138
UUCCACUUCAGUAUUGCCATT
1334
AL-DP-8095


 


AAGCAAUACUGAAGUGGAAACAA
943
GCAAUACUGAAGUGGAAACTT
1139
GUUUCCACUUCAGUAUUGCTT
1335
AL-DP-8096


 


AAAAUGUGUAGACAUUAUAAAAA
944
AAUGUGUAGACAUUAUAAATT
1140
UUUAUAAUGUCUACACAUUTT
1336
AL-DP-8097





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


Lower case letters: ribonucloetide bearing 2′-O-methyl substituent on ribose moiety


s: Indicates position of phosphorothioate internucleoside linkage


chol: cholesterol moiety conjugated to 3′ ribonucleotide.


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






Testing of siRNA Targeting HPV E1 Gene Expression
Unmodified and chemically modified dsRNA were tested to identify their relative abilities to reduce the expression level of mRNA encoding HPV E1 gene in a cell.
The assay conditions employed were as follows: C33A cells were obtained from ATCC. Sequences encoding HPV16 E6 and E1 were cloned into the pNAS-055 vector (Husken et al., Nucleic Acids Research, 31:e102, 2003), for expression as YFP fusion transcripts. The resulting plasmids were transfected into C33A cells, and stable lines expressing these fusion transcripts were derived by Zeocin selection, as per the manufacturer's protocol (Invitrogen). For transfection with siRNA against HPV16 E6 or HPV16 E1, respective cells were seeded at a density of 2.0×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30 nM, 3 nM or 300 pm as indicated) was carried out in a single dose with lipofectamine 2000® (Invitrogen) as described by the manufacturer.
24 hours after transfection cells were lysed and fusion YFP mRNA expression levels were quantified with the Quantigene Explore Kit (Panomics, Inc. (Fremont, Calif.) (formerly Genospectra, Inc.)) using a probe directed against YFP, according to the standard protocol. Fusion-YFP mRNA levels were normalized to GAP-DH mRNA. For each siRNA four individual datapoints were collected. siRNA duplexes unrelated to the HPV16 E1 or E6 genes were used as control. The activity of a given siRNA duplex was expressed as percent fusion-YFP mRNA concentration in treated cells relative to concentration of the same transcript in cells treated with the control siRNA duplex.
Table 6 shows the results of testing the E1 dsRNA of the invention.
















% mRNA





remaining




after



Duplex
treatement



dsRNA
at 300 pM
S.D.




















ND-9061
41.45
10.69



ND-9062
30.67
10.43



ND-9063
61.87
22.99



ND-9064
40.79
22.73



ND-9065
68.58
28.46



ND-9066
23.51
7.60



ND-9067
37.13
13.60



ND-9068
34.50
17.21



ND-9069
40.61
12.42



ND-9070
32.61
8.73



ND-9071
30.68
11.65



ND-9072
24.38
7.47



ND-9073
76.28
15.06



ND-9074
29.11
10.42



ND-9075
27.20
11.56



ND-9076
42.06
17.88



ND-9077
51.19
9.09



ND-9078
43.42
16.63



ND-9079
25.79
4.85



ND-9080
29.33
5.67



ND-9081
36.66
4.51



ND-9082
48.67
10.47



ND-9083
39.51
12.70



ND-9084
44.28
7.54



ND-9085
55.73
9.77



ND-9086
28.90
7.93



ND-9087
28.88
5.47



ND-9088
45.35
11.67



ND-9089
49.13
12.46



ND-9090
41.76
5.88



ND-9091
31.35
9.16



ND-9092
23.79
8.74



ND-9093
47.62
9.89



ND-9094
91.33
29.84



ND-9095
43.33
8.69



ND-9096
63.53
11.44



ND-9097
30.51
4.48



ND-9098
40.76
10.57



ND-9099
37.61
9.94



ND-9100
106.18
30.69



ND-9101
37.75
16.37



ND-9102
41.98
14.66



ND-9103
98.17
14.30



ND-9104
29.61
11.44



ND-9105
29.71
6.48



ND-9106
51.42
14.12



ND-9107
78.38
28.72



ND-9108
34.69
4.19



ND-9109
97.63
14.18



ND-9110
47.58
7.48



ND-9111
65.14
15.02



ND-9112
30.24
7.33



ND-9113
31.69
10.80



ND-9114
108.54
7.17



ND-9115
87.16
14.74



ND-9116
56.35
14.69



ND-9117
33.79
8.42



ND-9118
65.12
19.60



ND-9119
33.37
12.37



ND-9120
70.98
18.74



ND-9121
39.37
10.06



ND-9122
33.24
14.79



ND-9123
20.37
7.53



ND-9124
30.47
5.18



ND-9125
26.22
5.56



ND-9126
29.86
5.15



ND-9127
84.95
22.37



ND-9128
35.14
6.10



ND-9129
49.41
15.75



ND-9130
51.54
12.31



ND-9131
45.51
7.96



ND-9132
81.48
16.52



ND-9133
46.79
13.27



ND-9134
63.22
32.12



ND-9135
118.82
19.88



ND-9136
47.83
12.16



ND-9137
65.11
15.44



ND-9138
92.31
36.27



ND-9139
42.01
10.70



ND-9140
40.54
7.24



ND-9141
101.31
24.39



ND-9142
33.83
7.06



ND-9143
86.43
16.50



ND-9144
33.94
11.74



ND-9145
41.93
12.85



ND-9146
118.24
29.81



ND-9147
69.90
30.13



ND-9148
40.74
6.28



ND-9149
65.26
10.10



ND-9150
36.62
4.85



ND-9151
27.83
4.48



ND-9152
88.99
9.86



ND-9153
66.45
33.75



ND-9154
45.42
8.86



ND-9155
63.55
8.36



ND-9156
53.00
7.71



ND-9157
32.74
7.39



ND-9158
102.06
26.87



ND-9159
59.47
10.16



ND-9160
31.23
7.52



ND-9161
84.78
36.89



ND-9162
24.83
5.17



ND-9163
26.64
5.90



ND-9164
77.97
10.06



ND-9165
59.95
25.75



ND-9166
69.74
8.15



ND-9167
23.04
5.43



ND-9168
46.16
12.02



ND-9169
62.24
11.73



ND-9170
92.69
14.72



ND-9171
46.55
6.56



ND-9172
49.39
16.23



ND-9173
98.36
37.53



ND-9174
44.90
13.73



ND-9175
69.98
18.22



ND-9176
60.73
13.02



ND-9177
70.93
10.18



ND-9178
62.53
7.70



ND-9179
76.68
31.77



ND-9180
66.35
10.48



ND-9181
78.42
12.70



ND-9182
72.09
28.88



ND-9183
58.97
28.59



ND-9184
97.06
8.62



ND-9185
85.29
16.92



ND-9186
77.52
18.17



ND-9187
60.16
36.16



ND-9188
58.61
39.92



ND-9189
69.35
30.11



ND-9190
71.87
36.13



ND-9191
81.64
18.99



ND-9192
52.76
14.33



ND-9193
25.18
8.23



ND-9194
50.69
12.78



ND-9195
40.01
10.21



ND-9196
47.41
15.85



ND-9197
94.68
24.60



ND-9198
103.12
27.52



ND-9199
50.82
15.18



ND-9200
97.72
24.20



AL-DP-8042
117.14
34.54



AL-DP-8043
131.44
38.69



AL-DP-8044
28.60
11.52



AL-DP-8045
120.81
36.35



AL-DP-8046
93.19
17.57



AL-DP-8047
66.27
5.06



AL-DP-8048
33.70
8.18



AL-DP-8049
34.31
7.16



AL-DP-8050
60.60
19.36



AL-DP-8051
66.49
12.36



AL-DP-8052
45.46
12.49



AL-DP-8053
121.92
29.06



AL-DP-8054
45.00
4.56



AL-DP-8055
51.64
9.55



AL-DP-8056
35.51
4.67



AL-DP-8057
45.89
8.82



AL-DP-8058
38.47
4.44



AL-DP-8059
34.97
7.85



AL-DP-8060
66.44
14.39



AL-DP-8061
52.17
12.80



AL-DP-8062
100.52
25.88



AL-DP-8063
43.83
8.22



AL-DP-8064
26.25
5.84



AL-DP-8065
107.74
32.53



AL-DP-8066
94.13
13.45



AL-DP-8067
107.09
17.49



AL-DP-8068
48.99
10.40



AL-DP-8069
68.14
19.39



AL-DP-8070
60.42
11.52



AL-DP-8071
71.76
13.75



AL-DP-8072
62.25
6.16



AL-DP-8073
31.33
7.21



AL-DP-8074
47.97
11.55



AL-DP-8075
51.35
14.67



AL-DP-8076
50.40
17.25



AL-DP-8077
38.99
8.15



AL-DP-8078
50.93
11.54



AL-DP-8079
32.27
10.82



AL-DP-8080
33.91
10.48



AL-DP-8081
31.45
6.72



AL-DP-8082
26.41
7.99



AL-DP-8083
86.75
6.66



AL-DP-8084
112.73
25.79



AL-DP-8085
112.33
22.53



AL-DP-8086
39.84
12.22



AL-DP-8087
104.24
29.47



AL-DP-8088
59.29
13.99



AL-DP-8089
114.08
24.06



AL-DP-8090
35.69
6.75



AL-DP-8091
47.28
12.14



AL-DP-8092
92.85
19.28



AL-DP-8093
102.59
15.83



AL-DP-8094
87.51
18.86



AL-DP-8095
27.99
8.27



AL-DP-8096
31.74
7.52



AL-DP-8097
40.29
9.18










Design of dsRNA Targeting HPV E6 Gene Expression

Table 7 sets forth dsRNA compositions of the invention.














TABLE 7





Target sequence of mRNA



antisense strand





from HPV E6 reference

Sense strand

(guide sequence)


sequence (sequence of
SEQ
(target sequence)
SEQ
having double TT
SEQ


total 19mer target
ID.
having double TT
ID.
overhang
ID.
duplex


site + AA at both ends)
NO.
overhang (5′-3′)
NO.
(5′-3′)
NO.
name







AAUCGGUGGACCGGUCGAUGUAA
1336
UCGGUGGACCGGUCGAUGUTT
1424
ACAUCGACCGGUCCACCGATT
1586
ND-8899



 


AAGGUCGGUGGACCGGUCGAUAA
1337
GGUCGGUGGACCGGUCGAUTT
1425
AUCGACCGGUCCACCGACCTT
1587
ND-8900


 


AACGGUGGACCGGUCGAUGUAAA
1338
CGGUGGACCGGUCGAUGUATT
1426
UACAUCGACCGGUCCACCGTT
1588
ND-8901


 


AAGUCGGUGGACCGGUCGAUGAA
1339
GUCGGUGGACCGGUCGAUGTT
1427
CAUCGACCGGUCCACCGACTT
1589
ND-8902


 


AAAUCAUCAAGAACACGUAGAAA
1340
AUCAUCAAGAACACGUAGATT
1428
UCUACGUGUUCUUGAUGAUTT
1590
ND-8903


 


AACAACAGUUACUGCGACGUGAA
1341
CAACAGUUACUGCGACGUGTT
1429
CACGUCGCAGUAACUGUUGTT
1591
ND-8904


 


AACAAUACAACAAACCGUUGUAA
1342
CAAUACAACAAACCGUUGUTT
1430
ACAACGGUUUGUUGUAUUGTT
1592
ND-8905


 


AAGCUGCAAACAACUAUACAUAA
1343
GCUGCAAACAACUAUACAUTT
1431
AUGUAUAGUUGUUUGCAGCTT
1593
ND-8906


 


AAGGUGGACCGGUCGAUGUAUAA
1344
GGUGGACCGGUCGAUGUAUTT
1432
AUACAUCGACCGGUCCACCTT
1594
ND-8907


 


AAAAAUUAGUGAGUAUAGACAAA
1345
AAAUUAGUGAGUAUAGACATT
1433
UGUCUAUACUCACUAAUUUTT
1595
ND-8908


 


AAUCAUCAAGAACACGUAGAGAA
1346
UCAUCAAGAACACGUAGAGTT
1434
CUCUACGUGUUCUUGAUGATT
1596
ND-8909


 


AAAUACAACAAACCGUUGUGUAA
1347
AUACAACAAACCGUUGUGUTT
1435
ACACAACGGUUUGUUGUAUTT
1597
ND-8910


 


AAUGGACCGGUCGAUGUAUGUAA
1348
UGGACCGGUCGAUGUAUGUTT
1436
ACAUACAUCGACCGGUCCATT
1598
ND-8911


 


AAUACAACAAACCGUUGUGUGAA
1349
UACAACAAACCGUUGUGUGTT
1437
CACACAACGGUUUGUUGUATT
1599
ND-8912


 


AAAGAUUCCAUAAUAUAAGGGAA
1350
AGAUUCCAUAAUAUAAGGGTT
1438
CCCUUAUAUUAUGGAAUCUTT
1600
ND-8913


 


AACAAGCAACAGUUACUGCGAAA
1351
CAAGCAACAGUUACUGCGATT
1439
UCGCAGUAACUGUUGCUUGTT
1601
ND-8914


 


AAGUUAAUUAGGUGUAUUAACAA
1352
GUUAAUUAGGUGUAUUAACTT
1440
GUUAAUACACCUAAUUAACTT
1602
ND-8915


 


AAUUUGCUUUUCGGGAUUUAUAA
1353
UUUGCUUUUCGGGAUUUAUTT
1441
AUAAAUCCCGAAAAGCAAATT
1603
ND-8916


 


AAACUUUGCUUUUCGGGAUUUAA
1354
ACUUUGCUUUUCGGGAUUUTT
1442
AAAUCCCGAAAAGCAAAGUTT
1604
ND-8917


 


AACUGCAAACAACUAUACAUGAA
1355
CUGCAAACAACUAUACAUGTT
1443
CAUGUAUAGUUGUUUGCAGTT
1605
ND-8918


 


AAAUGACUUUGCUUUUCGGGAAA
1356
AUGACUUUGCUUUUCGGGATT
1444
UCCCGAAAAGCAAAGUCAUTT
1606
ND-8919


 


AACGACCCAGAAACUUACCACAA
1357
CGACCCAGAAAGUUACCACTT
1445
GUGGUAACUUUCUGGGUCGTT
1607
ND-8920


 


AAUUACUGCGACGUGAGGUAUAA
1358
UUACUGCGACGUGAGGUAUTT
1446
AUACCUCACGUCGCAGUAATT
1608
ND-8921


 


AAGUUACUGCGACGUGAGGUAAA
1359
GUUACUGCGACGUGAGGUATT
1447
UACCUCACGUCGCAGUAACTT
1609
ND-8922


 


AAUGCGACGUGAGGUAUAUGAAA
1360
UGCGACGUGAGGUAUAUGATT
1448
UCAUAUACCUCACGUCGCATT
1610
ND-8923


 


AAGUCGAUGUAUGUCUUGUUGAA
1362
GUCGAUGUAUGUCUUGUUGTT
1449
CAACAAGACAUACAUCGACTT
1611
ND-8924


 


AACGACGUGAGGUAUAUGACUAA
1362
CGACGUGAGGUAUAUGACUTT
1450
AGUCAUAUACCUCACGUCGTT
1612
ND-8925


 


AAGACUUUGCUUUUCGGGAUUAA
1363
GACUUUGCUUUUCGGGAUUTT
1451
AAUCCCGAAAAGCAAAGUCTT
1613
ND-8926


 


AAUUAGGUGUAUUAACUGUCAAA
1364
UUAGGUGUAUUAACUGUCATT
1452
UGACAGUUAAUACACCUAATT
1614
ND-8927


 


AAUUACCACAGUUAUGCACAGAA
1365
UUACCACAGUUAUGCACAGTT
1453
CUGUGCAUAACUGUGGUAATT
1615
ND-8928


 


AAGCAACAGUUACUGCGACGUAA
1366
GCAACAGUUACUGCGACGUTT
1454
ACGUCGCAGUAACUGUUGCTT
1616
ND-8929


 


AAUGCUUUUCGGGAUUUAUGCAA
1367
UGCUUUUCGGGAUUUAUGCTT
1455
GCAUAAAUCCCGAAAAGCATT
1617
ND-8930


 


AAUUAGUGAGUAUAGACAUUAAA
1368
UUAGUGAGUAUAGACAUUATT
1456
UAAUGUCUAUACUCACUAATT
1618
ND-8931


 


AAUAAUUAGGUGUAUUAACUGAA
1369
UAAUUAGGUGUAUUAACUGTT
1457
CAGUUAAUACACCUAAUUATT
1619
ND-8932


 


AAGAUGUAUGUCUUGUUGCAGAA
1370
GAUGUAUGUCUUGUUGCAGTT
1458
CUGCAACAAGACAUACAUCTT
1620
ND-8933


 


AACCGGUCGAUGUAUGUCUUGAA
1371
CCGGUCGAUGUAUGUCUUGTT
1459
CAAGACAUACAUCGACCGGTT
1621
ND-8934


 


AAGGAGCGACCCAGAAAGUUAAA
1372
GGAGCGACCCAGAAAGUUATT
1460
UAACUUUCUGGGUCGCUCCTT
1622
ND-8935


 


AAGAGCGACCCAGAAAGUUACAA
1373
GAGCGACCCAGAAAGUUACTT
1461
GUAACUUUCUGGGUCGCUCTT
1623
ND-8936


 


AAUGAGUAUAGACAUUAUUGUAA
1374
UGAGUAUAGACAUUAUUGUTT
1462
ACAAUAAUGUCUAUAGUCATT
1624
ND-8937


 


AAAAUACAACAAACCGUUGUGAA
1375
AAUACAACAAACCGUUGUGTT
1463
CACAACGGUUUGUUGUAUUTT
1625
ND-8938


 


AAGUAUGUCUUGUUGCAGAUCAA
1376
GUAUGUCUUGUUGCAGAUCTT
1464
GAUCUGCAACAAGACAUACTT
1626
ND-8939


 


AACUUUGCUUUUCGGGAUUUAAA
1377
CUUUGCUUUUCGGGAUUUATT
1465
UAAAUCCdGAAAAGCAAAGTT
1627
ND-8940


 


AAAUUAGUGAGUAUAGACAUUAA
1378
AUUAGUGAGUAUACACAUUTT
1466
AAUGUCUAUACUCACUAAUTT
1628
ND-8941


 


AAAAGAUUCCAUAAUAUAAGGAA
1379
AAGAUUCCAUAAUAUAAGGTT
1467
CCUUAUAUUAUGGAAUCUUTT
1629
ND-8942


 


AAGGUCGAUGUAUGUCUUGUUAA
1380
GGUCGAUGUAUGUCUUGUUTT
1468
AACAAGACAUACAUCGACCTT
1630
ND-8943


 


AACAUCAAGAACACGUAGAGAAA
1381
CAUCAAGAACACGUAGAGATT
1469
UCUCUACGUGUUCUUGAUGTT
1631
ND-8944


 


AAAACAGUUACUGCGACGUGAAA
1382
AACAGUUACUGCGACGUGATT
1470
UCACGUCGCAGUAACUGUUTT
1632
ND-8945


 


AAACAGUUACUGCGACGUGAGAA
1383
ACAGUUACUGCGACGUGAGTT
1471
CUCACGUCGCAGUAACUGUTT
1633
ND-8946


 


AAGUGUGAUUUGUUAAUUAGGAA
1384
GUGUGAUUUGUUAAUUAGGTT
1472
CCUAAUUAACAAAUCACACTT
1634
ND-8947


 


AAAUCAAGAACACGUAGAGAAAA
1385
AUCAAGAACACGUAGAGAATT
1473
UUCUCUACGUGUUCUUGAUTT
1635
ND-8948


 


AAUUUCGGGAUUUAUGCAUAGAA
1386
UUUCGGGAUUUAUGCAUAGTT
1474
CUAUGCAUAAAUCCCGAAATT
1636
ND-8949


 


AAACCCACAGGAGCGACCCAGAA
1387
ACCCACAGGAGCGACCCAGTT
1475
CUGGGUCGCUCCUGUGGGUTT
1637
ND-8950


 


AAAGAUGGGAAUCCAUAUGCUAA
1388
AGAUGGGAAUCCAUAUGCUTT
1476
AGCAUAUGGAUUCCCAUCUTT
1638
ND-8951


 


AAUAGUGAGUAUAGACAUUAUAA
1389
UAGUGAGUAUAGACAUUAUTT
1477
AUAAUGUCUAUACUCACUATT
1639
ND-8952


 


AAUGUGUGAUUUGUUAAUUAGAA
1390
UGUGUGAUUUGUOAAUUAGTT
1478
CUAAUUAACAAAUCACACATT
1640
ND-8953


 


AAUUAAUUAGGUGUAUUAACUAA
1391
UUAAUUAGGUGUAUUAACUTT
1479
AGUUAAUACACCUAAUUAATT
1641
ND-8954


 


AAAUAUGACUUUGCUUUUCGGAA
1392
AUAUGACUUUGCUUUUCGGTT
1480
CCGAAAAGCAAAGUCAUAUTT
1642
ND-8955


 


AACGGUCGAUGUAUGUCUUGUAA
1393
CGGUCGAUGUAUGUCUUGUTT
1481
ACAAGACAUACAUCGACCGTT
1643
ND-8956


 


AACAGGACCCACAGGAGCGACAA
1394
CAGGACCCACAGGAGCGACTT
1482
GUCGCUCCUGUGGGUCCUGTT
1644
ND-8957


 


AAUUUUCGGGAUUUAUGCAUAAA
1395
UUUUCGGGAUUUAUGCAUATT
1483
UAUGCAUAAAUCCCGAAAATT
1645
ND-8958


 


AAAAACAACUAUACAUGAUAUAA
1396
AAACAACUAUACAUGAUAUTT
1484
AUAUCAUGUAUAGUUGUUUTT
1646
ND-8959


 


AAUCCAUAUGCUGUAUGUGAUAA
1397
UCCAUAUGCUGUAUGUGAUTT
1485
AUCACAUACAGCAUAUGGATT
1647
ND-8960


 


AAUAUUCUAAAAUUAGUGAGUAA
1398
UAUUCUAAAAUUAGUGAGUTT
1486
ACUCACUAAUUUUAGAAUATT
1648
ND-8961


 


AAUAUGGAACAACAUUAGAACAA
1399
UAUGGAACAACAUUAGAACTT
1487
GUUCUAAUGUUGUUCCAUATT
1649
ND-8962


 


AAGUCUUGUUGCAGAUCAUCAAA
1400
GUCUUGUUGCAGAUCAUCATT
1488
UGAUGAUCUGCAACAAGACTT
1650
ND-8963


 


AAUAUUAACUGUCAAAAGCCAAA
1401
UAUUAACUGUCAAAAGCCATT
1489
UGGCUUUUGACAGUUAAUATT
1651
ND-8964


 


AAACCAAAAGAGAACUGCAAUAA
1402
ACCAAAAGAGAACUGCAAUTT
1490
AUUGCAGUUCUCUUUUGGUTT
1652
ND-8965


 


AAAAUUAGUGAGUAUAGACAUAA
1403
AAUUAGUGAGUAUAGACAUTT
1491
AUGUCUAUACUCACUAAUUTT
1653
ND-8966


 


AACAGAUCAUCAAGAACACGUAA
1404
CAGAUCAUCAAGAACACGUTT
1492
ACGUGUUCUUGAUGAUCUGTT
1654
ND-8967


 


AAUAUGCAUAGUAUAUAGAGAAA
1405
UAUGCAUAGUAUAUAGAGATT
1493
UCUCUAUAUACUAUGCAUATT
1655
ND-8968


 


AAAGAGAUGGGAAUCCAUAUGAA
1406
AGAGAUGGGAAUCCAUAUGTT
1494
CAUAUGGAUUCCCAUCUCUTT
1656
ND-8969


 


AAAGUGAGUAUAGACAUUAUUAA
1407
AGUGAGUAUAGACAUUAUUTT
1495
AAUAAUGUCUAUACUCACUTT
1657
ND-8970


 


AAUUCUAAAAUUAGUGAGUAUAA
1408
UUCUAAAAUUAGUGAGUAUTT
1496
AUACUCACUAAUUUUAGAATT
1658
ND-8971


 


AAAUGCAUAGUAUAUAGAGAUAA
1409
AUGCAUAGUAUAUAGAGAUTT
1497
AUCUCUAUAUACUAUGCAUTT
1659
ND-8972


 




ucGGuGGAccGGucGAuGuTsT
1498
AcAUCGACCGGUCcACCGATsT
1660
ND-8987


 




GGucGGuGGAccGGucGAuTsT
1499
AUCGACCGGUCcACCGACCTsT
1661
ND-8988


 




cGGuGGAccGGucGAuGuATsT
1500
uAcAUCGACCGGUCcACCGTsT
1662
ND-8989


 




GucGGuGGAccGGucGAuGTsT
1501
cAUCGACCGGUCcACCGACTsT
1663
ND-8990


 




AucAucAAGAAcAcGuAGATsT
1502
UCuACGUGUUCUUGAUGAUTsT
1664
ND-8991


 




cAAcAGuuAcuGcGAcGuGTsT
1503
cACGUCGcAGuAACUGUUGTsT
1665
ND-8992


 




cAAuAcAAcAAAccGuuGuTsT
1504
ACAACGGUUUGUUGuAUUGTsT
1666
ND-8993


 




GcuGcAAAcAAcuAuAcAuTsT
1505
AUGuAuAGUUGUUUGcAGCTsT
1667
ND-8994


 




GGuGGAccGGucGAuGuAuTsT
1506
AuAcAUCGACCGGUCCACCTsT
1668
ND-8995


 




AAAuuAGuGAGuAuAGAcATsT
1507
UGUCuAuACUcACuAAUUUTsT
1669
ND-8996


 




ucAucAAGAAcAcGuAGAGTsT
1508
CUCuACGUGUUCUUGAUGATsT
1670
ND-8997


 




AuAcAAcAAAccGuuGuGuTsT
1509
AcAcAACGGUUUGUUGuAUTsT
1671
ND-8998


 




uGGAccGGucGAuGuAuGuTsT
1510
AcAuAcAUCGACCGGUCCATsT
1672
ND-8999


 




uAcAAcAAAccGuuGuGuGTsT
1511
cAcAcAACGGUUUGUUGuATsT
1673
ND-9000


 




AGAuuccAuAAuAuAAGGGTsT
1512
CCCUuAuAUuAUGGAAUCUTsT
1674
ND-9001


 




cAAGcAAcAGuuAcuGcGATsT
1513
UCGcAGuAACUGUUGCUUGTsT
1675
ND-9002


 




GuuAAuuAGGuGuAuuAAcTsT
1514
GUuAAuAcACCuAAUuAACTsT
1676
ND-9003


 




uuuGcuuuucGGGAuuuAuTsT
1515
AuAAAUCCCGAAAAGcAAATsT
1677
ND-9004


 




AcuuuGcuuuucGGGAuuuTsT
1516
AAAUCCCGAAAAGcAAAGUTsT
1678
ND-9005


 




cuGcAAAcAAcuAuAcAuGTST
1517
cAUGuAuAGUUGUUUGcAGTST
1679
ND-9006


 




AuGAcuuuGcuuuucGGGATsT
1518
UCCCGAAAAGcAAAGUcAUTsT
1680
ND-9007


 




cGAcccAGAAAGuuAccAcTsT
1519
GUGGuAACUUUCUGGGUCGTsT
1681
ND-9008


 




uuAcuGcGAcGuGAGGuAuTsT
1520
AuACCUcACGUCGcAGuAATsT
1682
ND-9009


 




GuuAcuGcGAcGuGAGGuATsT
1521
uACCUcACGUCGcAGuAACTsT
1683
ND-9010


 




uGcGAcGuGAGGuAuAuGATsT
1522
UcAuAuACCUcACGUCGcATsT
1684
ND-9011


 




GucGAuGuAuGucuuGuuGTsT
1523
cAAcAAGAcAuAcAUCGACTsT
1685
ND-9012


 




cGAcGuGAGGuAuAUGAcuTsT
1524
AGUcAuAuACCUcACGUCGTsT
1686
ND-9013


 




GAcuuuGcuuuucGGGAuuTsT
1525
AAUCCCGAAAAGcAAAGUCTsT
1687
ND-9014


 




uuAGGuGuAuuAAcuGucATsT
1526
UGAcAGUuAAuAcACCuAATsT
1688
ND-9015


 




uuAccAcAGuuAuGcAcAGTsT
1527
CUGUGcAuAACUGUGGuAATsT
1689
ND-9016


 




GcAAcAGuuACuGcGAcGuTsT
1528
ACGUCGcAGuAACUGUUGCTsT
1690
ND-9017


 




uGcuuuucGGGAuuuAuGcTsT
1529
GcAuAAAUCCCGAAAAGcATsT
1691
ND-9018


 




uuAGuGAGuAuAGAcAuuATsT
1530
uAAUGUCuAuACUcACuAATsT
1692
ND-9019


 




uAAuuAGGuGuAuuAAcuGTsT
1531
cAGUuAAuAcACCuAAUuATsT
1693
ND-9020


 




GAuGuAuGucuuGuuGcAGTsT
1532
CUGcAAcAAGAcAuAcAUCTsT
1694
ND-9021


 




ccCGucGAuGuAuGucuuGTsT
1533
cAAGAcAuAcAUCGACCGGTsT
1695
ND-9022


 




GGAGcGAcCCAGAAAGuuATsT
1534
uAACUUUCUGGGUCGCUCCTsT
1696
ND-9023


 




GAGcGAcccAGAAAGuuAcTsT
1535
GuAACUUUCUGGGUCGCUCTsT
1697
ND-9024


 




uGAGuAuAGAcAuuAuuGuTsT
1536
AcAAuAAUGUCuAuACUcATsT
1698
ND-9025


 




AAuAcAAcAAAccGuuGuGTsT
1537
cAcAACGGUUUGUUGuAUUTsT
1699
ND-9026


 




GuAuGucuuGuuGcAGAucTsT
1538
GAUCUGcAAcAAGAcAuACTsT
1700
ND-9027


 




cuuuGcuuuucGGGAuuuATsT
1539
uAAAUCCCGAAAAGcAAAGTsT
1701
ND-9028


 




AuuAGuGAGuAuAGAcAuuTsT
1540
AAUGUCuAuACUcACuAAUTsT
1702
ND-9029


 




AAGAuuccAuAAuAuAAGGTsT
1541
CCUuAuAUuAUGGAAUCUUTsT
1703
ND-9030


 




GGucGAuGuAuGucuuGuuTsT
1542
AAcAAGAcAuAcAUCGACCTsT
1704
ND-9031


 




cAucAAGAAcAcGuAGAGATsT
1543
UCUCuACGUGUUCUUGAUGTsT
1705
ND-9032


 




AAcAGuuAcuGcGAcGuGATsT
1544
UcACGUCGcAGuAACUGUUTsT
1706
ND-9033


 




AcAGuuAcuGcGAcGuGAGTsT
1545
CUcACGUCGcAGuAACUGUTsT
1707
ND-9034


 




GuGuGAuuuGuuAAuuAGGTsT
1546
CCuAAUuAAcAAAUcAcACTsT
1708
ND-9035


 




AucAAGAAcAcGuAGAGAATsT
1547
UUCUCuACGUGUUCUUGAUTsT
1709
ND-9036


 




uuucGGGAuuuAuGcAuAGTsT
1548
CuAUGcAuAAAUCCCGAAATsT
1710
ND-9037


 




AcccAcAGGAGcGAcccAGTsT
1549
CuGGGUCGCUCCuGuGGGUTsT
1711
ND-9038


 




AGAuGGGAAuccAuAuGcuTsT
1550
AGcAuAUGGAUUCCcAUCUTsT
1712
ND-9039


 




uAGuGAGuAuAGAcAuuAuTsT
1551
AuAAUGUCuAuACUcACuATsT
1713
ND-9040


 




uGuGuGAuuuGuuAAuuAGTsT
1552
CuAAUuAAcAAAUcAcAcATsT
1714
ND-9041


 




uuAAuuAGGuGuAuuAAcuTsT
1553
AGUuAAuAcACCuAAUuAATsT
1715
ND-9042


 




AuAuGAcuuuGcuuuucGGTsT
1554
CCGAAAAGcAAAGUcAuAUTsT
1716
ND-9043


 




cGGucGAuGuAuGucuuGuTsT
1555
AcAAGAcAuAcAUCGACCGTsT
1717
ND-9044


 




cAGGAcccAcAGGAGcGAcTsT
1556
GUCGCUCCuGuGGGUCCUGTsT
1718
ND-9045


 




uuuucGCGAuuuAuGcAuATsT
1557
uAUGcAuAAAUCCCGAAAATsT
1719
ND-9046


 




AAAcAACuAuAcAuGAuAuTsT
1558
AuAUcAUGuAuAGUUGUUUTsT
1720
ND-9047


 




uccAuAuGcuGuAuGuGAuTsT
1559
AUcAcAuAcAGcAuAUGGATsT
1721
ND-9048


 




uAuUcuAAAAuuAGuGAGuTsT
1560
ACUCACuAAUUUuAGAAuATsT
1722
ND-9049


 




uAuGGAAcAAcAuuAGAAcTsT
1561
GUUCuAAUGUUGUUCcAuATsT
1723
ND-9050


 




GucuuGuuGcAGAucAucATsT
1562
UGAUGAUCUGCAAcAAGACTsT
1724
ND-9051


 




uAuuAAcuGucAAAAGccATsT
1563
UGGCUUUUGACAGUuAAuATsT
1725
ND-9052


 




AccAAAAGAGAAcuGcAAuTsT
1564
AUUGcAGUUCUCUUUUGGUTsT
1726
ND-9053


 




AAuuAGuGAGuAuAGAcAuTsT
1565
AUGUCuAuACUcACuAAUUTsT
1727
ND-9054


 




cAGAucAucAAGAAcAcGuTsT
1566
ACGuGUUCUuGAuGAUCuGTsT
1728
ND-9055


 




uAuGCAuAGuAuAuAGAGATsT
1567
UCUCuAUAuACuAUGcAuATsT
1729
ND-9056


 




AGAGAuGGGAAucCAuAuGTsT
1568
cAuAUGGAUUCCcAUCUCUTsT
1730
ND-9057


 




AGuGAGuAuAGAcAuuAuuTsT
1569
AAuAAUGUCuAuACUcACUTsT
1731
ND-9058


 




uucuAAAAuuAGuGAGuAuTsT
1570
AuACUcACuAAUUUuAGAATsT
1732
ND-9059


 




AuGCAuAGuAuAuAGAGAuTsT
1571
AUCUCuAuAuACuAUGcAUTsT
1733
ND-9060


 


AAGUGAUUUGUUAAUUAGGUGAA
1410
GUGAUUUGUUAAUUAGGUGTT
1572
CACCUAAUUAACAAAUCACTT
1734
AL-DP-7778


 


AAUGAUUUGUUAAUUAGGUGUAA
1411
UGAUUUGUUAAUUAGGUGUTT
1573
ACACCUAAUUAACAAAUCATT
1735
AL-DP-7779


 


AAGAUUUGUUAAUUAGGUGUAAA
1412
GAUUUGUUAAUUAGGUGUATT
1574
UACACCUAAUUAACAAAUCTT
1736
AL-DP-7780


 


AAAUUUGUUAAUUAGGUGUAUAA
1413
AUUUGUUAAUUAGGUGUAUTT
1575
AUACACCUAAUUAACAAAUTT
1737
AL-DP-7781


 


AAUGUGAUUUGUUAAUUAGGUAA
1414
UGUGAUUUGUUAAUUAGGUTT
1576
ACCUAAUUAACAAAUCACATT
1738
AL-DP-7782


 


AAUGUAUGGAACAACAUUAGAAA
1415
UGUAUGGAACAACAUUAGATT
1577
UCUAAUGUUGUUCCAUACATT
1739
AL-DP-7783


 


AAGUAUGGAACAACAUUAGAAAA
1416
GUAUGGAACAACAUUAGAATT
1578
UUCUAAUGUUGUUCCAUACTT
1740
AL-DP-7784


 


AAUGUGUACUGCAAGCAACAGAA
1417
UGUGUACUGCAAGCAACAGTT
1579
CUGUUGCUUGCAGUACACATT
1741
AL-DP-7803


 


AAACUGCGACGUGAGGUAUAUAA
1418
ACUGCGACGUGAGGUAUAUTT
1580
AUAUACCUCACGUCGCAGUTT
1742
7804


 


AAGAGGUAUAUGACUUUGCUUAA
1419
GAGGUAUAUGACUUUGCUUTT
1581
AAGCAAAGUCAUAUACCUCTT
1743
AL-DP-7805


 


AAAUGCUGUAUGUGAUAAAUGAA
1420
AUGCUGUAUGUGAUAAAUGTT
1582
CAUUUAUCACAUACAGCAUTT
1744
AL-DP-7807


 


AAUUUAUUCUAAAAUUAGUGAAA
1421
UUUAUUCUAAAAUUAGUGATT
1583
UCACUAAUUUUAGAAUAAATT
1745
AL-DP-7808


 


AACUGCGACGUGAGGUAUAUGAA
1422
CUGCGACGUGAGGUAUAUGTT
1584
CAUAUACCUCACGUCGCAGTT
1746
AL-DP-7810


 


AAACCGUUGUGUGAUUUGUUAAA
1423
ACCGUUGUGUGAUUUGUUATT
1585
UAACAAAUCACACAACGGUTT
1747
AL-DP-7812





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


Lower case letters: ribonucloetide bearing 2′-O-methyl substituent on ribose moiety


s: Indicates position of phosphorothioate internucleoside linkage


chol: cholesterol moiety conjugated to 3′ ribonucleotide.


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






Testing of siRNA Targeting HPV E6 Gene Expression
Unmodified and chemically modified dsRNA were tested to identify their relative abilities to reduce the expression level of mRNA encoding HPV E6 gene in a cell.
The assay conditions employed were as follows: C33A cells were obtained from ATCC. Sequences encoding HPV16 E6 and E1 were cloned into the pNAS-055 vector (Husken et al., Nucleic Acids Research, 31:e102, 2003), for expression as YFP fusion transcripts. The resulting plasmids were transfected into C33A cells, and stable lines expressing these fusion transcripts were derived by Zeocin selection, as per the manufacturer's protocol (Invitrogen). For transfection with siRNA against HPV16 E 6 or HPV16 E1, respective cells were seeded at a density of 2.0×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30 nM, 3 nM or 300 pm as indicated) was carried out in a single dose with lipofectamine 20000 (Invitrogen) as described by the manufacturer.
24 hours after transfection cells were lysed and fusion YFP mRNA expression levels were quantified with the Quantigene Explore Kit (Panomics, Inc. (Fremont, Calif.) (formerly Genospectra, Inc.)) using a probe directed against YFP, according to the standard protocol. Fusion-YFP mRNA levels were normalized to GAP-DH mRNA. For each siRNA four individual datapoints were collected. siRNA duplexes-unrelated to the HPV16 E1 or E6 genes were used as control. The activity of a given siRNA duplex was expressed as percent fusion-YFP mRNA concentration in treated cells relative to concentration of the same transcript in cells treated with the control siRNA duplex.
Table 8 shows the results of testing the E6 dsRNA of the invention.













TABLE 8








Mean

Mean





activity

activity




remaining

remaining



duplex
after 30 nM

after 300 pM



name
treatment
sd
treatment
sd






















ND-8899
15.23
3.19
31.29
9.57



ND-8900
11.61
2.88
26.80
10.23



ND-8901
10.88
3.54
24.77
5.19



ND-8902
20.19
7.36
43.46
6.89



ND-8903
10.38
2.51
22.95
5.47



ND-8904
13.71
4.67
22.11
5.50



ND-8905
13.81
4.29
24.69
4.62



ND-8906
8.35
2.17
24.23
6.62



ND-8907
13.88
3.12
36.94
6.13



ND-8908
14.47
3.48
45.15
7.92



ND-8909
19.99
3.67
49.36
9.80



ND-8910
36.96
9.77
74.18
15.82



ND-8911
18.66
4.19
45.51
6.82



ND-8912
47.42
6.99
76.11
12.97



ND-8913
55.53
16.75
76.63
15.44



ND-8914
9.69
2.50
19.91
6.63



ND-8915
49.02
7.97
93.38
6.83



ND-8916
11.88
2.94
49.78
8.49



ND-8917
14.00
2.04
50.36
8.31



ND-8918
13.70
3.43
29.01
6.51



ND-8919
10.31
2.44
42.51
10.89



ND-8920
10.29
2.72
25.20
9.73



ND-8921
20.23
3.71
37.17
10.15



ND-8922
11.64
2.31
24.95
8.99



ND-8923
12.43
1.97
24.39
8.13



ND-8924
15.19
4.52
32.09
7.01



ND-8925
14.24
1.87
34.21
5.61



ND-8926
10.17
2.85
19.04
3.68



ND-8927
20.77
4.89
41.40
9.23



ND-8928
95.02
20.87
92.24
15.19



ND-8929
17.51
5.27
19.86
6.81



ND-8930
13.58
2.65
61.16
11.03



ND-8931
13.78
2.00
37.55
8.11



ND-8932
105.07
21.10
91.19
12.68



ND-8933
14.88
3.07
43.06
8.64



ND-8934
13.03
3.75
24.32
5.92



ND-8935
13.19
2.88
21.87
4.17



ND-8936
10.04
1.94
21.98
6.92



ND-8937
15.39
3.44
42.70
11.35



ND-8938
55.90
5.56
93.49
10.41



ND-8939
11.51
2.04
29.57
10.38



ND-8940
12.80
2.94
29.67
6.73



ND-8941
19.46
2.91
43.13
3.64



ND-8942
96.02
29.93
85.34
4.57



ND-8943
13.44
3.90
19.03
5.18



ND-8944
14.35
2.09
20.03
4.68



ND-8945
11.45
1.98
23.80
8.36



ND-8946
15.43
2.27
43.12
13.34



ND-8947
13.32
2.20
53.58
18.04



ND-8948
12.85
3.18
23.22
6.79



ND-8949
86.23
23.43
75.99
7.17



ND-8950
29.49
7.99
47.19
14.70



ND-8951
10.51
2.85
20.21
6.23



ND-8952
12.10
2.74
28.82
9.06



ND-8953
41.13
11.23
77.64
9.46



ND-8954
46.52
8.41
81.61
14.93



ND-8955
38.40
8.46
83.38
16.32



ND-8956
12.13
2.23
21.94
9.54



ND-8957
28.39
6.18
53.84
12.53



ND-8958
36.41
9.92
55.67
8.77



ND-8959
33.95
11.23
63.63
5.53



ND-8960
13.63
2.39
26.49
9.24



ND-8961
80.42
18.39
89.13
8.61



ND-8962
33.00
4.18
82.57
9.01



ND-8963
16.67
1.85
28.39
9.50



ND-8964
14.17
2.58
43.74
14.37



ND-8965
17.23
5.15
48.03
10.97



ND-8966
23.01
5.10
53.86
10.10



ND-8967
18.68
5.13
23.30
6.20



ND-8968
10.99
1.83
28.22
9.33



ND-8969
13.75
2.67
32.62
10.53



ND-8970
11.02
2.68
29.14
10.73



ND-8971
21.71
3.11
57.75
10.54



ND-8972
17.10
3.20
52.10
10.84



ND-8987
40.36
7.25
91.12
9.07



ND-8988
20.54
5.27
30.76
12.23



ND-8989
36.60
7.23
74.41
8.61



ND-8990
17.55
8.33
61.02
11.13



ND-8991
11.29
2.87
19.03
5.98



ND-8992
14.49
3.37
44.53
14.74



ND-8993
18.45
5.75
48.07
6.79



ND-8994
13.16
1.80
25.92
7.94



ND-8995
52.21
5.93
90.43
4.86



ND-8996
32.77
6.96
57.54
7.12



ND-8997
14.45
1.50
20.63
4.28



ND-8998
137.83
33.37
90.09
14.65



ND-8999
82.01
13.74
85.69
10.39



ND-9000
69.77
21.32
83.16
14.34



ND-9001
54.71
18.91
74.70
8.87



ND-9002
12.15
2.05
22.98
6.98



ND-9003
76.52
11.49
98.54
7.10



ND-9004
62.23
16.29
84.38
8.22



ND-9005
38.12
6.77
64.57
6.57



ND-9006
12.96
3.15
26.03
4.76



ND-9007
18.24
4.88
42.16
7.87



ND-9008
21.06
4.60
20.01
6.00



ND-9009
35.15
5.62
79.96
7.01



ND-9010
13.71
2.83
53.80
12.21



ND-9011
38.04
3.56
60.45
10.19



ND-9012
44.63
37.28
67.30
8.30



ND-9013
13.31
1.81
31.12
6.40



ND-9014
12.69
3.66
27.50
7.48



ND-9015
16.26
3.61
21.18
4.80



ND-9016
29.49
8.14
66.50
15.07



ND-9017
16.98
2.22
27.17
7.64



ND-9018
35.62
7.31
86.49
7.60



ND-9019
23.48
2.57
60.66
13.05



ND-9020
113.04
21.57
88.75
12.94



ND-9021
38.45
5.44
68.21
9.53



ND-9022
14.21
2.86
53.78
13.38



ND-9023
21.84
3.72
41.95
11.93



ND-9024
117.68
33.94
86.00
6.55



ND-9025
86.38
19.82
81.09
9.82



ND-9026
113.52
9.02
95.62
10.60



ND-9027
13.61
2.09
51.98
15.63



ND-9028
14.49
4.02
45.08
11.80



ND-9029
20.16
3.25
39.00
8.28



ND-9030
104.95
34.72
76.74
10.03



ND-9031
19.90
6.09
26.32
9.90



ND-9032
16.43
3.38
19.10
5.44



ND-9033
100.99
24.54
86.16
11.95



ND-9034
13.77
2.84
33.36
13.56



ND-9035
13.54
1.58
57.07
19.24



ND-9036
12.91
3.20
21.78
6.03



ND-9037
30.90
8.30
74.12
12.35



ND-9038
121.49
24.79
87.65
7.07



ND-9039
10.19
3.13
23.32
9.60



ND-9040
11.45
2.34
22.86
8.27



ND-9041
33.73
8.63
82.99
13.62



ND-9042
18.21
3.81
60.07
13.85



ND-9043
36.15
3.87
71.81
12.23



ND-9044
13.77
3.59
30.27
10.81



ND-9045
56.81
19.55
85.99
9.99



ND-9046
26.03
6.18
51.21
10.14



ND-9047
100.23
24.53
85.98
5.59



ND-9048
21.82
4.07
44.44
12.82



ND-9049
82.93
21.46
87.79
7.07



ND-9050
18.51
3.33
40.70
10.96



ND-9051
22.80
3.37
42.44
14.86



ND-9052
12.61
3.78
37.58
13.35



ND-9053
19.88
4.32
53.11
3.23



ND-9054
33.65
8.32
59.71
6.42



ND-9055
22.61
7.41
27.44
7.04



ND-9056
16.61
3.38
34.34
13.22



ND-9057
25.51
6.29
51.45
10.10



ND-9058
27.60
4.56
54.99
13.52



ND-9059
23.83
4.36
84.76
13.88



ND-9060
17.12
3.29
44.54
15.68



AL-DP-7778
19.35
8.95
63.31
14.21



AL-DP-7779
41.30
9.51
65.96
7.82



AL-DP-7780
24.01
7.52
59.43
8.85



AL-DP-7781
13.69
3.41
53.58
9.31



AL-DP-7782
31.35
5.31
65.84
10.41



AL-DP-7783
14.46
2.85
38.92
10.30



AL-DP-7784
13.52
1.52
25.09
7.89



AL-DP-7803
39.68
4.75
66.72
11.32



7804
12.56
3.96
26.81
6.28



AL-DP-7805
13.92
2.22
35.87
8.95



AL-DP-7807
35.54
4.95
70.94
11.01



AL-DP-7808
81.47
9.77
96.18
10.87



AL-DP-7810
15.14
2.12
37.66
16.19



AL-DP-7812
12.89
1.99
25.18
12.05










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

## Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human E6AP gene in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said E6AP, inhibits expression of said E6AP gene by at least 40% as compared to a control cell.

2. The dsRNA of claim 1, wherein the second sequence comprises SEQ ID NO:313.

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

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

5. The dsRNA of claim 4, wherein said modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

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

7. A cell comprising the dsRNA of claim 1.

8. A pharmaceutical composition for inhibiting the expression of the E6AP gene in an organism, comprising the dsRNA of claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the first sequence comprises SEQ ID NO:157 and the second comprises SEQ ID NO:313.

10. A method for inhibiting the expression of the E6AP gene in a cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the E6AP gene, thereby inhibiting expression of the E6AP gene in the cell.

11. A vector for inhibiting the expression of the E6AP gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of claim 1.

12. A cell comprising the vector of claim 11.

13. The dsRNA of claim 1, wherein said contact is performed in vitro at 30 nM or less.

14. The dsRNA of claim 2, wherein the second sequence consists of SEQ ID NO:313.

15. The dsRNA of claim 2, wherein the first sequence comprises SEQ ID NO:157 and the second sequence comprises SEQ ID NO:313.

16. The dsRNA of claim 2, wherein the first sequence consists of SEQ ID NO:157 and the second sequence consists of SEQ ID NO:313.

17. The dsRNA of claim 1, wherein the second sequence consists of SEQ ID NO:313.

18. The dsRNA of claim 4, wherein said modified nucleotide is a 2′-O-methyl modified nucleotide.

19. The dsRNA of claim 4, wherein said modified nucleotide is a 5′-phosphorothioate group.

20. The pharmaceutical composition of claim 8, wherein the first sequence consists of SEQ ID NO:157 and the second consists of SEQ ID NO:313.