CA2406485A1 - Hpv-specific short-mers - Google Patents

Abstract

The invention provides oligonucleotides capable of inhibiting human papillomavirus (HPV) replication. More specifically, the invention provides oligonucleotides complementary to the translational start site of the HPV E1 open reading frame which inhibit HPV replication through interaction with a nucleic acid or a protein target. Oligonucleotides of the invention contain at least 4 deoxyribonucleotides and/or ribonucleotides and may include a variet y of modifications to the internucleotide linkages or the mononucleotides. Furthermore, the invention provides pharmaceutical compositions and methods for treating disorders or diseases associated with HPV infection.

Description

HPV-SPECIFIC SHORT-MERS
BACKGROUND OF THE INVENTION
1. Field of the Invention This invention relates to the human papillomavirus (HPV). More specifically, this invention relates to the inhibition and treatment of human papillomavirus infections, including the treatment and prevention of human papillomavinis-associated disorders or diseases. The invention provides short, synthetic oligonucleotides which are useful for inhibiting replication of HPV.

2. Background Human papillomaviruses (HPV) are DNA viruses that infect epithelial cells resulting in a range of lesions from benign skin and genital warts (condyloma acuminate) and epidermodysplasia verruciformis (EV) to respiratory or laryngeal papillornatosis and cervical carcinoma. Cervical cancer is the second most prevalent type of cancer affecting women with 400,00 to 500,000 newly reported cases and 200,000 deaths each year (Parkin, D.M. et al. (1993) Int. J. Cafzcer 54: 594-606).
Neonates may also be infected with HPV during passage through the mother's birth canal leading to laryngeal papillomas, or benign epithelial tumors of the larynx.
Papillomas develop in HPV infected infants by the age of two necessitating the need for multiple surgeries to remove the benign papillomas which may occlude the airway.
There are at least 70 different types of human papillomaviruses based on DNA
sequence diversity as measured by liquid hybridization (Pfister et al. (1994) htteYVirol. 37: 143-149). Each HPV type exhibits host specificity. Several HPV
types infect genital epithelia and represent the most prevalent etiologic agents of sexually transmitted viral disease. The genital HPV types can be further subdivided into "high-risk" types that are associated with the development of neoplasms, most commonly HPV-16 and HPV-18; and "low-risk" types that are rarely associated with malignancy, most commonly HPV-6 and HPV-l l . The malignant types may integrate into the genome of the host cell, thereby eliminating the requirement for viral DNA replication gene products. In contrast, the benign types, most commonly HPV-6 and HPV-11, rely on viral proteins El and E2 for replication of the episomal genome. Two HPV types, HPV-6 and HPV-11, are commonly associated with laryngeal papillomas, or benign epithelial tumors of the larynx.
Human papillomaviruses are nonenveloped DNA viruses containing a circular, double stranded, 7,900 base pair DNA genome that can be divided into three distinct functional domains: the upstream regulatory region (LTRR), which contains the origin of viral DNA replication and enhancers and promoters involved in transcription; the L
region that encodes the structural proteins, L1 and L2; and the E region that encodes genes required for vegetative functions. The HPV genome encodes for eight viral proteins, El, E2, E4, E5, E6, E7, L1 and L2, (shown schematically in FIG. 1) that are translated from complex families of alternatively spliced mRNAs.
Most HPV types require the activity of two virally encoded proteins, El and E2, for initiation of viral DNA replication (IJstav et al. (1991) EMBO J., 10:
449-457;
Chiang et al. (1992) Pr~oc. Natl. Acad. Sci. (USA) 89: 5799-5803; Del Vecchio, A.M.
et al. (1992) J. Virol. 66: 5949-5958; Sandier, A.B. et al. (1993) J. Yirol.
67: 5079-5087; Scheffner, M. et al. (1994) In Human Pathogenic Papillomaviruses (Ed.
Zur hausen, H.) pp. 83-100, Heidelberg, Springer-Verlag) and episornal maintenance of the viral genome. However, in certain in vitro experiments it has been shown that only the activity of the El protein is essential for viral replication (Gopalakrishnan et al. (1994) Proc. Natl. Acad. Sci. (USA) 91: 9597-9601). E1 is an ATP-hydrolyzing DNA helicase which is though to be involved in unwinding DNA at the viral origin during replication of the viral genome by the human host cell DNA replication complex (Hughes et al. (1993) Nucleic Acids Res. 21: 5817-5823; Chow et al.
(1994) Intervirol. 37: 150-158; Jerkins, O. et al. (1996) J. Gera. Yirol. 77:1805-1809; Conger, I~.L. et al. (1999) J. Biol. Cherra. 274: 2696-2705). E2 is involved in the regulation of HPV transcriptional activity through facilitation of the assembly of transcriptional complexes containing host proteins (Ham, J. et al. (1991) Trends Biochern.
Sci. 16:
440-444; Liu, J.-S. et al. (1995) .I. Biol. Chern. 270: 27283-27291).
The E4 protein associates with the intermediate-filament network of the host cell and is the most abundant gene product expressed by the papillomaviruses (Dorrbar, J. et al. (1986) EMBO J. 5: 355-362; Dorrbar, J. et al. Nature 352:
824-827).
The E5, E6 and E7 gene products encode transforming proteins (Androphy, E.J.
et al.
(1987) EMBO J. 6: 989-992; Bedel, M.A, et al. (1989) J. Virol. 63: 1247-1255;
Matlashewski, E. et al. (1987) EMBO J. 6: 1741-1746; Vousden, K.H. et al.
(1988) Oncogene Res. 3: 167-175). E5 is a highly hydrophobic protein which interacts with the epidermal growth factor receptor (Chen, S.-L. et al. (1990) J. Yirol. 64:

3233; Leechanachai, P. et al. (1992) Oncogene 7: 19-25; Leptak, C. et al.
(1991) J.
Virol. 65: 7078-7083; Pim, D. et al. (1992) Oncogene 7: 27-32; Straight, S.W.
et al.
(1993) J. Virol. 67: 4521-4532). In some HPV types, the E6 and E7 proteins interact with the tumor suppressor proteins p53 (Lechner, M.S. et al. (1992) EMBO J.
11:
3045-3052; Werness, B.A. et al. (1990) Science 248: 76-79) and retinoblastoma (Dyson, N. et al. (1989) Science 243: 934-937; Gage, J.R. et al. J. Yirol. 64:
723-730), respectively. Both E6 and E7 interact with a number of cellular proteins which influence the outcome of infection. L 1 and L2 are common to all HPV types and encode for capsid proteins (Broker, T.R. et al. (1986) Cancer Cells 4: 17-36;
zur Hausen, H. et al. (1987) In The Papovaviridai. Vol. 2 The Papillomaviruses (eds.
Salzman, N. and Howley, P.M.) pp. 245-263, New York, Plenum Press; Pfister, H.
(1987) Obstet. Gynecol. Clin. No~tlt Ana. 14: 349-361).
Current treatment for HPV infection is extremely limited. There are at present no approved HPV-specific antiviral therapeutics. Management normally involves physical destruction of the wart by surgical, cryosurgical, chemical, or laser removal of infected tissue. Nonanogenital warts are transmitted by skin-to-skin contact while anogenital warts are usually transmitted sexually. Both types of warts produce much morbidity but rarely undergo malignant transformation. They are commonly treated with surgical or cytodestructive therapy, but immunomodulatory agents, such as imiquimod, have been proven to be very effective in anogenital warts and are being evaluated in nonanogenital warts (Severson J., et al., .J. Cutan. Med. Sung.
(2001) Jan;S(1):43-60). Topical anti-metabolites such as 5-fluorouracil and podophyllum preparations have also been used (Reichman in Harrison's Principles of Internal Medicine, 13th Ed. (Isselbacher et al., eds.) McGraw-Hill, Inc., NY (1993) pp.

803). However, reoccurrence after these procedures is common, and subsequent repetitive treatments progressively destroy healthy tissue. Interferon has so far been the only treatment with an antiviral mode of action, but its limited effectiveness restricts its use (Cowsert (1994) hatervirol. 37: 226-230; Bornstein et al.
(1993) Obstetrics Gyraecol. Sur. 4504: 252-260; Browder et al. (1992) Anna.
Pharnaacother.
26: 42-45.
Other types of HPV have marked oncogenic potential such that over 99% of all cervical cancers and over 50% of other anogenital cancers are due to infection with oncogenic HPV. Many cofactors, such as cigarette smoking, genetics, and helper viruses, have potential roles in HPV oncogenesis, but their relative contributions are poorly understood (Severson J., et al., J. Cutafa. Med. Surg. (2001) Jan;S(1):43-60).
Recently, research directed at development of HPV antiviral compounds has focused on developing HPV specific antisense oligonucleotides. Antisense oligonucleotides can modulate gene expression by binding to target single-stranded nucleic acid molecules according to the Watson-Crick rule or to double stranded nucleic acids by the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H; or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression.
Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85: 7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (Proc. Natl. Acad. Sci. (ZISA) (1989) 86:

7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed.
Pederson et al. (U.S. PatentNos. 5,149,797 and 5,220,007) discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Agrawal et al. (WO 94/02498) discloses hybrid oligonucleotides having regions of deoxyribonucleotides and 2'-O-methyl-ribonucleotides.
The mechanism of DNA replication is conserved among papillomaviruses. Of all papillomavirus proteins, E1 is the most conserved. It is an ATPase and helicase and has sequence homology to the ATPase domain of the simian virus 40 (SV40) T
antigen, the initiator for SV40 origin sequence (ori) replication. As does the T antigen, the E1 protein binds to the on and unwinds DNA in the presence of the host single-stranded DNA binding protein RPA and topoisomerase I. The human papillomavirus (HPV) and bovinepapillomavirus type 1 (BPV-1) El proteins are thought to function as a helicase at the replication fork, since each is required during elongation. The BPV-1 El protein is known to interact with the 180-kDa catalytic subunit of the host DNA polymerase a., thereby bringing host replication proteins to the unwound ori.
Because of the homologous nature of the on and of E1 and E2 proteins, proteins from one virus type can efficiently replicate either a homologous or heterologous viral ori.
(Nianxiang Zou et al., J. Virol., (1998), 72(4):43436-3441).
A limited number of antisense oligonucleotides have been designed which inhibit the expression of HPV. For example, oligonucleotides specific for various regions of HPV El and E2 mRNA have been prepared (see, e.g., U.S. 5,364,758, WO
91/08313, WO 93/20095, and WO 95/04748).
A need still remains for the development of oligonucleotides that are capable of inhibiting the replication and expression of human papillomavirus whose uses are accompanied by a successful prognosis and low or no cellular toxicity.

SUMMARY OF THE INVENTION
In order to design a therapeutic compound against human papillomaviruses, the E1 gene of HPV types 6 (Gen Bank HPV6b accession no. M14119) and 11 (Gen Bank HPV 11 accession no. X00203) has been targeted. Types 6 and 11 together are associated with over 90% of cases of non-malignant genital warts. A 46 nucleotide region (from -17 to +29 of the E1 open reading frame) centered on the initiation site for protein translation has been examined in detail. This region is conserved in a number of clinical isolates of HPV types 6 and 11. The entire open reading frame of the gene (from -17 to +1950) has also been investigated. This entire region shows high sequence identity between HPV type 6 and HPV type 11. Further HPV strains which are targeted in the present invention, are HPV 16, 18, 31, 45 and 58.
We have now found that oligonucleotides directed to the +1 to +20 region of the E1 gene are particularly useful for inhibiting HPV replication. More particularly, we have found that oligonucleotides directed to this region that are as short as 4 nucleotides can effectively inhibit HPV replication. These short oligonucleotides, or short-mers, have a specific sequence related effect which may be acting to inhibit HPV replication through multiple mechanisms. Oligonucleotides of the invention may be acting through interaction with a nucleic acid or protein target, or may be acting through both types of interactions.
The present invention provides synthetic oligonucleotides complementary to the region spanning +1 to +20 of the translational start site of the HPV E1 protein, or a portion thereof. More particularly, the invention provides oligonucleotides that are modified so as to increase their stability or their HPV inhibitory activity.
Such modifications may include, for example, modifications of the internucleoside linkages, sugar, base, capped ends and chimeric or hybrid oligonucleotides.
The invention further provides pharmaceutical compositions and methods for treatment of HPV infections, including treatment and prevention of HPV-associated disorders or diseases.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the HPV genome.
FIG. 2 A-E shows Southern blot hybridizations of total DNA isolated from CIN-612 9E raft culture cells hybridized with an HPV3lb specific probe to determine the level of HPV replication in the cultures. The CIN-612 9E cultures were treated with 25 p,M of oligonucleotides of the present invention and the level of inhibition of viral replication was compared to a mock treated culture.
FIG. 2 A-B shows Southern blot hybridizations of total DNA isolated from CIN-612 9E raft culture cells hybridized with an HPV3lb specific probe to determine the level of HPV replication in the cultures. The CIN-612 9E cultures were treated with 2.5 pM of oligonucleotides of the present invention and the level of inhibition of viral replication was compared to a mock treated culture.
DETAILED DESCRIPTION OF THE INVENTION
As discussed above, we have discovered synthetic oligonucleotides that have significant HPV inhibitory activity.
The invention provides synthetic oligonucleotides complementary to a nucleic acid spanning the translational start site of human papillomavirus gene El.
For purposes of the invention, the nucleic acid spanning the translational start site of human papillomavirus gene E1 is intended to indicate the region of the E1 gene from nucleotide +1 to +20 (for example, nucleotides 832-851 of the HPV-6b genome), or a portion thereof. This region has the sequence as set forth in SEQ ID NO: 35 (5'-atg gcg gac gat tca ggt ac-3') and the oligonucleotide complementary to this region has the sequence as set forth in SEQ ID NO: 36 (5'-gX acc Xga aXc gXc cgc caX-3'), wherein X may be thymidine or uracil and any nucleotide may be substituted with mosme.
For purposes of the invention, an oligonucleotide sequence that is complementary to a nucleic acid is intended to mean that an unmodified version of the oligonucleotide would be capable of binding to the nucleic acid sequence under physiological conditions, e.g., interaction between an oligonucleotide and a single stranded nucleic acid by Watson-Crick base pairing or 'Wobble' base pairing.
For purposes of the invention, oligonucleotides that have HPV inhibitory activity is intended to mean oligonucleotides that are capable of interfering with or disrupting HPV replication at some point in the viral life cycle through a variety of possible mechanisms. For example, the oligonucleotides may be inhibiting HPV
replication through interaction with a nucleic acid or protein target, or may be acting through both types of target. Oligonucleotides acting through a nucleic acid target may be binding to single stranded DNA, double stranded DNA or rnRNA and disrupting the function of the target by preventing the binding of factors required for normal transcription, splicing, or translation, or by triggering the enzymatic destruction of mRNA by RNase H, etc. Oligonucleotides acting through a protein target may be binding to receptors or any other type of protein, such as, for example, DNA polymerase, transcription factors, etc.
Oligonucleotides of the present invention have a specific sequence related effect. This is intended to mean that the sequence of the oligonucleotide, including the linear arrangement of the mononucleotides and the tertiary structure of the oligonucleotide as a whole as determined by the sequence of mononucleotides and any modifications to the nucleotides or the internucleotide linkages, produces a specific HPV inhibitory effect. However, this is not meant to limit the invention to a mechanism that is reliant upon base pairing between the oligonucleotide and a nucleic acid. The specific sequence related effect may be occurring through a variety of mechanisms as discussed above.
Synthetic oligonucleotides of the invention comprise sequence complementary to the region of the HPV E1 open reading frame spanning nucleotides +1 to +20, or a portion thereof. Preferably, the oligonucleotides comprise from about 4 and up to about 20 mononucleotides, more preferably from about 4 to about 12, 14, 16 or mononucleotides. Preferred oligonucleotides of the invention include those that contain 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides.
More preferably, oligonucleotides of the invention comprise the sequence set forth in Table 1 which follows, or a portion thereof. Most preferably, oligonucleotides of the invention comprise the sequences set forth in SEQ ID NO: 1-3, 2S and 34.
Preferred synthetic oligonucleotides comprise at least one, and preferably more than one, modification. Modifications include, for example, modifications of the internucleotide linkage, the base or the sugar moiety, capped ends and chimeric or hybrid oligonucleotides.
Synthetic oligonucleotides include chemically synthesized polymers of deoxyribonucleotide and/or ribonucleotide monomers connected by internucleotide linkages. Oligonucleotides may be constructed entirely of deoxyribonucleotides, entirely of ribonucleotides or of a combination of deoxyribonucleotides and ribonucleotides, including hybrid and inverted hybrid oligonucleotides. Hybrid oligonucleotides contain a core region of deoxyribonucleotides interposed between flanking regions of ribonucleotides. Inverted hybrids contain a core region of ribonucleotides interposed between flanking regions of deoxyribonucleotides.
Synthetic oligonucleotides of the invention may be connected by standard phosphodiester internucleotide linkages between the 5' group of one mononucleotide pentose ring and the 3' group of an adjacent mononucleotide. Such linkages could also be established using different sites of connection, including 5' to 5', 3' to 3', 2' to 5' and 2' to 2', or any combination thereof. In addition to phosphodiester linkages, the mononucleotides may also be connected by alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester linkages, or any combination thereof. Preferably, an oligonucleotide of the invention comprises at least one phosphorothioate internucleotide linkage, more preferably, all linkages in the oligonucleotide are phosphorothioate internucleotide linkages.
Oligonucleotides of the invention may be constructed such that all mononucleotides are connected by the same type of internucleotide linkages or by combinations of different internucleotide linkages, including chimeric or inverted chimeric oligonucleotides. Chimeric oligonucleotides have a phosphorothioate core region interposed between methylphosphonate or phosphoramidate flanking regions.

5 Inverted chimeric oligonucleotides have a nonionic core region (e.g.
alkylphosphonate and/or phosphoramidate and/or phosphotriester internucleoside linkage) interposed between phosphorothioate flanking regions.
Synthetic oligonucleotides of the invention may be constructed of adenine, 10 cytosine, guanine, inosine, thymidine or uracil mononucleotides. Preferred oligonucleotides are constructed from mononucleotides which contain modifications to the base and/or sugar moiety of the mononucleotide. Modifications to the base or sugar include covalently attached substituents of alkyl, carbocyclic aryl, heteroaromatic or heteroalicyclic groups having from 1 to 3 separate or fused rings and 1 to 3 N, O or S atoms, or a heterocyclic structure.
Alkyl groups preferably contain from 1 to about 1 S carbon atoms, more preferably from 1 to about 12 carbon atoms and most preferably from 1 to about carbon atoms. Specific examples of alkyl groups include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl etc.
Aralkyl groups include the above-listed alkyl groups substituted by a carbocyclic aryl group having 6 or more carbons, for example, phenyl, naphthyl, phenanthryl, anthracyl, etc.
Cycloalkyl groups preferably have from 3 to about 8 ring carbon atoms, e.g.
cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, 1,4-methylenecyclohexane, adamantyl, cyclopentylmethyl, cyclohexylmethyl, 1- or 2-cyclohexylethyl and 1-, 2-or 3-cyclohexylpropyl, etc.
Exemplary heteroaromatic and heteroalicyclic group include pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

Preferred modifications to the sugar include modifications to the 2' position of the ribose moiety which include but are not limited to 2'-O-substituted with an -G-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl, or allyl group having 2-6 carbon atoms wherein such -O-alkyl, aryl or allyl group may be unsubstituted or may be substituted (e.g., with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carballcoxyl, or amino groups), or wherein the 2'-O-group is substituted by an amino, or halogen group.
None of these substitutions are intended to exclude the native 2'-hydroxyl group in case of ribose or 2'-H- in the case of deoxyribose.
Preferred modified oligonucleotides include 2'-O-methyl ribonucleotides (2'-OMe) and 5-methylated deoxycytosine (5-Me-dC). Oligonucleotides of the invention may be constructed entirely of unmodified mononucleotides, entirely of mononucleotides containing a particular modification, or of a variety mononucleotides containing different modifications. Particularly preferred oligonucleotides comprises at least one, preferably one to five 2'-O-methyl ribonucleotides at the 3' end of the oligonucleotide. Moreover, the oligonucleotide may further comprise at least one, preferably one to five 2'-O-methyl ribonucleotides at the 5'-end.
Sugar groups of the mononucleotides may be natural or modified (e.g.
synthetic) and in an open chain or ring form. Sugar groups may be comprised of mono-, di-, oligo- or poly-saccharides wherein each monosaccharide unit comprises from 3 to about 8 carbons, preferably from 3 to about 6 carbons, containing polyhydroxy groups or polyhydroxy and amino groups. Non-limiting examples include glycerol, ribose, fructose, glucose, glucosamine, mannose, galactose, maltose, cellobiose, sucrose, starch, amylose, amylopectin, glycogen and cellulose. The hydroxyl and amino groups are present as free or protected groups containing e.g.
hydrogens and/or halogens. Preferred protecting groups include acetonide, t-butoxy carbonyl groups, etc. Monosaccharide sugar groups may be of the L or D
configuration and a cyclic monosaccharide unit may contain a 5 or 6 membered ring of the a, or (3 conformation. Disaccharides may be comprised of two identical or two dissimilar monosaccharide units. Oligosaccharides may be comprised of from 2 to 10 monosaccharides and may be homopolymers, heteropolymers or cyclic polysugars.
Polysaccharides may be homoglycans or heteroglycans and may be branched or unbranched polymeric chains. The di-, oligo- and poly-saccharides may be comprised of 1 ~ 4, 1 ~ 6 or a mixture of 1 -~ 4 and 1 -j 6 linkages. The sugar moiety may be attached to the link group through any of the hydroxyl or amino groups of the carbohydrate.
Other modifications include those which are internal or are at the ends) of the oligonucleotide molecule and include additions to the molecule at the internucleoside phosphate linkages, such as cholesteryl, cholesterol, or diamine compounds with varying numbers of carbon residues between the two amino groups, and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome. Additional linkers including non-nucleoside linkers include, but are not limited to, polyethylene glycol of varying lengths, e.g., triethylene glycol, monoethylene glycol, hexaethylene glycol, (Ma et al. (1993) Nucleic Acids Res.
21:
2585-2589; Benseler et al. (1993) J. Am. Chem. Soc. 115: 8483-8484), hexylamine, and stilbene (Letsinger et al, (1995) J. Anz. Clzeni. Soc. 117: 7323-7328) or any other commercially available linker including abasic linkers or commercially available asymmetric and symmetric linkers (CloneTech, Palo Alto, California) (e.g., Glen Research Product Catalog, Sterling, VA).
Additionally oligonucleotides capped with ribose at the 3' end of the oligonucleotide may be subjected to NaI04 oxidation/reductive amination.
Amination may include but is not limited to the following moieties, spermine, spermidine, Tris(2-aminoethyl) amine (TAEA), DOPE, long chain alkyl amines, crownethers, coenzyme A, NAD, sugars, peptides, dendrimers.
Oligonucleotides may also be capped with a bulky substituent at their 3' and/or 5' end(s), or have a substitution in one or both nonbridging oxygens per nucleotide. Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule (reviewed in Agrawal et al. (1992) Treads Biotechnol. 10: 152-158).
Some non-limited examples of capped species include 3'-O-methyl, 5'-O-methyl, 2'-O-methyl, and any combination thereof.
Preferred oligonucleotides of the invention have sequences selected from the group of SEQ ID NOs: 1-37 as set forth in Table 1, including modifications thereof.
Particularly preferred oligonucleotides have sequences selected from the group consisting of SEQ ID NOs: 1, 2, 4, 1 l, 21 and 26, including the internucleotide linkage composition and further modifications as set forth in Table 1. Most preferred oligonucleotides have sequences selected from the group consisting of SEQ ID
NOs:
1,2,3,28,34:
Synthetic oligonucleotides of the invention can be prepared by art recognized methods. For example, nucleotides can be covalently linked using art-recognized techniques such as phosphoramidite, H-phosphonate chemistry, or methylphosphoramidite chemistry (see, e.g., Goodchild (1990) Bioconjugate Claem. 2:
165-187; Uhlmann et al. (1990) Chern. Rev. 90: 543-584; Caruthers et al.
(1987) Meth. Enzymol. 154: 287-313; U.S. Patent No. 5,149,798) which can be carried out manually or by an automated synthesizer and then processed (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10: 152-158). Oligonucleotides with phosphorothioate linkages can be prepared using methods well known in the field such as phosphorarnidite (see, e.g., Agrawal et al. (1988) Proc. Natl. Acad. Sci.
(USA) 85:
7079-7083) or H-phosphonate (see, e.g., Froehler (1986) Tet~~alaedron Lett.
27: 5575-5578) chemistry. The synthetic methods described in Bergot et al. (J.
Chromatog.
(1992) 559: 35-42) can also be used. Oligonucleotides with other types of modified internucleotide linkages can be prepared according to known methods (see, e.g., Goodchild (1990) Bioconjugate Chem. 2: 165-187; Agrawal et al. (1988) Proc.
Natl.
Acad. Sci. (USA) 85: 7079-7083; Uhlmann et al. (1990) Chena. Rev. 90: 534-583;
and Agrawal et al. (1992) Trends Biotechnol. 10: 152-158).
In other aspects, the invention provides a pharmaceutical composition. The pharmaceutical composition is a physical mixture of at least one, and preferably two or more HPV-specific oligonucleotides with the same or different sequences, modification(s), and/or lengths. In some embodiments, this pharmaceutical formulation also includes a physiologically or pharmaceutically acceptable carrier.
Specific embodiments include a therapeutic amount of a lipid carrier.
The oligonucleotides of the present invention are suitable for use as therapeutically active compounds, especially for use in the control or prevention of human papillomavirus infection.
In this aspect of the invention, a therapeutic amount of a pharmaceutical composition containing HPV-specific synthetic oligonucleotides is administered to a cell to inhibit human papillomavirus replication. In a similar aspect, the oligonucleotides of the present invention can be used for treating human papillomavirus infection comprising the step of administering to an infected animal or cell a therapeutic amount of a pharmaceutical composition containing at least one HPV-specific oligonucleotide, and in some embodiments, at least two HPV-specific oligonucleotides. In some preferred embodiments, the method includes administering at least one oligonucleotide, or at least two oligonucleotides, having a sequence set forth in Table 1 or in the Sequence Listing as SEQ ID NOS: 1-37 including modifications thereof.
In all methods involving the administration of oligonucleotide(s) of the invention, at least one, and preferably two or more identical or different oligonucleotides may be administered simultaneously or sequentially as a single treatment episode in the form of separate pharmaceutical compositions.
More specifically, the invention includes methods of treatment of a mammal susceptible to (prophylactic treatment) or suffering from a disease associated with viruses of the human papillomavirus family. Examples of clinical conditions which are caused by such viruses are benign skin and genital warts (condyloma acuminate), epidermodysplasia verruciformis (EV), respiratory or laryngeal papillomatosis and cervical carcinoma. Methods in of the present invention comprise administration of a therapeutically effective amount of one or more compounds of the invention to virally infected cells, such as mammalian cells, particularly human cells.

5 Administration of compounds of the invention may be made by a variety of suitable routes including oral, topical (including transdermal, buccal or sublingal), nasal and parenteral (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection) with oral or parenteral being generally preferred.
It also will be appreciated that the preferred method of administration and dosage amount 10 may vary with, for example, the condition and age of the recipient.
Compounds of the invention may be used in therapy in conjunction with other pharmaceutically active medicaments, such as another anti-viral agent, or an anti-cancer agent. Additionally, while one or more compounds of the invention may be 15 administered alone, they also may be present as part of a pharmaceutical composition in mixture with conventional excipient, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, oral or other desired administration and which do not deleteriously react with the active compounds and are not deleterious to the recipient thereof. Suitable pharmaceutically acceptable Garners include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the active compounds.
For parenteral application, particularly suitable are solutions, preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories. Ampules are convenient unit dosages.
For enteral application, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch. A syrup, elixir or the like can be used wherein a sweetened vehicle is employed. Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.

Therapeutic compounds of the invention also may be incorporated into liposomes. The incorporation can be carned out according to known liposome preparation procedures, e.g. sonication and extrusion. Suitable conventional methods of liposome preparation are also disclosed in e.g. A.D. Bangham et al., J.
Mol. Biol., 23: 238-252 (1965); F. Olson et al., Bioclzim. Biophys. Acta, 557: 9-23 (1979); F.
Szoka et al., Proc. Nat. Acad. Sci., 75:4194-4198 (1978); S. Kim et al., Biochim.
Biophys. Acta, 728: 339-348 (1983); and Mayer et al., Biochirn. Biop7~ys.
Acta, 858:
161-168 (1986).
It will be appreciated that the actual preferred amounts of active compounds used in a given therapy will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests.
All documents mentioned herein are incorporated herein by reference.
The present invention is further illustrated by the following examples. These examples are provided to aid in the understanding of the invention and are not to be construed as limitations thereof.
Example 1: Synthesis of Oligonucleotides.
Oligonucleotides were synthesized using standard phosphoramidite chemistry (Beaucage (1993) Meth. Mol. Biol. 20: 33-61) on either an ABI 394 DNA/RNA
synthesizer (Perkin-Elmer, Foster City, CA), a Pharmacia Gene Assembler Plus (Pharmacia, Uppsala, Sweden) or a Gene Assembler Special (Pharmacia, Uppsala, Sweden) using the manufacturers' standard protocols and custom methods. The custom methods served to increase the coupling time from 1.5 min to 12 min for the 2'-O-methyl RNA amidites. The Pharmacia synthesizers required additional drying of the arnidites, activating reagent and acetonitrile. This was achieved by the addition of 3 1~ molecular sieves (EM Science, Gibbstown, NJ) before installation on the machine.

DNA (3-cyanoethyl phosphoramidites were purchased from Cruachem (Glasgow, Scotland). The DNA support was 500 ~ pore size controlled pore glass (CPG) (PerSeptive Biosystems, Cambridge, MA) derivatized with the appropriate 3' base with a loading of between 30 to 40 mmole per gram. 2'-O-methyl RNA (3-cyanoethyl phosphoramidites and CPG supports (50010 were purchased from Glen Research (Sterling, VA). For synthesis of random sequences, the DNA
phosphoramidites were mixed by the synthesizer according to the manufacturer's protocol (Pharmacia, Uppsala, Sweden).
All 2'-O-methyl RNA-containing oligonucleotides were synthesized using ethylthiotetrazole (American International Chemical (AIC), Natick, MA) as the activating agent, dissolved to 0.25 M with low water acetonitrile (Aldrich, Milwaukee, WI). Some of the DNA-only syntheses were done using 0.25 M
ethylthiotetrazole, but most were done using 0.5 M 1-H-tetrazole (AIC). The thiosulfurizing reagent used in all the PS oligonucleotides was 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage Reagent, R.I. Chemical, Orange, CA, or AIC, Natick, MA) as a 2% solution in low water acetonitrile (w/v).
The cholesteryl CPG (chol) and polyethylene glycol (PEG), 5'-amino-modifier [C6NH2] and cholesteryl (chol) phosphoramidites used to synthesize oligos with linkers which were used in accordance with manufacturer's instructions (Glen Research, Sterling, VA) The 3'-NHZ Cap is a 3'-(3-amino 2-propanol) conjugate which was prepared with 3'-amino modifier C3 CPG according to manufacturer's instructions (Glen Research, Sterling, VA).
For oxidation, Redox or amination of oligonucleotide phosphorothioates containing a ribonucleotide at the 3' terminus the synthesis was carried out as follows.
Oligonucleotide phosphorothioate (1 mM) containing a ribonucleotide at the 3' terminus was oxidized with NaI04 (1.2 mM) for 30 minutes on ice in 0.1 M
sodium acetate pH 4.75 to yield the 3'-dialdehyde (Ox.) product. For addition of amines, 6 equivalents of amine in 0.2 M sodium phosphate buffer (pH 8) was added to the oxidized oligonucleotide at room temperature for 30 minutes followed by addition of 30 equivalents of NaCNBH3. The solution was left overnight at room temperature.
The product was purified by preparative polyacrylamide gel Electrophoresis on a 20%
denaturing gel. The same procedure was carried out in the absence of amine to yield the 3' diol (Ox/Red.) product.
After completion of synthesis, the CPG was air dried and transferred to a 2 mL
screw-cap microfuge tube. The oligonucleotide was deprotected and cleaved from the CPG with 2 mL ammonium hydroxide (25-30%). The tube was capped and incubated at room temperature for 20 minutes, then incubated at 55°C for 7 hours.
After deprotection was completed, the tubes were removed from the heat block and allowed to cool to room temperature. The caps were removed and the tubes were microcentrifuged at 10,000 rpm for 30 minutes to remove most of the ammonium hydroxide. The liquid was then transferred to a new 2 mL screw cap microcentrifuge tube and lyophilized on a Speed Vac concentrator (Savant, Farmingdale, NY).
After drying, the residue was dissolved in 400 ~,L of 0.3 M NaCI and the DNA was precipitated with 1.6 mL of absolute EtOH. The DNA was pelleted by centrifugation at 14,000 rpm for 15 minutes, the supernatant decanted, and the pellet dried.
The DNA was precipitated again from 0.1 M NaCl as described above. The final pellet was dissolved in 500 p,L HZO and centrifuged at 14,000 rpm for 10 minutes to remove any solid material. The supernatant was transferred to another microcentrifuge tube and the amount of DNA was determined spectrophotometrically. The concentration was determined by the optical density at 260 nM. The E26o for the DNA portion of the oligonucleotide was calculated by using OLIGSOL (Lautenberger (1991) Biotechhiques 10: 778-780). The E26o of the 2'-O-methyl portion was calculated by using OLIGO 4.0 Primer Extension Software (NBI, Plymouth, MN).
Oligonucleotide purity was checked by polyacrylamide gel Electrophoresis (PAGE) and UV shadowing. 0.2 ODz~o units were loaded with 95% formamide/H20 and Orange G dye onto a 20% denaturing polyacrylamide gel (20 cm x 20 cm). The gel was run until the Orange G dye was within one inch of the bottom of the gel. The band was visualized by shadowing with shortwave UV light on a thin layer chromatography plate (Kieselgel 60 F254, EM Separations, Gibbstown, NJ).

Some oligonucleotides were synthesized without removing the 5'-trityl group (trityl-on) to facilitate reverse-phase HPLC purification. Trityl-on oligonucleotides were dissolved in 3 mL water and centrifuged at 6000 rpm for 20 minutes. The supernatant was filtered through a 0.45 micron syringe filter (Gelman Scientific, Ann Arbor, MI) and purified on a 1.5 x 30 cm glass liquid chromatography column (Spectrum, Houston, TX) packed with C-18 p,Bondapak chromatography matrix (Waters, Franklin, MA) using a 600E HPLC (Waters, Franklin, MA). The oligonucleotide was Eluted at 5 mL/min with a 40 minute gradient from 14-32%
acetonitrile (Baxter, Burdick and,Jackson Division, Muskegon, MI) in 0.1 M
ammonium acetate (J.T. Baker, Phillipsburg, NJ), followed by 32% acetonitrile for 12 minutes. Peak detection was done at 260 nm using a Dynamax UV-C absorbance detector (Rainin, Emeryville, CA).
The HPLC purified trityl-on oligonucleotide was evaporated to dryness and the trityl group was removed by incubation in 5 mL 80% acetic acid (EM
Science, Gibbstown, NJ) for 15 minutes. After evaporating the acetic acid, the oligonucleotide was dissolved in 3 mL 0.3 M NaCI and ethanol precipitated. The precipitate was isolated by centrifugation and precipitated again with ethanol from 3 mL 0.1 M
NaCI.
The precipitate was isolated by centrifugation and dried on a Savant Speed Vac (Savant, Farmingdale, NY). Quantitation and PAGE analysis were performed as described above for ethanol precipitated oligonucleotides.
Standard phosphoramidite chemistry was applied in the synthesis of oligonucleotides containing methylphosphonate linkages using two Pharmacia Gene Assembler Special DNA synthesizers. One synthesizer was used for the synthesis of phosphorothioate portions of oligonucleotides using (3-cyanoethyl phosphoramidites method discussed above. The other synthesizer was used for introduction of methylphosphonate portions. Reagents and synthesis cycles that had been shown advantageous in methylphosphonate synthesis were applied (Hogrefe et al., in Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs (Agrawal, et al.) (1993) Humana Press Inc., Totowa, NJ). For example, 0.1 M
methyl phosphoramidites (Glen Research, Sterling, VA) were activated by 0.25 M
ethylthiotetrazole; 12 minute coupling time was used; oxidation with iodine (0.1 M) 5 in tetrahydrofuran/2,6-lutidine/water (74.75/25/0.25) was applied immediately after the coupling step; dimethylaminopyridine (DMAP) was used for the capping procedure to replace standard N-methylimidazole (NMI). The chemicals were purchased from Aldrich (Milwaukee, WI).
10 The work up procedure was based on a published procedure (Hogrefe et al.
(1993) Nucleic Acids Research 21: 2031-2038). The product was cleaved from the resin by incubation with 1 mL of ethanol/acetonitrile/ammonia hydroxide (45/45/10) for 30 minutes at room temperature. Ethylenediamine (1.0 mL) was then added to the mixture to deprotect at room temperature for 4.5 hours. The resulting solution and 15 two washes of the resin with 1 mL 50/50 acetonitrile/0.1 M triethylammonium bicarbonate (TEAB), pH 8, were pooled and mixed well. The resulting mixture was cooled on ice and neutralized to pH 7 with 6 N HCl in 20/80 acetonitrile/water (4-5 mL), then concentrated to dryness using the Speed Vac concentrator. The resulting solid residue was dissolved in 20 mL of water, and the sample desalted by using a 20 Sep-Pak cartridge. After passing the aqueous solution through the cartridge twice at a rate of 2 mL per minute, the cartridge was washed with 20 mL 0.1 M TEAB and the product Eluted with 4 mL 50% acetonitrile in 0.1 M TEAB at 2.mL per minute.
The Equate was evaporated to dryness by Speed Vac.
The crude product was purified by polyacrylamide gel Electrophoresis (PAGE) and desalted using a Sep-Pak cartridge. The oligonucleotide was ethanol precipitated from 0.3 M NaCI, then 0.1 M NaCI. The product was dissolved in p,L water and quantified by UV absorbance at 260 nm.
Example 2: Biological testing.
Human papillomaviruses (HPVs) have a tropism for squamous epithelium.
The life cycle of HPV is tightly linked to the differentiation state of the infected cells, a strict requirement which had previously made it difficult to study the virus in vitro.
The recently developed organotypic "raft" culture system, which supports the complete differentiation-specific replication cycle of HPV, concomitant with the production of infectious virion was used to study the effects of oligonucleotides of the present invention on HPV replication (Meyers, C. et al. (1992) Scieface 257:
971-973;

Meyers, C. et al. (1998) In Cell Biology: A laboratory handbook (eds. Celis, J.E.) pp.
513-520, Academic Press, Inc. Orlando, FL; Meyers, C. et al. (1992) Papillo~aavirus Res. 3: 1-3; Meyers, C. et al. (1993) In Current Topics in Microbiology and Immunology, Vol on Human pathogenic papillomaviruses (ed. zur Hausen) Springer-Verlag, NewYork; Bedell, M.A. et al. (1991) J. Virol. 65: 2254-2260; Meyers, C.
(1996) Virology 248: 218-230).
Cells and assembly of raft cultures: The CIN-612 cell line was established from a cervical intraepithelial neoplasia (CIN) type I biopsy and contains HPV3lb DNA, of which the 9E clonal derivative maintains episomal copies of HPV3lb genome at approximately 50 copies per cell. CIN-612 9E cells were maintained in monolayer culture with E medium containing 5% fetal bovine serum in the presence of mitomycin C-treated J2 3T3 feeder cells. J2 3T3 cells were maintained in DMEM
containing 5% newborn calf serum. Dermal equivalents were prepared in 6 well culture dishes using rat tail type I collagen, culture media and fibroblasts.
The fibroblasts condition the system and provide necessary stromal influence for epithelial growth and stratification. CIN-612 9E epithelial cells were counted and 600,000 cells were seeded onto the collagen plugs submerged under E medium. Epithelial cells were allowed to reach confluence, media was removed, and the collagen matrices were lifted onto stainless steel grids. Subsequent feeding of the epithelium was via diffusion of E medium from below the matrix. Epithelial tissues were allowed to stratify and differentiate at the air-liquid interface over a 10-day period.
At the end of the 10 days, the epithelium was harvested by separating from the collagen layer and stored in -20°C until further manipulations.
Treatment of raft cultures with 2.5 N,M and 25 p.M HPV/L1-HPV/L25 and ORI
1001 (SEQ ID NOs: 1-37): Raft cultures were fed every other day with E medium containing the test compounds. Each oligonucleotide (SEQ ID NOs: 1-37) was dissolved in sterile water to obtain 5 mM stock solutions. For each oligonucleotide tested 12 mL E medium was added to a 15 mL Falcon tube. Each stock solution of HPV/L1-HPV/L25 and ORI 1001 (SEQ ID NOs: 1-37) was then added to the medium to obtain a final concentration of 25 N.M. Another set of raft cultures were treated with HPV/Ll-HPV/L7 and ORI 1001 (SEQ ID NOs: 1-7 and 26) at a final concentration of 2.5 p.M. Media was removed from the raft cultures via aspiration and freshly prepared medium was then added being careful not to get any fluid on top of the raft tissues, as it retards epithelial stratification. Mock raft cultures were fed with E media only.
Nucleic acid extractions. Nucleic Acid extractions were preformed as previously described (Ozbun, M.A. et al. (1998) Virology 248: 218-230). Total cellular DNA was harvested by incubating raft tissues overnight at 55°C
in 10 mM
Tris-HCl, pH 7.5, 25 mM EDTA, 0.2% SDS and 50 mg/mL RNase A. 100 mg/mL
Proteinase K was then added and further incubated for 4 hours. The DNA was sheared by passaging 10 times through an 18-gauge needle. The solution was extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and then extracted once with an equal volume of chloroform-isoamyl alcohol (24:1).
The DNA was ethanol precipitated using 0.3 M sodium acetate. The DNA was dried and dissolved in TE and sample concentrations were established by determining optical densities. Concentrations were verified by electrophoresis through agarose gels and staining with ethidium bromide.
Southern blotting and hybridization. Southern blotting to detect HPV3lb DNA was preformed as previously described (Ozbun, M.A. et al. (1998) Virology 248: 218-230). Total cellular DNA samples (5 p,g) were digested overnight with ~Ybal, which linearizes the HPV3lb genome at nucleotide 4998. Total cellular DNA
samples were separated on 0.8% agarose gels. The DNA was transferred to GeneScreen Plus membranes (New England Nuclear Research Products, Boston, Massachusetts), which were handled according to the manufacturer's instructions.
For probe preparation plasmid pBS-HPV31 was digested with EcoRI to release the complete HPV31 genome, and the HPV31 sequences were purified from plasmid sequences by agarose gel electrophoresis and Gene Clean (Bio101, Vista California).
DNA sequences were labeled with [a-32P] dCTP (3,000 Ci/mmol; DuPont NEN), using a Random Primed DNA labeling kit (Boehringer Mannheim Corp., Indianapolis, Indiana) according to the manufacturer's instructions. Labeled probe was separated from unincorporated nucleotides by centrifugation through a Sephadex G-50 column (Boehringer Mannheim Corp.). Hybridization was carried out with cpm per mL of probe. Membranes were washed to remove nonspecific hybridization and then exposed to film. The intensity of HPV3lb replication in treated samples was then compared to the mock treated control DNA to determine the impact of each treatment of viral replication. Densitometric analysis of the Southern blots was performed to numerically compare the effect of the compounds on HPV3lb replication to untreated control samples. The levels of replication were also compared to standard copy number controls to numerically determine the effect of the treatments on the number of HPV3lb genome per cell.
Histochemical analysis. To determine the impact of the drug treatments on raft tissue differentiation and morphology, tissue histologic analysis was performed.
Raft cultures were grown in the presence of various treatments for 10 days, harvested, fixed in 4% paraformaldehyde, and embedded in paraffin. 4- ~M cross-sections were prepared. Sections were stained with hematoxylin and eosin.
RESULTS
Results from the Southern Blotting analysis can be seen in Table 1 and Figure 2.
Table 1 shows the effect of each of the oligonucleotides (SEQ I.D NOs: 1.-37) on the replication of HPV3lb presented as relative densitometric analysis as compared to the level of HPV3lb replication which occurred in mock treated cells.
The oligonucleotides varied in their ability to decrease HPV3lb replication as compared to the mock treated cells. Particularly potent oligonucleotides at 25 ~.M
were HPV/L1, HPV/L2, HPV/L4, HPV/Ll 1, HPV/L21 and ORI 1001 (SEQ ID NOs:
l, 2, 4, 11, 21 and 26). HPV/L1 (SEQ ID NO: 1) showed the greatest inhibitory effect of HPV3lb replication at both concentrations tested, achieving close to a 3-fold reduction in viral replication at 25 ~,M and close to a 5-fold reduction in viral replication at 2.5 wM. Some of the compounds actually enhanced the replication of HPV3lb compared to mock controls.
FIG 2. A-E: C1N-612 9E raft cultures were treated with 25 ~.M (final concentration) of oligonucleotides of the present invention HPV/Ll-HPV/L25 and ORI 1001 (SEQ ID NOs: 1-37) and their effect on HPV3lb genome replication was determined. Southern blot hybridization of total DNA isolated from treated CIN-9E raft cultures is shown. 5 ~g of total DNA from each raft was digested with Hin.dIII

to linearize episomal HPV3lb DNA, electrophoresed on a 0.8% agarose gel, transferred to nylon membrane, followed by hybridization with a HPV3lb specific probe. Lane 1 of each compound tested consists of uncut DNA. Lane 2 of each compound tested consists of HindIIl digested episomal DNA. Form I (FI) indicates supercoiled DNA, Form II (FII) indicates nicked circular DNA and Form III
(FIII) indicates linearized DNA.

Table 1. Effect of Oligonucleotides of the present invention (SEQ ID NOs: 1-34) on HPV3lb replication in raft cultures. Oligonucleotides were tested at 2.5 mM and 25 mM
final concentrations.
Effects of treated cells were compared to mock treated cells in a densitometric analysis of Southern blots of total DNA isolated from the raft cells probed with an HPV3lb specific probe.
SEQ Oligo Relative ID Compound Seguence Length Density NO 2.5 pM
25 p,M

Mock 1 1 1 HPV/L1 TCC GCC AU-3 8 0.218 0.350 2 HPV/L2 TCC GCC AT-3 8 1.474 0.65 3 HPV/L3 5 ~GTA CCT GAA 9 0.702 0.995 4 HPV/L4 5 ~GT'A CCT G 7 1.659 0.503 5 HPVlLS GAA TCG TCC GCC 12 0.699 0.98 6 HPV/L6 A TCG TCC GCC AU-3 12 0.825 1.83 7 HPV/L7 A CCT GAA TCG TCC GCC AU-3 18 1.249 0.714 8 HPV/L8 CT GAA TCG TCC GCC AU-3 16 ND 0.653 9 HPV/L9 GAA TCG TCC GCC AU-3 14 ND 1.02 10 HPV/L10 CG TCC GCC AU-3 10 ND 1.819 11 HPV/L11 TCG TCC GCC AU-3 11 ND 0.5 12 HPV/L12 UCG UCC GCC AU-3 11 ND 2.187 13 HPV/L13 TCG TCC GCC AT-3 11 ND 3.65 14 HPV/L14 C GCC AU-3 6 ND 1.232 15 HPV/L15 3 ~GTA CCT GAA TCG TCC GCC 18 ND 0.76 16 HPV/L16 3 ~GTA CCT GAA TCG TCC 15 ND 1.056 17 HPV/L17 5 -GTA CCT GAA TGG TC-3 14 ND 0.836 18 HPV/L18 5 -GTA CCT GAA TCG ~ 12 ND 1.79 19 HPV/L19 5 ~GTA CCT GAA T 10 ND 1.344 20 HPV/L20 5 ~GTA CCT GA 8 ND 1.504 21 HPVlL21 5 ~GTA CCT 6 ND 0.605 22 HPV/L22 A CCT GAA TCG TCC GCC 16 ND 0.844 23 HPV/L23 CT GAA TCG TCC 11 ND 0.976 24 HPV/L24 GAA TCG TC 8 ND 0.772 25 HPV/L25 A TCG TC 6 ND 1.208 26 ORI-1001 3 ~GTA CCT GAA TCG TCC GCC 20 0.716 0.533 27 ORI-2027 5'-GCC AU-3 28 ORI-2028 5'-CC AU-3 29 ORI-2029 S -CAU-3' ORI-2030 5'TCC GCCA-3' 31 ORI-2031 5'TCC GCC-3' 32 ORI-2032 5'TCC _GC-3' 33 ORI-2033 3 TCC _G-3' 34 ORI-2034 5'-CCT ACCAU-3' 36 5 ~GX ACC XGA AXC GXC CGC

37 5 -GTA CCU GAA-3' ~

ND = not determined X = thymidine or uracil and any nucleotide may be substituted with inosine.
underlined nucleotides are 2'-O-methyl ribonucleotides, all other nucleotides are unmodified deoxyribonucleotides.
15 Internucleotide linkages are phosphorothioate The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention as set forth in the following claims.

SEQUENCE LISTING
<110> ORIGENIX TECHNOLOGIES, INC.
<120> HPV-SPECIFIC SHORT-MFRS
c130> 49622-PCT (71956) <140> PCT/US01/40501 <141> 2001-04-11 c150> 60/195,996 c151> 2000-11-04 <160> 37 <170> PatentIn Ver. 2.1 <210> 1 <211> 8 <212> DNA
<213> Artificial Sequence <220>
c223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1 tccgccau 8 <210> 2 <211> 8 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 tccgccat 8 <210> 3 <211> 9 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide ~~TB~TIT~J'I'E SHEET ~R~TLE ~~~

<400> 3 gtacctgaa <210> 4 <211> 7 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 gtacctg <210> 5 <211> 12 <212 > DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 gaatcgtccg cc . 12 <210> 6 <211> 12 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 atcgtccgcc au 12 <210> 7 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 acctgaatcg tccgccau 18 <210> 8 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide c220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 8 ctgaatcgtc cgccau 16 <210> 9 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide c400> 9 gaatcgtccg ccau 14 <210> 10 <211> 10 <212> DNA
<213> Artificial Sequence ' <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 cgtccgccau 10 <210> 11 <211> 11 <212> ANA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220> , <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 11 tcgtccgcca a 11 <210> 12 <211> 11 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 12 ucguccgcca a 11 <210> 13 <211> 11 <212 > DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 13 tcgtccgcca t 11 <210> 14 <211> 6 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 14 cgccau <210> 15 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 15 gtacctgaat cgtccgcc 18 <210> 16 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 16 gtacctgaat cgtcc 15 <210> 17 <211> 14 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 17 gtacctgaat cgtc 14 <210> 18 <211> 12 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 gtacctgaat cg 12 <210> 19 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> is gtacetgaat 10 <210> 20 <211> 8 <212 > DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 20 gtacctga <210> 21 <211> 6 <212> DNA
c213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 21 gtacct g <210> 22 <211> 16 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 acctgaatcg tccgoc 16 <210> 23 <211> 11 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide c400> 23 ctgaatcgtc c 11 <210a 24 <211> 8 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 24 gaatcgtc 8 <210> 25 <211> 6 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 atcgtc 6 <210> 26 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 26 gtacctgaat cgtccgccau 20 <210> 27 <211> 5 <212> RNA
<213~ Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 27 gccau 5 <210> 28 <211> 4 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 28 ccau 4 <210> 29 <211> 3 <212> RNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 29 cau 3 <210> 30 <211> 7 <222> DNA
<213>'Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 30 tccgcca c210> 31 <211> 6 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 31 tccgcc <210> 32 <211> 5 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 32 tccgc <210> 33 <211> 4 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 33 tccg <210> 34 <211> 8 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 34 cctaccau <210> 35 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 35 atggcggacg attcaggtac 20 <210> 36 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <220>
<221> modified_base <222> (2) <223> Thymidine or uracil and may be substituted with inosine <220>
<221> modified_basa <222> (6) <223> Thymidine or uracil and may be substituted with inosine c220>
<221> modified_base <222> (10) <223> Thymidine or uracil and may be substituted with inosine <220>
<221> modified_base <222> (13) <223> Thymidine or uracil and may be substituted with inosine <220>
<221> modified_base <222> (20) <223> Thymidine or uracil and may be substituted with inosine <400> 36 gnaccngaan cgnccgccan 20 c210> 37 <211> 9 <212> DNA
<213> Artificial. Sequence <220>
<223> Description of Combined DNA/RNA Molecule:
Synthetic oligonucleotide <220>
<223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 37 gtaccugaa

Claims (43)

What is claimed is:

1. An oligonucleotide complementary to a portion of a human papillomavirus E1 open reading frame contained within nucleotide +1 to nucleotide +20, and including at least 4 nucleotides.

2. The oligonucleotide of claim 1, wherein the oligonucleotide is modified.

3. The oligonucleotide of claim 2, wherein at least one modification occurs in the internucleotide linkage.

4. The oligonucleotide of claim 3, wherein the modification comprises at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester linkages, and combinations thereof.

5. The oligonucleotide of claim 4, wherein the modification comprises at least one phosphorothioate internucleotide linkage.

6. The oligonucleotide of claim 5, wherein all of the internucleotide linkages are phosphorothioate internucleotide linkages.

7. The oligonucleotide of claim 2 which comprises at least one deoxyribonucleotide.

8. The oligonucleotide of claim 2 which comprises at least one ribonucleotide.

9. The oligonucleotide of claim 2 which comprises at least one deoxyribonucleotide and at least one ribonucleotide.

10. The oligonucleotide of claim 8 comprising at least one 2'-O-methyl nucleotide.

11. The oligonucleotide of claim 2 comprising at least one inosine nucleotide.

12. The oligonucleotide of claim 2 which comprises at least one modification to the sugar moiety of at least one mononucleotide.

13. The oligonucleotide of claim 2 which comprises at least one modification to the base moiety of at least one mononucleotide.

14. The oligonucleotide of claim 2 which comprises a cap at the 5' end.

15. The oligonucleotide of claim 2 which comprises a cap at the 3' end.

16. The oligonucleotide of claim 2 which comprises a cap the 5' end and the 3' end.

17. The oligonucleotide of claim 2 which comprises the sequence of SEQ ID
NO: 36 or a portion thereof.

18. The oligonucleotide of claim 2 which comprises a sequence selected from the group consisting of any one or more of SEQ ID NOs: 1 through 37.

19. The oligonucleotide of claim 2 which comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 11, 21, 26, 28 and 34.

20. The oligonucleotide of claim 2 which comprises SEQ ID NO: 1.

21. The oligonucleotide of claim 2 which comprises SEQ ID NO: 2.

22. The oligonucleotide of claim 2 which comprises SEQ ID NO: 3.

23. The oligonucleotide of claim 2 which comprises SEQ ID NO: 28.

24. The oligonucleotide of claim 2 which comprises SEQ ID NO: 34.

25. The oligonucleotide of claim 2 which is capable of inhibiting the replication of HPV.

26. The oligonucleotide of claim 25, wherein the inhibition is due to interaction of the oligonucleotide with a nucleic acid.

27. The oligonucleotide of claim 25, wherein the inhibition is due to interaction of the oligonucleotide with a protein.

28. The oligonucleotide of claim 27 wherein the protein is a receptor.

29. The oligonucleotide of any one of claims 1 through 28 wherein the oligonucleotide has a total of 4 nucleotides.

30. The oligonucleotide of any one of claims 1 through 28 wherein the oligonucleotide has a total of 18 nucleotides.

31. The oligonucleotide of any one of claims 1 through 28 wherein the oligonucleotide has a total of from 6 to 18 nucleotides.

32. An oligonucleotide comprising a sequence of any one of SEQ ID NOS.
1 through 25 or SEQ ID NOS. 27-37.

33. An oligonucleotide that consists of a sequence of SEQ ID NOS. 1 through 25 or SEQ ID NOS. 27-37.

34. The oligonucleotide of any one of claims 1 through 33 wherein the oligonucleotide is a synthetic oligonucleotide.

35. A pharmaceutical composition comprising at least one oligonucleotide of any one of claims 1 through 34 and a pharmaceutically acceptable carrier.

36. A method for treating a mammal infected with a papillomavirus comprising administering to the mammal at least one oligonucleotide or composition of any one of claims 1 through 35.

37. The method of claim 36 wherein the mammal is a human.

38. The method of claim 36 or 37 wherein the oligonucleotide is administered in combination with another anti-viral compound.

39. A method for treating a mammal suffering from an HPV-associated disorder or disease comprising administering to the mammal an oligonucleotide or composition of any one of claims 1 through 35.

40. The method of claim 39 wherein the mammal is a human.

41. The method of claim 39 or 40 wherein the HPV-associated disorder or disease is benign skin warts, genital warts, epidermodysplasia verruciformis, respiratory papillomatosis, laryngeal papillomatosis or cervical carcinoma.

42. The method of claim 41 wherein the HPV-associated disorder or disease is associated with infection by HPV strains 6, 11, 16, 18, 31, 45, or 58.

43. A synthetic oligonucleotide capable of inhibiting HPV replication by a mechanism other than an antisense mechanism.