WO2001077294A2 - Oligonucleotides meres specifiquement courts de hpv - Google Patents

Oligonucleotides meres specifiquement courts de hpv Download PDF

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Publication number
WO2001077294A2
WO2001077294A2 PCT/US2001/040501 US0140501W WO0177294A2 WO 2001077294 A2 WO2001077294 A2 WO 2001077294A2 US 0140501 W US0140501 W US 0140501W WO 0177294 A2 WO0177294 A2 WO 0177294A2
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oligonucleotide
hpv
seq
oligonucleotides
replication
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PCT/US2001/040501
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English (en)
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WO2001077294A3 (fr
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Radhakrishnan Iyer
Yi Jin
Wenqiang Zhou
Arlene Roland
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Origenix Technologies, Inc.
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Priority to AU2001257604A priority Critical patent/AU2001257604A1/en
Priority to JP2001575148A priority patent/JP2004501614A/ja
Priority to EP01931140A priority patent/EP1315507A4/fr
Priority to CA002406485A priority patent/CA2406485A1/fr
Publication of WO2001077294A2 publication Critical patent/WO2001077294A2/fr
Publication of WO2001077294A3 publication Critical patent/WO2001077294A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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 papillomavirus- associated disorders or diseases.
  • the invention provides short, synthetic oligonucleotides which are useful for inhibiting replication of HPV.
  • HPV Human papillomaviruses
  • 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. Cancer 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.
  • HPV types There are at least 70 different types of human papillomaviruses based on DNA sequence diversity as measured by liquid hybridization (Pfister et al. (1994) Intervirol. 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-11. The malignant types may integrate into the genome of the host cell, thereby eliminating the requirement for viral DNA replication gene products.
  • HPV-6 and HPV-11 rely on viral proteins El and E2 for replication of the episomal genome.
  • 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 (URR), which contains the origin of viral DNA replication and enhancers and promoters involved in transcription; the L region that encodes the structural proteins, LI 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, LI and L2, (shown schematically in FIG. 1) that are translated from complex families of alternatively spliced mRNAs.
  • El 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; Jenkins, O. et al. (1996) J.
  • 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 Biochem. Sci. 16: 440-444; Liu, J.-S. et al. (1995) J. Biol. Chem. 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) EMBOJ. 6: 989-992; Bedel, M.A. et al. (1989) J. Virol. 63: 1247-1255; Matlashewski, E. et al. (1987) EMBOJ. 6: 1741-1746; Vousden, K.H.
  • E5 is a highly hydrophobic protein which interacts with the epidermal growth factor receptor (Chen, S .-L. et al. (1990) J. Virol. 64: 3226- 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).
  • 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
  • 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. Surg. (2001) Jan;5(l):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. 801- 803).
  • 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) Intervirol. 37: 226-230; Bornstein et al. (1993)
  • HPV HPV
  • oncogenic potential such that over 99% of all cervical cancers and over 50% of other anogenital cancers are due to infection with oncogenic HPV.
  • 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.
  • 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. (USA) (1989) 86: 7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
  • chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed.
  • Pederson et al. U.S. Patent Nos. 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. discloses hybrid oligonucleotides having regions of deoxyribonucleotides and 2'-0-methyl-ribonucleotides.
  • El 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 El protein binds to the ori and unwinds DNA in the presence of the host single- stranded DNA binding protein RPA and topoisomerase I.
  • SV40 simian virus 40
  • the human papillomavirus (HPV) and bovine papillomavirus 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 ⁇ , thereby bringing host replication proteins to the unwound ori.
  • oligonucleotides which inhibit the expression of HPV.
  • 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).
  • oligonucleotides directed to the +1 to +20 region of the El 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 El 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 HPV31b specific probe to determine the level of HPV replication in the cultures.
  • the CIN-612 9E cultures were treated with 25 ⁇ 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 HP V3 lb specific probe to determine the level of HPV replication in the cultures.
  • the CIN-612 9E cultures were treated with 2.5 ⁇ M of oligonucleotides of the present invention and the level of inhibition of viral replication was compared to a mock treated culture.
  • the invention provides synthetic oligonucleotides complementary to a nucleic acid spanning the translational start site of human papillomavirus gene El .
  • the nucleic acid spanning the translational start site of human papillomavirus gene El is intended to indicate the region of the El 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 tea ggt ac-3') and the oligonucleotide complementary to this region has the sequence as set forth in SEQ ID NO: 36 (5'-gX ace Xga aXc gXc cgc caX-3'), wherein X may be thymidine or uracil and any nucleotide may be substituted with inosine.
  • 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.
  • 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.
  • 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 mRNA 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 El open reading frame spanning nucleotides +1 to +20, or a portion thereof.
  • the oligonucleotides comprise from about 4 and up to about 20 mononucleotides, more preferably from about 4 to about 12, 14, 16 or 18 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, 28 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.
  • 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.
  • 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, 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 18 carbon atoms, more preferably from 1 to about 12 carbon atoms and most preferably from 1 to about 6 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.
  • 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.
  • 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'-0-substituted with an -O- 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, carbalkoxyl, or amino groups), or wherein the 2'-0-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'-0-methyl ribonucleotides (2'-
  • 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'-0-methyl ribonucleotides at the 3' end of the oligonucleotide. Moreover, the oligonucleotide may further comprise at least one, preferably one to five 2'-0-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.
  • 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 ⁇ or ⁇ 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 - 6 linkages.
  • the sugar moiety may be attached to the link group through any of the hydroxyl or amino groups of the carbohydrate.
  • modifications include those which are internal or are at the end(s) 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.
  • oligonucleotides capped with ribose at the 3' end of the oligonucleotide may be subjected to NaI0 4 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) Trends Biotechnol. 10: 152-158). Some non-limited examples of capped species include 3'-0-methyl, 5'-0-methyl, 2'-0- 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, 11, 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.
  • nucleotides can be covalently linked using art-recognized techniques such as phosphoramidite, H-phosphonate chemistry, or methylphosphoramidite chemistry (see, e.g., Goodchild (1990) Bioconjugate Chem. 2: 165-187; Uhlmann et al. (1990) Chem. 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.
  • Oligonucleotides with phosphorothioate linkages can be prepared using methods well known in the field such as phosphoramidite (see, e.g., Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:
  • 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.
  • this pharmaceutical formulation also includes a physiologically or pharmaceutically acceptable carrier. Specific embodiments include a therapeutic amount of a lipid carrier.
  • 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.
  • a therapeutic amount of a pharmaceutical composition containing HPV-specific synthetic oligonucleotides is administered to a cell to inhibit human papillomavirus replication.
  • 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.
  • 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.
  • 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.
  • 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.
  • viruses of the human papillomavirus family examples include 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.
  • 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 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 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.
  • 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 carriers 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.
  • 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.
  • solutions preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories.
  • Ampules are convenient unit dosages.
  • talc and/or carbohydrate carrier binder or the like are particularly suitable 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 carried out according to known liposome preparation procedures, e.g. sonication and extrusion. Suitable conventional methods of liposome preparation are also disclosed in e.g.
  • 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'-0-methyl RNA amidites. The Pharmacia synthesizers required additional drying of the amidites, activating reagent and acetonitrile.
  • DNA ⁇ -cyanoethyl phosphoramidites were purchased from Cruachem (Glasgow, Scotland).
  • the DNA support was 500 A 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.
  • CPG pore size controlled pore glass
  • 2'-0-methyl RNA ⁇ - cyanoethyl phosphoramidites and CPG supports 500 A were purchased from Glen Research (Sterling, VA).
  • the DNA phosphoramidites were mixed by the synthesizer according to the manufacturer's protocol (Pharmacia, Uppsala, Sweden).
  • 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 thiosulfurizmg reagent used in all the PS oligonucleotides was 3H-l,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).
  • cholesteryl CPG chol
  • PEG polyethylene glycol
  • 5'-amino- modifier [C 6 NH 2 ]
  • cholesteryl (chol) phosphoramidites used to synthesize oligos with linkers which were used in accordance with manufacturer's instructions (Glen Research, Sterling, VA)
  • the 3'-NH 2 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).
  • Oligonucleotide phosphorothioate (1 mM) containing a ribonucleotide at the 3' terminus was oxidized withNaI0 4 (1.2 mM) for 30 minutes on ice in 0.1 M sodium acetate pH 4.75 to yield the 3'-dialdehyde (Ox.) product.
  • 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 NaCNBH 3 . 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.
  • 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.
  • 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 NaCl 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 ⁇ L H 2 0 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 E 26 o for the DNA portion of the oligonucleotide was calculated by using OLIGSOL (Lautenberger (1991) Biotechniques 10: 778-780). The E 26 o of the 2'-0-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 OD 26 o units were loaded with 95% formamide/H 2 0 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.
  • PAGE polyacrylamide gel Electrophoresis
  • 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 ⁇ 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).
  • 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,
  • the oligonucleotide was dissolved in 3 mL 0.3 M NaCl and ethanol precipitated. The precipitate was isolated by centrifugation and precipitated again with ethanol from 3 mL 0.1 M NaCl. 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 ⁇ -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.
  • the resulting mixture was cooled on ice and neutralized to pH 7 with 6 N HC1 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 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
  • oligonucleotide was ethanol precipitated from 0.3 M NaCl, then 0.1 M NaCl.
  • the product was dissolved in 400 ⁇ L water and quantified by UV absorbance at 260 nm.
  • Example 2 Biological testing.
  • HPVs Human papillomaviruses
  • HPVs Human papillomaviruses
  • 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) Science 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) Papillomavirus
  • the CIN-612 cell line was established from a cervical intraepithelial neoplasia (CIN) type I biopsy and contains HPV31b DNA, of which the 9E clonal derivative maintains episomal copies of HP V3 lb 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.
  • 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/ L 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 HPV3 lb DNA was preformed as previously described (Ozbun, M.A. et al. (1998) Virology 248: 218-230). Total cellular DNA samples (5 ⁇ g) were digested overnight with Xbal, which linearizes the HPV3 lb 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.
  • 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 (BiolOl, Vista California). DNA sequences were labeled with [ ⁇ - 32 P] 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 10 6 cpm per mL of probe.
  • Membranes were washed to remove nonspecific hybridization and then exposed to film. The intensity of HPV31b 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 HPV31b 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 HPV3 lb genome per cell.
  • Table 1 shows the effect of each of the oligonucleotides (SEQ ID NOs: 1-37) on the replication of HP V3 lb presented as relative densitometric analysis as compared to the level of HPV31b replication which occurred in mock treated cells.
  • the oligonucleotides varied in their ability to decrease HPV3 lb replication as compared to the mock treated cells.
  • Particularly potent oligonucleotides at 25 ⁇ M were HPV/L1, HPV/L2, HPV/L4, HPV/L11, HPV/L21 and ORI 1001 (SEQ ID NOs: 1, 2, 4, 11, 21 and 26).
  • HPV/L1 (SEQ ID NO: 1) showed the greatest inhibitory effect of HP V3 lb 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 ⁇ M. Some of the compounds actually enhanced the replication of HPV31b compared to mock controls.
  • FIG. 1 CIN-612 9E raft cultures were treated with 25 ⁇ M (final concentration) of oligonucleotides of the present invention HPV/L1-HPV/L25 and ORI 1001 (SEQ ID NOs: 1-37) and their effect on HPV31b genome replication was determined.
  • Southern blot hybridization of total DNA isolated from treated CIN-612 9E raft cultures is shown. 5 ⁇ g of total DNA from each raft was digested with Hindlll to linearize episomal HPV31b DNA, electrophoresed on a 0.8% agarose gel, transferred to nylon membrane, followed by hybridization with a HPV31b specific probe. Lane 1 of each compound tested consists of uncut DNA. Lane 2 of each compound tested consists of Hindlll digested episomal DNA. Form I (FI) indicates supercoiled DNA, Form II (FII) indicates nicked circular DNA and Form III (Fill) indicates linearized DNA.
  • FI indicates supercoiled DNA
  • Form II FII
  • X thymidine or uracil and any nucleotide may be substituted with inosine.
  • underlined nucleotides are 2'-0-methyl ribonucleotides, all other nucleotides are unmodified deoxyribonucleotides.
  • Internucleotide linkages are phosphorothioate

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Abstract

La présente invention concerne des oligonucléotides capables d'inhiber la réplication du papillomavirus (HPV). Cette invention concerne plus spécifiquement des oligonucléotides complémentaires au site de départ de traduction de la phase de lecture ouverte du HPV E1 qui inhibe la réplication du HPV par interaction avec un acide nucléique ou avec une cible protéinique. Les oligonucléotides de cette invention contiennent au moins 4 désoxyribonucléotides et/ou ribonucléotides et peuvent inclure diverses modifications des liaisons internucléotidiques ou des mononucléotides. Cette invention concerne aussi des compositions pharmaceutiques et des techniques de traitement des pathologies ou des maladies associées à l'infection par le HPV.
PCT/US2001/040501 2000-04-11 2001-04-11 Oligonucleotides meres specifiquement courts de hpv WO2001077294A2 (fr)

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EP01931140A EP1315507A4 (fr) 2000-04-11 2001-04-11 Oligomers courts specifique au hpv
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP1399457A1 (fr) * 2001-06-07 2004-03-24 Isis Pharmaceuticals, Inc. Procedes de purification d'oligonucleotides
WO2011144216A1 (fr) * 2010-05-19 2011-11-24 Tartu Ulikool (University Of Tartu) Procédé et trousse pour l'identification de composés capables d'inhiber la réplication du papillomavirus humain

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WO1995004748A1 (fr) * 1993-08-09 1995-02-16 Isis Pharmaceuticals, Inc. Oligomeres servant a moduler des processus viraux
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA

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US5457189A (en) * 1989-12-04 1995-10-10 Isis Pharmaceuticals Antisense oligonucleotide inhibition of papillomavirus
US6509149B2 (en) * 1995-06-06 2003-01-21 Hybridon, Inc. HPV-specific oligonucleotides

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Publication number Priority date Publication date Assignee Title
EP0425995A2 (fr) * 1989-11-03 1991-05-08 Abbott Laboratories Utilisation des amorces oligomnucléotidiques conservées pour l'amplification des séquences d'ADN du virus papilloma humain
US5863717A (en) * 1989-11-03 1999-01-26 Abbott Laboratories Use of conserved oligonucleotide primers to amplify human papillomavirus DNA sequences
WO1995004748A1 (fr) * 1993-08-09 1995-02-16 Isis Pharmaceuticals, Inc. Oligomeres servant a moduler des processus viraux
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA

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LEWIS ET AL. ANTIVIRAL RESEARCH vol. 34, 1997, page A72, ABSTRACT NO. 106, XP002949116 *
ROBERTS ET AL. ANTIVIRAL RESEARCH vol. 34, 1997, page A71, ABSTRACT NO. 105, XP002949115 *
See also references of EP1315507A2 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1399457A1 (fr) * 2001-06-07 2004-03-24 Isis Pharmaceuticals, Inc. Procedes de purification d'oligonucleotides
EP1399457A4 (fr) * 2001-06-07 2006-08-23 Isis Pharmaceuticals Inc Procedes de purification d'oligonucleotides
WO2011144216A1 (fr) * 2010-05-19 2011-11-24 Tartu Ulikool (University Of Tartu) Procédé et trousse pour l'identification de composés capables d'inhiber la réplication du papillomavirus humain

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