JP2004501614A - HPV-specific shortmers - Google Patents

Abstract

The present invention provides an oligonucleotide capable of inhibiting the replication of human papillomavirus (HPV). More specifically, the present invention provides oligonucleotides complementary to the translation start site of the HPV EEl open reading frame that inhibit HPV replication through the interaction of nucleic acids or proteins with targets. The oligonucleotides of the present invention comprise at least four deoxyribonucleotides and / or ribonucleotides and may contain various modifications to internucleotide linkages or mononucleotides. Further, the present invention provides pharmaceutical compositions and methods for treating a disorder or disease associated with an HPV infection.

Description

ＮＤ＝決定されず。
Ｘ＝チミジンまたはウラシル、そして任意のヌクレオチドが、イノシンで置換され得る。
下線を付したヌクレオチドは、２’−Ｏ−メチルリボヌクレオチドであり、他のすべてのヌクレオチドは、未改変デオキシリボヌクレオチドである。
ヌクレオチド間結合は、ホスホロチオエートである。
【００７９】
本発明を、その好ましい実施形態を参照して、詳細に記載した。しかし本開示を考慮して、当業者が、添付の特許請求の範囲に示される本発明の意図および範囲内にある改変および改良をなし得ることが理解される。
【図面の簡単な説明】
【図１】図１は、ＨＰＶゲノムの模式図である。
【図２】図２Ａ〜Ｅは、培養物中におけるＨＰＶ複製のレベルを決定するためにＨＰＶ３１ｂ特異的プローブとハイブリダイズされた、ＣＩＮ−６１２　９Ｅ　ｒａｆｔ培養細胞から単離された全ＤＮＡのサザンブロットハイブリダイゼーションを示す。ＣＩＮ−６１２　９Ｅ培養物を、２５μＭの本発明のオリゴヌクレオチドで処理し、そしてウイルス複製の阻害レベルを偽性（Ｍｏｃｋ）処理培養物と比較した。
【図３】図３Ａ〜Ｂは、培養物中におけるＨＰＶ複製のレベルを決定するためにＨＰＶ３１ｂ特異的プローブとハイブリダイズされた、ＣＩＮ−６１２　９Ｅ　ｒａｆｔ培養細胞から単離された全ＤＮＡのサザンブロットハイブリダイゼーションを示す。ＣＩＮ−６１２　９Ｅ培養物を、２．５μＭの本発明のオリゴヌクレオチドで処理し、そしてウイルス複製の阻害レベルを偽性（Ｍｏｃｋ）処理培養物と比較した。[0001]
(Background of the Invention)
(1. Field of the Invention)
The present invention relates to human papillomavirus (HPV). More particularly, the present invention relates to the inhibition and treatment of human papillomavirus infection, including the treatment and prevention of disorders or diseases associated with human papillomavirus. The present invention provides short synthetic oligonucleotides that are useful for inhibiting HPV replication.
[0002]
(2. Background)
Human papillomavirus (HPV) infects epithelial cells, causing benign skin and genital warts (condyloma acuminatum) and verrucous epidermal dysplasia (EV) to respiratory or pharyngeal papillomatosis and cervix. It is a DNA virus that causes lesions that extend to cancer. Cervical cancer is the second most prevalent type of cancer affecting women, with 400,000 to 500,000 new cases reported each year and 200,000 deaths (Parkin). (1993) Int. J. Cancer 54: 594-606). Newborns can also become infected with HPV while passing through the mother's birth canal, leading to pharyngeal papillomas or benign epithelial tumors of the pharynx. Papillomas develop in HPV-infected infants up to two years of age and necessitate multiple surgeries to remove benign papillomas that can block the airways.
[0003]
Based on DNA sequence diversity as measured by liquid hybridization, there are at least 70 different types of human papillomavirus (Pfister et al. (1994) Intervirol. 37: 143-149). Each HPV type shows host specificity. Several HPV types infect the genital epithelium and represent the most prevalent pathogenesis of sexually transmitted virosis. Genital HPV types are further classified as “high-risk” types associated with the development of neoplasms (most commonly HPV-16 and HPV-18); and “low-risk” types rarely associated with malignancy (most In general, it can be subdivided into HPV-6 and HPV-11). The malignant form may eliminate the need for a viral DNA replication gene product by integrating into the genome of the host cell. In contrast, the benign forms (most commonly HPV-6 and HPV-11) rely on viral proteins E1 and E2 for episomal genome replication. Two HPV types, HPV-6 and HPV-11, are usually associated with pharyngeal papillomas or benign epithelial tumors of the pharynx.
[0004]
Human papillomavirus is a non-enveloped DNA virus containing a circular double-stranded 7,900 base pair DNA genome, which can be divided into three distinct functional domains: viral DNA replication. An upstream regulatory region (URR) containing the origin and enhancers and promoters involved in transcription; an L region encoding structural proteins L1 and L2; and an E region encoding genes required for trophic function. The HPV genome encodes eight viral proteins, El, E2, E4, E5, E6, E7, L1 and L2 (illustrated in FIG. 1), which comprise the complex of alternatively spliced mRNAs. Translated from family.
[0005]
Most HPV types are required for the initiation of viral DNA replication (Ustav et al. (1991) EMBO J., 10: 449-457; Chiang et al. (1992) Proc. Natl. Acad. Sci. (USA) 89: 5799- Del. Vecchio, AM et al. (1992) J. Virol. 66: 5949-5958; Sandler, AB et al. (1993) J. Virol. 67: 5079-5087; Scheffner, M. et al. ) In Human Pathogenic Papillomaviruses (Zur housen, H. eds. Pp. 83-100, Heidelberg, Springer-Verlag), and two proteins encoded by the virus for episomal maintenance of the viral genome. It requires the activity of E1 and E2 is a click quality. However, certain in vitro experiments have shown that only the activity of the E1 protein is essential for viral replication (Gopalakrishnan et al. (1994) Proc. Natl. Acad. Sci. (USA) 91: 9597-9601). El is an ATP hydrolyzing DNA helicase, which is thought to be involved in unwinding DNA at the viral origin during viral genome replication by the human host cell DNA replication complex (Hughes (1993) Nucleic Acids Res. 21: 5817-5823; Chow et al. (1994) Intervirol. 37: 150-158; Jenkins, O. et al. (1996) J. Gen. Virol. 77: 1805-1809; (L. et al. (1999) J. Biol. Chem. 274: 2696-2705). E2 is involved in regulating HPV transcriptional activity through facilitating assembly of transcription complexes containing host proteins (Ham, J. et al. (1991) Trends Biochem. Sci. 16: 440-444; Liu, J.- (1995) J. Biol. Chem. 270: 27283-27291).
[0006]
The E4 protein is associated with the intermediate filament network of host cells and is the most abundant gene product expressed by papillomavirus (Dorbar, 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, EJ et al. (1987) EMBO J. 6: 989-992; Bedel, MA et al. (1989) J. Virol. 63. 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 that 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, SW et al. (1993) J. Virol. 67: 4521-4532). In some HPV types, the E6 and E7 proteins are each tumor suppressor proteins, p53 (Lechner, MS et al. (1992) EMBO J. 11: 3045-3052; Werness, BA et al. (1990). ) Science 248: 76-79) and retinoblastoma (Dyson, N. et al. (1989) Science 243: 934-937; Gage, JR et al., J. Virol. 64: 723-730). I do. E6 and E7 both interact with a number of cellular proteins that affect the outcome of the infection. L1 and L2 are common to all HPV types and encode the capsid protein (Broker, TR et al. (1986) Cancer Cells 4: 17-36; zur Hausen, H. et al. (1987) ) In The Papovaviridai, Volume 2, The Papillomaviruses (Salzman, N. and Howley, eds., Pp. 245-263), New York, Plenum Press; Pfister, H. (1987) Obeyst. Am. 14: 349-361).
[0007]
Current treatments for HPV infection are very limited. Currently, there are no approved HPV-specific antiviral therapeutics. Management usually involves the physical destruction of the wart by surgical, cryosurgical, chemical or laser removal of the infected tissue. Nonanal genital warts are transmitted by skin-to-skin contact, while genital warts are usually sexually transmitted. Both types of warts cause much morbidity, but rarely cause malignant transformation. These are commonly treated with surgical or cryosurgery treatments, but immunomodulators such as imiquimod have proven to be very effective in anogenital warts. And is being evaluated in non-anogenital warts (Severson J., et al., J. Cutan. Med. Surg. (2001) Jan; 5 (1): 43-60). Topical antimetabolites such as 5-fluorouracil and podofilm preparations have also been used (Reichman, Harrison's Principles of Internal Medicine, 13th edition (eds. Isselbacher et al.) McGraw-Hill, Inc., NY (1993). Pp. 801-803). However, recurrences after these procedures are common, and subsequent repeated treatments gradually destroy healthy tissue. Interferon has so far been the only treatment with a mode of antiviral action, but its limited efficacy limits its use (Cowsert (1994) Intervirol. 37: 226-230; Bornstein et al. (1993) Obstrics Gynecol. Sur. 4504: 252-260; Browner et al. (1992) Ann. Pharmacother. 26: 42-45).
[0008]
Other types of HPV have significant oncogenic potential, so that more than 99% of all cervical cancers and more than 50% of other anogenital cancers result from infection with oncogenic HPV. Many cofactors, such as smoking, genetics, and helper viruses, have a potential role in HPV carcinogenesis, but their relative contributions are poorly understood (Severson J., et al., J. Cutan.Med.Surg. (2001) Jan; 5 (1): 43-60).
[0009]
In recent years, research on the development of HPV antiviral compounds has focused on the development of HPV-specific antisense oligonucleotides. Antisense oligonucleotides can regulate gene expression by binding to a target single-stranded nucleic acid molecule according to Watson-Crick's law, or by binding to double-stranded nucleic acid according to Hoogsteen's rule for base pairing. And, in doing so, can 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 inducing enzymatic destruction of mRNA by RNase H; or by destroying the target via a reactive group directly attached to the antisense oligonucleotide.
[0010]
Improved oligonucleotides having higher potency in inhibiting such viruses, pathogens, and selective gene expression have been developed more recently. Some of these oligonucleotides with modifications in 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) disclose that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates can be synthesized from conventional phosphodiester-linked oligodeoxynucleotides. Rather than being more effective at inhibiting HIV-1. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1989) 86: 7790-7794) disclose the advantages of oligonucleotide phosphorothioates in inhibiting HIV-1 in initially and chronically infected cells. I have.
[0011]
In addition, chimeric oligonucleotides having more than one type of internucleotide linkage in the oligonucleotide have been developed. (U.S. Pat. Nos. 5,149,797 and 5,220,007) describe a core sequence of oligonucleotide phosphodiesters or phosphorothioates flanked by nucleotide methylphosphonates or phosphoramidates. Disclosed are chimeric oligonucleotides having the formula: Agrawal et al. (WO 94/02498) disclose hybrid oligonucleotides having regions of deoxyribonucleotides and 2'-O-methyl ribonucleotides.
[0012]
The mechanism of DNA replication is conserved between papillomaviruses. Of all papillomavirus proteins, E1 is the most conserved. It is an ATPase and a helicase and has sequence homology to the ATPase domain of the simian virus 40 (SV40) T antigen, an initiator of replication of the SV40 origin sequence (ori). Like the T antigen, the E1 protein binds to ori and unwinds the DNA in the presence of the host single-stranded DNA binding protein RPA and topoisomerase I. The E1 protein of human papillomavirus (HPV) and bovine papillomavirus type 1 (BPV-1) is thought to function as a helicase at the replication fork. Because each is needed during elongation. The E1 protein of BPV-1 is known to interact with the 180-kDa catalytic subunit of DNA polymerase α of the host, thereby bringing the host unfolded protein to the unwound ori. Due to the homologous nature of the ori and E1 and E2 proteins, proteins from one virus type can efficiently replicate either the homologous virus or the heterologous virus ori (Nianxiang Zou et al., J. Virol., (1998), 72 (4): 43436-3441).
[0013]
A limited number of antisense oligonucleotides have been designed to inhibit HPV expression. For example, oligonucleotides specific to various regions of HPV E1 and E2 mRNA have been prepared (eg, US Pat. No. 5,364,758, WO 91/08313, WO 93/20095, and WO 95/04748). checking).
[0014]
There remains a need for the development of oligonucleotides that can inhibit the replication and expression of human papillomavirus, the use of which has a successful prognosis and low or no cytotoxicity.
[0015]
(Summary of the Invention)
To design therapeutic compounds against human papillomavirus, the E1 gene of HPV type 6 (GenBank HPV6b accession number M14119) and type 11 (GenBank HPV11 accession number X00203) were targeted. Both types 6 and 11 are associated with more than 90% of nonmalignant genital warts. A 46 nucleotide region centered on the start site for protein translation (positions -17 to +29 of the E1 open reading frame) was examined in detail. This region is conserved in many clinical isolates of HPV types 6 and 11. The entire open reading frame of this gene (positions -17 to +1950) was also studied. This entire region shows high sequence identity between HPV type 6 and HPV type 11. Additional HPV strains targeted in the present invention are HPV type 16, type 18, type 31, type 45 and type 58.
[0016]
The present inventors have now found that oligonucleotides directed to the region from position +1 to +20 of the E1 gene are particularly useful for inhibiting HPV replication. More specifically, the present inventors have found that oligonucleotides directed to this region, as short as 4 nucleotides, can efficiently inhibit HPV replication. These short oligonucleotides (i.e., short-mers) have specific sequence-related effects that can act to inhibit HPV replication through multiple mechanisms. Oligonucleotides of the invention may act through interaction of nucleic acids or proteins with targets, or may act through both types of interactions.
[0017]
The present invention provides a synthetic oligonucleotide complementary to a region extending from position +1 to position +20 of the translation initiation site of the HPV E1 protein, or a part thereof. More specifically, the present invention provides oligonucleotides that have been modified to increase stability or HPV inhibitory activity. Such modifications can include, for example, modifications of internucleoside linkages, sugars, bases, capped ends, and chimeric or hybrid oligonucleotides. The invention further provides pharmaceutical compositions and methods for the treatment of HPV infection, including the treatment and prevention of a disorder or disease associated with HPV.
[0018]
(Detailed description of the invention)
As discussed above, we have discovered synthetic oligonucleotides with significant HPV inhibitory activity.
[0019]
The present invention provides a synthetic oligonucleotide complementary to a nucleic acid spanning the translation initiation site of human papillomavirus gene E1. For the purposes of the present invention, the nucleic acid spanning the translation initiation site of human papillomavirus gene E1 is the region of the E1 gene from nucleotide +1 to +20 (eg, nucleotides 833 to 851 of the HPV-6b genome) or a portion thereof. It is intended to indicate This region has the sequence 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 set forth in SEQ ID NO: 36. (5′-gX acc Xga aXc gXc cgc caX-3 ′), where X can be thymidine or uracil, and any nucleotide can be replaced with inosine.
[0020]
For purposes of the present invention, an oligonucleotide sequence that is complementary to a nucleic acid is one in which the unmodified version of the oligonucleotide is capable of undergoing physiological conditions (eg, by Watson-Crick or “Wobble” base pairing). , Interaction between an oligonucleotide and a single-stranded nucleic acid).
[0021]
For the purposes of the present invention, oligonucleotides having HPV inhibitory activity are those that can interfere with HPV replication or disrupt HPV replication at some point in the viral life cycle through a variety of possible mechanisms. It is intended to mean. For example, an oligonucleotide may inhibit HPV replication through interaction of a nucleic acid or protein with a target, or may act through both types of targets. Oligonucleotides acting through a nucleic acid target can bind to single-stranded DNA, double-stranded DNA, or mRNA and prevent the binding of factors required for normal transcription, splicing, or translation, or By inducing enzymatic destruction of mRNA by RNase H and the like, the function of the target can be disrupted. Oligonucleotides acting through a protein target can bind to a receptor or any other type of protein (eg, such as a DNA polymerase, a transcription factor, etc.).
[0022]
The oligonucleotides of the present invention have specific sequence related effects. This is the sequence of the oligonucleotide (the linear sequence of mononucleotides, as well as the three-dimensional structure of the oligonucleotide as a whole as determined by the sequence of mononucleotides and any modifications to nucleotides or internucleotide linkages Is intended to mean producing a specific HPV inhibitory effect. However, this is not meant to limit the invention to mechanisms that rely on base pairing between the oligonucleotide and the nucleic acid. Specific sequence-related effects can occur through various mechanisms, as discussed above.
[0023]
The synthetic oligonucleotides of the invention comprise a sequence that is complementary to the region of the HPV E1 open reading frame, or a portion thereof, spanning nucleotide positions +1 to +20. Preferably, the oligonucleotide comprises from about 4 to about 20 mononucleotides, more preferably, from about 4 to about 12, 14, 16, or 18 mononucleotides. Preferred oligonucleotides of the present invention include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Or an oligonucleotide containing 19 nucleotides. More preferably, the oligonucleotides of the invention comprise the sequences shown in Table 1 below or a portion thereof. Most preferably, the oligonucleotide of the invention comprises the sequences shown in SEQ ID NOs: 1-3, 28 and 34.
[0024]
Preferred synthetic oligonucleotides contain at least one modification, and more preferably more than one modification. Modifications include, for example, modifications of internucleotide linkages, base or sugar moieties, capped ends, and chimeric or hybrid oligonucleotides.
[0025]
Synthetic oligonucleotides include chemically synthesized polymers of deoxyribonucleotides and / or ribonucleotides linked by internucleotide linkages. Oligonucleotides can be composed entirely of deoxyribonucleotides, all ribonucleotides, or a combination of deoxyribonucleotides and ribonucleotides, including hybrid oligonucleotides and reverse hybrid oligonucleotides. Hybrid oligonucleotides include a deoxyoligonucleotide core region inserted between adjacent regions of a ribonucleotide. Reverse hybrids include a core region of ribonucleotides inserted between adjacent regions of deoxyribonucleotides.
[0026]
The synthetic oligonucleotides of the invention can be linked by a standard phosphodiester nucleotide ring bond between the 5 'group of the pentose ring of one mononucleotide and the 3' group of an adjacent mononucleotide. Such linkages are also established using different linking sites, including 5 ′ → 5 ′, 3 ′ → 3 ′, 2 ′ → 5 ′, and 2 ′ → 2 ′, or any combination thereof. obtain. In addition to phosphodiester bonds, mononucleotides can also include alkyl phosphonate, phosphorothioate, phosphorodithioate, alkyl phosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamido, They may be linked by a date bond, or a carboxymethyl ester bond, or any combination thereof. Preferably, the oligonucleotide of the invention comprises at least one phosphorothioate internucleotide linkage, more preferably all linkages in the oligonucleotide are phosphorothioate internucleotide linkages.
[0027]
The oligonucleotides of the present invention can be constructed such that all mononucleotides are linked by the same type of internucleotide linkage or by a combination of different internucleotide linkages, including chimeric oligonucleotides or reverse chimeric oligonucleotides. Nucleotides. Chimeric oligonucleotides have a phosphorothioate core region inserted between adjacent regions of methylphosphonate or phosphoramidate. Reverse chimeric oligonucleotides have a nonionic core region (eg, an alkyl phosphonate and / or phosphoramidate and / or phosphotriester internucleoside linkage) inserted between phosphorothioate flanking regions.
[0028]
The synthetic oligonucleotides of the invention can be constructed from adenine, cytosine, guanine, inosine, thymidine or uracil mononucleotides. Preferred oligonucleotides are constructed from mononucleotides that contain modifications in the base and / or sugar moieties of the mononucleotide. Modifications to bases or sugars include alkyl, carbocyclic aryl, heteroaromatic or heteroaliphatic groups having from 1 to 3 separate or fused rings and from 1 to 3 N, O or S atoms. And a covalent substituent having a cyclic group or a heterocyclic structure.
[0029]
Alkyl groups preferably contain 1 to about 18 carbon atoms, more preferably 1 to about 12 carbon atoms, and most preferably 1 to about 6 carbon atoms. Particular examples of alkyl groups include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
[0030]
Aralkyl groups include those alkyl groups listed above substituted with a carbocyclic aryl group having 6 or more carbons (eg, phenyl, naphthyl, phenanthryl, anthracyl, and the like). .
[0031]
Cycloalkyl groups preferably have from 3 to about 8 cyclic 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.).
[0032]
Exemplary heteroaromatic and heteroalicyclic groups include pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and Pyrrolidinyl.
[0033]
Preferred modifications to the sugar include modifications to the 2 'position of the ribose moiety, including, but not limited to: -O containing 1 to 6 saturated or unsaturated carbon atoms. -2'-O-substitution with a lower alkyl group, or an O-aryl or an allyl group having 2 to 6 carbon atoms, wherein such an -O-alkyl, aryl or allyl group is It may be unsubstituted, substituted (e.g., with a halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxyl, or amino group), or The 2′-O— group is substituted by an amino or halogen group. Neither of these substitutions is intended to exclude either the native 2'-hydroxyl group in the case of ribose or the 2'-H- in the case of deoxyribose.
[0034]
Preferred modified oligonucleotides include 2'-O-methyl ribonucleotide (2'-OMe) and 5-methylated deoxycytosine (5-Me-dC). The oligonucleotides of the invention can be constructed entirely from unmodified mononucleotides, all constructed from mononucleotides containing a particular modification, or constructed from various mononucleotides containing different modifications. Certain preferred oligonucleotides comprise at least one, and preferably 1-5, 2'-O-methyl ribonucleotides at the 3 'end of the oligonucleotide. Furthermore, the oligonucleotide may further comprise at its 5 'end at least one, preferably 1 to 5 2'-O-methyl ribonucleotides.
[0035]
The sugar group of a mononucleotide can be natural or modified (eg, synthetic), and can be in linear or cyclic form. The sugar group may be composed of mono-, di-, oligo-, or poly-saccharides, wherein each monosaccharide unit contains 3 to about 8 carbons, preferably 3 to about 6 carbons, It contains a polyhydroxy group, or a polyhydroxy group and an amino group. 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 radicals or as protected groups containing, for example, hydrogen and / or halogen. Preferred protecting groups include acetonide, t-butoxycarbonyl group and the like. Monosaccharide sugar groups can be in the L or D configuration, and the cyclic monosaccharide unit can include an α-conformation or β-conformation 5- or 6-membered ring. A disaccharide can be composed of two identical monosaccharide units or two different monosaccharide units. Oligosaccharides can be composed of 2 to 10 monosaccharides and can be homopolymers, heteropolymers, or cyclic polysaccharides. Polysaccharides can be homoglycans or heteroglycans, and can be branched or unbranched polymer chains. Di-, oligo-, and poly-saccharides may be composed of 1 → 4 bonds, 1 → 6 bonds, or a mixture of 1 → 4 and 1 → 6 bonds. The sugar moiety can be linked to the linking group through any hydroxyl or amino group of the carbohydrate.
[0036]
Other modifications include those internal or at the end of the oligonucleotide molecule, and additions to the molecule at internucleoside phosphate linkages (eg, cholesteryl, cholesterol, or various numbers of carbon atoms between two amine groups). Diamine compounds with residues, and terminal ribose, deoxyribose, and phosphate modifications, which cleave or crosslink to paired strands, or other proteins that bind to the relevant enzyme or viral genome )). Additional linkers, including non-nucleoside linkers, include, but are not limited to, polyethylene glycols of various lengths such as 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. Am. Chem. Soc. 117: 7323). -7328), or any other commercially available linker, including abasic linkers, or commercially available asymmetric and symmetric linkers (CloneTech, Palo Alto, Cal.) fornia) (for example, Glen Research Product Catalog, Sterling, VA).
[0037]
An additional oligonucleotide capped with ribose at the 3 'end of the oligonucleotide is NaIO ₄ It can be subjected to oxidation and / or reductive amination. Aminations may include, but are not limited to, the following moieties: spermine, spermidine, Tris (2-aminoethyl) amine (TAEA), DOPE, long chain alkylamine, crown ether, coenzyme A, NAD, sugar, peptide, dendrimer.
[0038]
Oligonucleotides can also be capped at their 3 'and / or 5' ends with bulky substituents, or have one substitution at one or both non-bridging oxygens per nucleotide. . Such modifications may be at some or all of the internucleoside linkages and may be at either or both termini of the oligonucleotide, and / or internally to the molecule (Agrawal et al. (1992) Trends Biotechnol. 10: 152-158). Some non-limiting examples of capped species include 3'-O-methyl, 5'-O-methyl, 2'-O-methyl, and any combination thereof.
[0039]
Preferred oligonucleotides of the present invention have a sequence (including modifications thereof) selected from the group of SEQ ID NOs: 1-37 shown in Table 1. Particularly preferred oligonucleotides have a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 4, 11, 21 and 26 (including the internucleotide binding compositions and further modifications shown in Table 1). Most preferred oligonucleotides have a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 28, 34.
[0040]
Synthetic oligonucleotides of the invention can be prepared by art-recognized methods. For example, nucleotides can be phosphoramidite, H-phosphonate chemistry, or methylphosphoramidite chemistry (eg, Goodchild (1990) Bioconjugate Chem. 2: 165-187; Uhlmann et al. (1990) Chem. Rev. 90: 543-584. (1987) Meth. Enzymol. 154: 287-313; see US Patent No. 5,149,798), covalently using art-recognized techniques. Can be linked. These can be performed by manual or automated synthesizers and then processed (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10: 152-158). Oligonucleotides having phosphorothioate linkages can be obtained by methods well known in the art (eg, phosphoramidites (see, eg, Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 7079-7083)) or H-phosphonates (see, for example, Froehler (1986) Tetrahedron Lett. 27: 5575-5578) chemistry. The synthetic method described in Bergot et al. (J. Chromatog. (1992) 559: 35-42) may also be used. Oligonucleotides having other types of modified internucleotide linkages can be prepared according to known methods (eg, Goodchild (1990) Bioconjugate Chem. 2: 165-187; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 7079-7083; Uhlmann et al. (1990) Chem. Rev. 90: 534-583; and Agrawal et al. (1992) Trends Biotechnol. 10: 152-158).
[0041]
In another aspect, the invention provides a pharmaceutical composition. The pharmaceutical composition comprises at least one HPV-specific oligonucleotide, and preferably two or more HPV-specific oligonucleotides, having the same or different sequences, modifications, and / or lengths. It is a mixture. In some embodiments, the pharmaceutical formulation also includes a physiologically or pharmaceutically acceptable carrier. Certain embodiments include a therapeutic amount of a lipid carrier.
[0042]
The oligonucleotides of the present invention are suitable for use as therapeutically active compounds, particularly in the control or prevention of human papillomavirus infection.
[0043]
In this aspect of the invention, a therapeutic amount of a pharmaceutical composition comprising the HPV-specific synthetic oligonucleotide is administered to the cells to inhibit human papillomavirus replication. In a similar aspect, the oligonucleotides of the invention can be used to treat human papillomavirus infection. Here, administering a therapeutic amount of a pharmaceutical composition comprising at least one HPV-specific oligonucleotide, and in some embodiments, at least two HPV-specific oligonucleotides, to an infected animal or cell. . In some preferred embodiments, the method comprises at least one oligonucleotide, or at least two oligonucleotides, having the sequence shown in Table 1 or the Sequence Listing as SEQ ID NOs: 1-37, including modifications thereof. Is administered.
[0044]
In all methods involving the administration of the oligonucleotides of the invention, at least one, and preferably two or more, identical or different oligonucleotides are administered in a single treatment dose in the form of separate pharmaceutical compositions. Can be administered simultaneously or sequentially.
[0045]
More specifically, the present invention relates to a method of treating a mammal susceptible to a disease associated with a human papillomaviridae virus (prophylactic treatment) or a mammal suffering from a disease associated with a human papillomaviridae virus. Treatment method. Examples of clinical conditions caused by such viruses include benign skin and genital warts (condyloma acuminatum), wart-like epidermal dysplasia (EV), respiratory or pharyngeal papillomatosis, and cervix It is cancer. The methods of the invention involve administering a therapeutically effective amount of one or more compounds of the invention to cells infected with the virus (eg, mammalian cells, especially human cells).
[0046]
Administration of the compounds of the invention can be oral, topical (including transdermal, buccal, or sublingual), nasal, and parenteral (intraperitoneal, subcutaneous, intravenous, intradermal). (Including injection, or intramuscular injection), and oral or parenteral is generally preferred. It is also understood that preferred modes of administration and dosages can vary with, for example, the condition and age of the recipient.
[0047]
The compounds of the present invention may be used in therapy in combination with other pharmaceutically active medicaments, such as another antiviral or anticancer agent. In addition, one or more compounds of the present invention may be administered alone, but they may also be suitable for conventional excipients (ie, suitable for parenteral, oral, or other administration as desired, and active A pharmaceutically acceptable organic or inorganic carrier material that does not deleteriously react with the compound and is not harmful to the recipient thereof). Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, vegetable oil, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, Viscous paraffins, aromatic oils, fatty acid monoglycerides and diglycerides, petroetral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone and the like. The pharmaceutical preparations can be sterilized and, if desired, auxiliaries which do not deleteriously react with the active compound (e.g. lubricating agents, preservatives, stabilizing agents, wetting agents, emulsifying agents, affecting osmotic pressure). (Eg, salts, buffers, colorants, flavors, and / or fragrances).
[0048]
Particularly suitable for parenteral application are solutions, preferably oily or aqueous solutions, and suspensions, emulsions or implants, including suppositories. Ampules are conventional unit dosages.
[0049]
Particularly suitable for enteral application are tablets, dragees, or capsules, such as with talc and / or carbohydrate carrier binders, which are preferably lactose and / or corn starch and / or potato starch. is there. Syrups, elixirs and the like can be used, wherein a sweetened vehicle is used. Sustained-release compositions containing these can be formulated wherein the active ingredient is protected with a differentially degradable coating, for example, by microencapsulation, multiple coatings, and the like.
[0050]
Therapeutic compounds of the present invention can also be incorporated into liposomes. Incorporation can be performed according to known liposome preparation procedures, such as sonication and extrusion. Suitable conventional methods of liposome preparation are also disclosed below: D. Bangham et al. Mol. Biol. , 23: 238-252 (1965); Olson et al., Biochim. Biophys. Acta, 557: 9-23 (1979); Szoka et al., Proc. Nat. Acad. Sci. , 75: 4194-4198 (1978); Kim et al., Biochim. Biophys. Acta, 728: 339-348 (1983); and Mayer et al., Biochim. Biophys. Acta, 858: 161-168 (1986).
[0051]
It will be understood that the actual preferred amount of active compound used in a given treatment will vary according to the particular compound employed, the particular composition formulated, the mode of application, the particular site of administration and the like. Optimal dosing rates for a given dosing protocol can be readily ascertained by those skilled in the art using conventional dosage determination tests.
[0052]
All documents referred to herein are hereby incorporated by reference.
[0053]
The present invention is further illustrated by the following examples. These examples are provided to aid the understanding of the present invention and should not be construed as limiting the invention.
[0054]
(Example 1: Synthesis of oligonucleotide)
Oligonucleotides were purchased from an ABI 394 DNA / RNA synthesizer (Perkin-Elmer, Foster City, Calif.), Pharmacia Gene Assembler Plus (Pharmacia, Uppsala, Sweden, or from Gene Assembl, Sweden) or Gene Assembler, Sweden. Synthesized using standard phosphoramidite chemistry (Beaucage (1993) Meth. Mol. Biol. 20: 33-61) using standard protocols and custom methods from vendors. The custom method served to increase the coupling time for 2'-O-methyl RNA amidite from 1.5 minutes to 12 minutes. The Pharmacia synthesizer required further drying of the amidite, activating reagent and acetonitrile. This was achieved by adding a 3Å molecular sieve (EM Science, Gibbstown, NJ) before mounting on the machine.
[0055]
DNA β-cyanoethyl phosphoramidite was purchased from Cruchem (Glasgow, Scotland). The DNA support was 500 ° pore size controlled pore glass (CPG) derivatized with the appropriate 3 ′ base (PerSeptive Biosystems, Cambridge, Mass.) With a loading between 30 and 40 mmoles per gram. 2′-O-methyl RNA β-cyanoethyl phosphoramidite and CPG support (500 °) were purchased from Glen Research (Sterling, VA). For random sequence synthesis, the DNA phosphoramidites were mixed on a synthesizer according to the manufacturer's protocol (Pharmacia, Uppsala, Sweden).
[0056]
All 2'-O-methyl RNA-containing oligonucleotides were purified from ethylthiotetrazole (American International Chemical (AIC), Natick, NY) dissolved in low water acetonitrile (Aldrich, Milwaukee, WI) at 0.25M. MA) as an activator. Some DNA-only synthesis was performed with 0.25 M ethylthiotetrazole, but most with 0.5 M 1-H-tetrazole (AIC). The thiosulfurizing reagent used in all PS oligonucleotides was 3H-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage Reagent, 2% solution in low-water acetonitrile (w / v)). RI Chemical, Orange, CA, or AIC, Natick, MA).
[0057]
Cholesteryl CPG (chol) and polyethylene glycol (PEG), 5'-amino-modifier [C] used to synthesize oligos with linkers ₆ NH ₂ ] And cholesteryl phosphoramidite were used according to the manufacturer's instructions (Glen Research, Sterling, VA).
[0058]
3'-NH ₂ Cap is a 3 '-(3-amino-2-propanol) conjugate prepared using the 3'-amino modifier C3 CPG according to the manufacturer's instructions (Glen Research, Sterling, VA).
[0059]
For the oxidation, redox, or amination of oligonucleotide phosphorothioates containing a ribonucleotide at the 3 'end, the synthesis was performed as follows.
[0060]
Oligonucleotide phosphorothioate containing ribonucleotides at the 3 'end (1 mM) was added in 0.1 M sodium acetate (pH 4.75) for 30 minutes on ice with NaIO ₄ (1.2 mM) to give the 3'-dialdehyde (Ox.) Product. For the addition of the amine, 6 equivalents of amine in 0.2 M sodium phosphate buffer (pH 8) are added to the oxidized oligonucleotide for 30 minutes at room temperature, followed by 30 equivalents of NaCNBH ₃ Was added. The solution was left overnight at room temperature. This product was purified by preparative polyacrylamide gel electrophoresis on a 20% denaturing gel. The same procedure was performed in the absence of an amine to give the 3 'diol (Ox / Red.) Product.
[0061]
After completion of the synthesis, the CPG was air-dried and transferred to a 2 mL screw-cap microcentrifuge tube. The oligonucleotide was deprotected and cleaved from CPG with 2 mL of ammonium hydroxide (25-30%). The tube was capped and incubated at room temperature for 20 minutes, then at 55 ° C. for 7 hours. After deprotection was completed, the tube was removed from the heat block and allowed to cool to room temperature. The cap was removed and the tube was 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 μl of 0.3 M NaCl and the DNA was precipitated with 1.6 mL of anhydrous EtOH. The DNA was pelleted by centrifugation at 14,000 rpm for 15 minutes, the supernatant was decanted, and the pellet was dried. This DNA was again precipitated from 0.1 M NaCl as described above. 500 μl of H ₂ Dissolved in O and centrifuged at 14,000 rpm for 10 minutes to remove any solid material. The supernatant was transferred to another microfuge tube and the amount of DNA was determined spectrophotometrically. The concentration was determined by the optical density at 260 nM. E for the DNA portion of the oligonucleotide ₂₆₀ Was calculated by OLIGSOL (Lautenberger (1991) Biotechniques 10: 778-780). E of the 2'-O-methyl moiety ₂₆₀ Was calculated by using OLIGO 4.0 Primer Extension Software (NBI, Plymouth, MN).
[0062]
Oligonucleotide purity was checked by polyacrylamide gel electrophoresis (PAGE) and UV shadowing. 0.2 OD ₂₆₀ The unit is 95% formamide / H ₂ Loaded on a 20% denaturing polyacrylamide gel (20 cm × 20 cm) with O and Orange G dye. The gel was run until the Orange G dye was within one inch of the low side of the gel. Bands were visualized by short wave UV light shadowing on thin layer chromatography plates (Kieselgel 60 F254, EM Separations, Gibbstown, NJ).
[0063]
Some oligonucleotides were synthesized without removing the 5'-trityl group (trityl-on) to facilitate reverse phase HPLC purification. The trityl-one oligonucleotide was 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 using a 600E HPLC (Waters, Franklin, Mass.) Using a C-18 μBondapak chromatography matrix (Waters, Franklin, Mass.). ) Was purified on a 1.5 x 30 cm glass liquid chromatography column (Spectrum, Houston, TX). Oligonucleotides were run at 5 mL / min using a 40-min gradient of 14-32% acetonitrile (Baxter, Burdick and Jackson Division, Muskegon, MI) in 0.1 M ammonium acetate (JT Baker, Phillipsburg, NJ). Elution was followed by elution with 32% acetonitrile for 12 minutes. Peak detection was performed at 260 nm using a Dynamax UV-C absorbance detector (Rainin, Emeryville, CA).
[0064]
The HPLC purified trityl-one oligonucleotide was evaporated to dryness and the trityl group was removed by incubation in 5 mL of 80% acetic acid (EM Science, Gibbstown, NJ) for 15 minutes. After evaporation of the acetic acid, the oligonucleotide was dissolved in 3 mL of 0.3 M NaCl and ethanol precipitated. The precipitate was isolated by centrifugation and again precipitated from 3 mL of 0.1 M NaCl with ethanol. The precipitate was isolated by centrifugation and dried on a Savant Speed Vac (Savant, Farmingdale, NY). Quantitative and PAGE analyzes were performed on the ethanol-precipitated oligonucleotides as described above.
[0065]
Standard phosphoramidite chemistry was applied to the synthesis of oligonucleotides containing methylphosphonate linkages using two Pharmacia Gene Assembler Special DNA synthesizers. One synthesizer was used for the synthesis of the phosphorothioate moiety of the oligonucleotide using the β-cyanoethyl phosphoramidite method discussed above. The other synthesizer was used for the introduction of the methylphosphonate moiety. Reagents and synthetic cycles which have been shown to be advantageous in methylphosphonate synthesis have been applied (Hogreffe et al., Methods in Molecular Biology, Volume 20: Protocols for Oligonucleotides and Analogs (Agrawall et al., 1992). Totowa, NJ). For example, 0.1 M methyl phosphoramidite (Glen Research, Sterling, VA) is activated with 0.25 M ethylthiotetrazole; using a coupling time of 12 minutes; tetrahydrofuran / 2,6-lutidine / Oxidation with iodine (0.1 M) in water (74.75 / 25 / 0.25) was applied immediately after this coupling step; dimethylaminopyridine (DMAP) was used for the capping procedure. Replaced the standard N-methylimidazole (NMI). These chemicals were purchased from Aldrich (Milwaukee, WI).
[0066]
The working procedure was based on a published procedure (Hogreffe et al. (1993) Nucleic Acids Research 21: 2031-2038). The product was removed from the resin by incubation with 1 mL ethanol / acetonitrile / ammonium hydroxide (45/45/10) for 30 minutes at room temperature. Then, ethylenediamine (1.0 mL) was added to the mixture and deprotected at room temperature for 4.5 hours. The resulting solution and 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 with 6N HCl in 20/80 acetonitrile / water (4-5 mL) to pH 7, then concentrated to dryness using a Speed Vac concentrator. The resulting solid residue was dissolved in 20 mL water and the sample was desalted by using a Sep-Pak cartridge. After two passes of the aqueous solution through the cartridge at a rate of 2 mL / min, the cartridge is washed with 20 mL of 0.1 M TEAB and the product is washed with 4 mL of 50% acetonitrile in 0.1 M TEAB. Elution was at 2 mL / min. The eluate was evaporated to dryness with a Speed Vac.
[0067]
The crude product was purified by polyacrylamide gel electrophoresis (PAGE) and desalted using a Sep-Pak cartridge. Oligonucleotides were ethanol precipitated from 0.3 M NaCl and then ethanol precipitated from 0.1 M NaCl. The product was dissolved in 400 μL water and quantified by UV absorbance at 260 nm.
[0068]
(Example 2: Biological test)
Human papillomavirus (HPV) has tropism for squamous epithelium. The life cycle of HPV is closely linked to the state of differentiation (strict requirements) of infected cells, which previously made it difficult to study this virus in vitro. Using the recently developed organotypic “raft” culture system, which supports a complete differentiation-specific replication cycle of HPV that accompanies the production of infectious virions, the oligonucleotides of the invention against HPV replication are used. The effect was studied (Meyers, C. et al. (1992) Science 257: 971-973; Meyers, C. et al. (1998) Cell Biology: A laboratory handbook (Celis, JE.), Pp. 513-520, Academic. Press, Inc. Orlando, FL; Meyers, C. et al. (1992) Papillomavirus Res. 3: 1-3-3; Meyers, C. et al. (1993) Current Topics in Microbiology and Immunology. , Vol on Human pathogenic papillomaviruses (ed. By zur Hausen) Springer-Verlag, New York; Bedell, MA, et al. (1991) J. Virol. 65: 2254-2260; Meyers, C.i. 230).
[0069]
Cell and raft culture assembly: The CIN-612 cell line was established from a cervical intraepithelial neoplasia (CIN) type I biopsy. The CIN-612 cell line contains HPV31b DNA, of which the 9E clonal derivative maintains approximately 50 episomal copies of the HPV31b genome per cell. CIN-612 9E cells were maintained in monolayer culture using E medium containing 5% fetal calf serum in the presence of mitomycin C-treated J2 3T3 feeder cells. J2 3T3 cells were maintained in DMEM containing 5% newborn calf serum. Skin equivalents were prepared in 6-well culture dishes using rat tail type I collagen, culture medium, and fibroblasts. Fibroblasts adapt this lineage and exert the interstitial effects required for epithelial proliferation and stratification. CIN-612 9E epithelial cells were counted and 600,000 cells were seeded on collagen plugs soaked under E medium. Epithelial cells were allowed to reach confluence, media was removed, and the collagen matrix was placed on a stainless steel grid. Subsequent feeding of the epithelium was via diffusion of E medium from under the matrix. Epithelial tissue was stratified and differentiated at the air-liquid interface over a period of 10 days. After 10 days, the epithelium was harvested by separating from the collagen layer and stored at -20 C until further manipulation.
[0070]
Treatment of raft cultures with 2.5 μM and 25 μM HPV / L1-HPV / L25 and ORI 1001 (SEQ ID NOs: 1-37): feed raft cultures every other day with E-medium containing test compound did. Each oligonucleotide (SEQ ID NOs: 1-37) was dissolved in sterile water to obtain a 5 mM stock solution. For each oligonucleotide tested, 12 mL of 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 this medium to give a final concentration of 25 μM. Another set of raft cultures was treated with a final concentration of 2.5 μM HPV / L1 to HPV / L7 and ORI 1001 (SEQ ID NOS: 1 to 7 and 26). Media was removed from the raft culture via aspiration and then freshly prepared media was added carefully so as not to place any liquid on top of the raft tissue. This is because placing a liquid on top delays the stratification of the epithelium. Sham raft cultures were fed with E medium only.
[0071]
Nucleic acid extraction. Extraction of nucleic acids was performed as previously described (Ozbun, MA et al. (1998) Virology 248: 218-230). Total cellular DNA was collected by incubating raft tissue overnight at 55 ° C. in 10 mM Tris-HCl, pH 7.5, 25 mM EDTA, 0.2% SDS and 50 mg / mL RNaseA. Then 100 mg / mL Proteinase K was added and incubated for another 4 hours. The DNA was sheared by passing it 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 once with an equal volume of chloroform-isoamyl alcohol (24: 1). DNA was ethanol precipitated using 0.3 M sodium acetate. The DNA was dried and dissolved in TE, and the sample concentration was confirmed by determining the optical density. The concentration was confirmed by electrophoresis through an agarose gel and staining with ethidium bromide.
[0072]
Southern blotting and hybridization. Southern blotting for detection of HPV31b DNA was performed as previously described (Ozbun, MA, et al. (1998) Virology 248: 218-230). A sample (5 μg) of total cellular DNA was digested with XbaI overnight. XbaI linearizes the HPV31b genome at nucleotide position 4998. Total cellular DNA samples were separated on a 0.8% agarose gel. The DNA was transferred to a GeneScreen Plus membrane (New England Nuclear Research Products, Boston, Mass.) And the membrane was handled according to the manufacturer's instructions. For probe preparation, plasmid pBS-HPV31 was digested with EcoRI to release the complete HPV31 genome. The HPV31 sequence was purified from the plasmid sequence by agarose gel electrophoresis and Gene Clean (BiolOl, Vista California). The DNA sequence was analyzed using the Random Primed DNA labeling kit (Boehringer Mannheim Corp., Indianapolis, Indiana) according to the manufacturer's instructions. ³² P] dCTP (3,000 Ci / mmol; DuPont NEN). The labeled probe was separated from unincorporated nucleotides by centrifugation through a Sephadex G-50 column (Boehringer Mannheim Corp.). Hybridization was performed at 10 per mL probe. ⁶ Performed at cpm. The membrane was washed to remove non-specific hybridization and then exposed to film. The intensity of HPV31b replication in the treated samples was then compared to mock-treated control DNA to determine the effect of each treatment on virus replication. Densitometric analysis of Southern blots was performed to numerically compare the effect of compounds on HPV31b replication on untreated control samples. Replication levels were also compared to a standard copy number control to numerically determine the effect of treatment on the number of HPV31b genomes per cell.
[0073]
Histochemical analysis. Histological analysis of the tissues was performed to determine the effect of drug treatment on the differentiation and morphology of the raft tissues. Raft cultures were grown for 10 days in the presence of various treatments, harvested, fixed in 4% paraformaldehyde, and embedded in paraffin. A 4 μM cross section was prepared. Sections were stained with hematoxylin and eosin.
[0074]
(result)
The results from the Southern blot analysis can be seen in Table 1 and FIG.
[0075]
Table 1 shows the effect of each oligonucleotide (SEQ ID NOs: 1-37) on HPV31b replication, expressed as a relative densitometric analysis when compared to HPV31b replication levels generated in mock-treated cells. These oligonucleotides varied in their ability to reduce HPV31b replication when compared to mock-treated cells. Particularly potent oligonucleotides at 25 μM 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 highest inhibitory effect of HPV31b replication at both concentrations tested, at 25 μM achieving a nearly １ ／ -fold reduction in viral replication, and at 2.5 μM Approximately 1 / 5-fold reduction in virus replication was achieved. Some compounds did, in fact, enhance replication of HPV31b compared to sham controls.
[0076]
FIG. AE: CIN-612 9E raft cultures are treated with 25 μM (final concentration) oligonucleotides of HPV / L1-HPV / L25 and ORI 1001 (SEQ ID NOs: 1-37) of the invention and HPV31b genome replication To determine its effect. 2 shows Southern blot hybridization of total DNA isolated from treated CIN-612 9E raft cultures. 5 μg of total DNA from each raft was digested with HindIII to linearize episomal HPV31b DNA, electrophoresed on a 0.8% agarose gel, transferred to a nylon membrane, and then hybridized with an HPV31b-specific probe. . Lane 1 for each compound tested consists of uncleaved DNA. Lane 2 for each compound tested consists of HindIII digested episomal DNA. Form I (FI) indicates supercoiled DNA, Form II (FII) indicates nicked circular DNA, and Form III (FIII) indicates linearized DNA.
[0077]
Table 1. Effect of oligonucleotides of the invention (SEQ ID NOs: 1-34) on HPV31b replication in raft cultures. Oligonucleotides were tested at final concentrations of 2.5 mM and 25 mM. The effect of the treated cells was compared to mock-treated cells in a Southern blot densitometric analysis of total DNA isolated from raft cells probed with an HPV31b-specific probe.
[0078]
[Table 1]

ND = not determined.
X = thymidine or uracil, and any nucleotide can be replaced with inosine.
Underlined nucleotides are 2'-O-methyl ribonucleotides, and all other nucleotides are unmodified deoxyribonucleotides.
The internucleotide linkage is a phosphorothioate.
[0079]
The invention has been described in detail with reference to preferred embodiments thereof. It is understood, however, that in light of the present disclosure, those skilled in the art may make modifications and improvements which fall within the spirit and scope of the invention as set forth in the appended claims.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of the HPV genome.
FIGS. 2A-E show Southern blots of total DNA isolated from CIN-612 9E raft cultured cells hybridized with HPV31b-specific probes to determine the level of HPV replication in culture. Shows hybridization. CIN-612 9E cultures were treated with 25 μM of the oligonucleotides of the invention and the level of inhibition of viral replication was compared to mock-treated cultures.
FIGS. 3A-B are Southern blots of total DNA isolated from CIN-612 9E raft cultures hybridized with HPV31b-specific probes to determine the level of HPV replication in culture. Shows hybridization. CIN-612 9E cultures were treated with 2.5 μM of the oligonucleotides of the invention and the level of inhibition of viral replication was compared to mock-treated cultures.

Claims (43)

Oligonucleotides that are complementary to the portion of the human papillomavirus E1 open reading frame comprised between nucleotides +1 to +20 and comprise at least 4 nucleotides. 2. The oligonucleotide according to claim 1, wherein said oligonucleotide is modified. 3. The oligonucleotide of claim 2, wherein at least one modification occurs in an internucleotide linkage. The modification is an alkyl phosphonate bond, phosphorothioate bond, phosphorodithioate bond, alkylphosphonothioate bond, phosphoramidate bond, carbamate bond, carbonate bond, phosphate triester bond, acetamidodate bond, carboxymethyl ester bond, The oligonucleotide according to claim 3, comprising at least one internucleotide bond selected from the group consisting of: and a combination thereof. 5. The oligonucleotide of claim 4, wherein said modification comprises at least one phosphorothioate internucleotide linkage. The oligonucleotide of claim 5, wherein all internucleotide linkages are phosphorothioate internucleotide linkages. 3. The oligonucleotide according to claim 2, comprising at least one deoxyribonucleotide. 3. The oligonucleotide according to claim 2, comprising at least one ribonucleotide. 3. The oligonucleotide according to claim 2, comprising at least one deoxyribonucleotide and at least one ribonucleotide. 9. The oligonucleotide according to claim 8, comprising at least one 2'-O-methyl nucleotide. 3. The oligonucleotide according to claim 2, comprising at least one inosine nucleotide. 3. The oligonucleotide of claim 2, comprising at least one modification to the sugar moiety of at least one mononucleotide. 3. The oligonucleotide according to claim 2, comprising at least one modification to the base portion of at least one mononucleotide. 3. The oligonucleotide according to claim 2, comprising a cap at the 5 'end. 3. The oligonucleotide according to claim 2, comprising a cap at the 3 'end. 3. The oligonucleotide according to claim 2, comprising caps at the 5 'and 3' ends. 3. The oligonucleotide according to claim 2, comprising the sequence of SEQ ID NO: 36 or a part thereof. The oligonucleotide according to claim 2, comprising a sequence selected from the group consisting of any one or more of SEQ ID NOs: 1 to 37. 3. The oligonucleotide according to claim 2, comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 11, 21, 26, 28 and 34. 3. The oligonucleotide according to claim 2, comprising SEQ ID NO: 1. 3. The oligonucleotide according to claim 2, comprising SEQ ID NO: 2. 3. The oligonucleotide according to claim 2, comprising SEQ ID NO: 3. 3. The oligonucleotide according to claim 2, comprising SEQ ID NO: 28. 3. The oligonucleotide according to claim 2, comprising SEQ ID NO: 34. 3. The oligonucleotide of claim 2, which is capable of inhibiting HPV replication. 26. The oligonucleotide of claim 25, wherein said inhibition results from an interaction between said oligonucleotide and a nucleic acid. 26. The oligonucleotide of claim 25, wherein said inhibition is due to an interaction between said oligonucleotide and a protein. 28. The oligonucleotide of claim 27, wherein said protein is a receptor. 29. The oligonucleotide according to any one of claims 1 to 28, wherein said oligonucleotide has a total of 4 nucleotides. 29. The oligonucleotide according to any one of claims 1 to 28, wherein said oligonucleotide has a total of 18 nucleotides. 29. The oligonucleotide according to any one of claims 1-28, wherein said oligonucleotide has a total of 6-18 nucleotides. An oligonucleotide comprising the sequence set forth in any one of SEQ ID NOs: 1-25 or SEQ ID NOs: 27-37. An oligonucleotide consisting of the sequence of SEQ ID NOS: 1 to 25 or SEQ ID NOs: 27 to 37. The oligonucleotide according to any one of claims 1 to 33, wherein the oligonucleotide is a synthetic oligonucleotide. A pharmaceutical composition comprising at least one oligonucleotide according to any one of claims 1 to 34 and a pharmaceutically acceptable carrier. 36. A method of treating a mammal infected with a papilloma virus, comprising administering to the mammal at least one oligonucleotide or composition according to any one of claims 1 to 35. . 37. The method of claim 36, wherein said mammal is a human. 38. The method of claim 36 or 37, wherein the oligonucleotide is administered in combination with another antiviral compound. A method for treating a mammal suffering from a disorder or disease associated with HPV, comprising administering to the mammal an oligonucleotide or a composition according to any one of claims 1 to 35. Including, methods. 40. The method of claim 39, wherein said mammal is a human. 41. The HPV-related disorder or disease is benign cutaneous wart, genital wart, wart-like epidermal dysplasia, respiratory papillomatosis, pharyngeal papillomatosis or cervical cancer. The method described in. 42. The method of claim 41, wherein the HPV-related disorder or disease is associated with infection by HPV6, HPV11, HPV16, HPV18, HPV31, HPV45, or HPV58. A synthetic oligonucleotide capable of inhibiting HPV replication by a mechanism other than the antisense mechanism.

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