Oligonucleotides specific for human papillomavirus

The invention relates to human papillomavirus. In particular, the invention relates to the inhibition, treatment and diagnosis of human papillomavirus-related diseases by means of synthetic oligonucleotides complementary to human papillomavirus nucleic acid.

Human papillomaviruses (HPV) comprise a group of at least 70 types based on a difference in DNA sequence as measured by liquid hybridization (Pfister et al., 1994, Intervirol. 37: 143-149). These envelope-less DNA viruses infect epithelial cells, resulting in a range of diseases ranging from benign cutaneous and genital warts (Condyloma acuminata) and epidermodysplasia verruciformis (EV) to respiratory or laryngeal papillomatosis and cervical carcinoma. Each virus type shows host specificity.

Multiple types of HPV infect genital epithelia and are the most common etiological agents of sexually transmitted viral diseases. Genital HPV types are also most commonly associated with the development of neoplasms in HPV-16 and HPV-16 and high-risk types HPV-18, and "low-risk" types, which are rarely associated with malignancy, most commonly HPV-6 and HPV-11. The malignant types can integrate into the genome of the host cell and therefore avoid the requirement for viral DNA replication gene products. In contrast, the benign types, most commonly HPV-6 and HPV-11, require the viral proteins E1 and E2 for replication of the episomal genome.

The current treatment for HPV infections is extremely limited. There are currently no approved HPV-specific antiviral therapeutics. Treatment usually involves the physical destruction of the wart by surgical, cryo-operative, chemical or laser-mediated removal of infected tissue. Topical anti-metabolites such as 5'-fluorouracil and podophyllum preparations have also been used (Reichman in Harrison's Principles of Internal Medicine, 13th ed. Edition (Isselbacher et al., Ed.), McGraw-Hill, Inc., NY (1993), pp. 801-803). However, regression occurs after this treatment procedure frequently and repeated treatments gradually destroy healthy tissue. Interferon treatment has been the only treatment with an antiviral mode of action to date, but its use is limited by its limited efficacy (Cowsert (1994), Intervirol 37: 226-230, Bornstein et al (1993), Obstetrics Gynecol. Sur. 4504: 252-260; Browder et al. (1992) Ann. Pharmacother. 26: 42-45).

Two HPV types, HPV-6 and HPV-11, are commonly associated with laryngeal papillomas or benign epithelial laryngeal tumors. Newborns can be infected with a genital papillomavirus at the time of passage through the mother's birth canal. At the age of two, papillomas have developed and the infected children undergo multiple surgeries to remove the benign papillomas that can block the airway. At the moment, there is no treatment for childhood laryngeal papillomatosis. There is therefore a great need for a specific antiviral for the treatment of a human papillomavirus infection.

New chemotherapeutic agents capable of modulating cellular and foreign gene expression have been developed (see Zamecnik et al., (1978), Proc Natl Acad Sci. (USA) 75: 280-284). These substances, called antisense oligonucleotides, bind to single-stranded target nucleic acid molecules according to a Watson-Crick base pairing or to double-stranded nucleic acids by Hoogsteen base pairing. They destroy the function of the target through one or more mechanisms. They prevent the binding of factors necessary for normal transcription, splicing or translation, activate the enzymatic destruction of mRNA by RNase H or destroy the target by means of reactive groups directly linked to the antisense oligonucleotide.

Improved oligonucleotides have recently been developed and have greater efficacy in inhibiting such viruses, pathogens, and selective gene expression. Some of these oligonucleotides, which have modifications in their internucleotide linkages, were more effective than their unmodified counterparts. Agrawal et al. (Proc Natl Acad Sci. (USA) (1988) 85: 7079-7083) describe e.g. For example, oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates inhibit more potent HIV-1 than common phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (Proc Natl Acad Sci. (1989) 86: 7790-7794) describe the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.

In addition, chimeric oligonucleotides with more than one type of internucleotide linkages within the oligonucleotide have been developed. Pederson et al. (U.S. Patent Nos. 5,149,797 and 5,220,007) describe chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methyl phosphonates or phosphoramidates. Agrawal et al. (WO 94/02498) describe hybrid oligonucleotides with regions of deoxyribonucleotides and 2'-O-methylribonucleotides.

A limited number of antisense oligonucleotides have been designed that inhibit the expression of HPV. For example, oligonucleotides specific for different regions of HPV E1 and E2 mRNA have been prepared (see, e.g., U.S. Patent 5,364,758, WO 91/08313, WO 93/20095, and WO 95/04748).

The development of oligonucleotides which are capable of inhibiting the replication and expression of human papillomavirus and whose use is associated with successful treatment and little or no cellular toxicity is still required.

Brief description of the drawings

The above and other objects of the invention, various features thereof, and the invention itself may be better understood by the following description and drawings.

Fig. 1 is a schematic representation of the HPV genome.

Figure 2 is a graph of the antisense activity of 20mer PS oligonucleotides in stably transfected cells and the corresponding RNase H activity.

Figure 3 is a graph of a transient transfection luciferase assay used to demonstrate the antisense activity of the oligonucleotides of the present invention.

Figure 4 is a graph showing antisense inhibition of HPV luciferase expression in transiently transfected CHO cells treated with various concentrations of PS HPV1, HPV2 or HPV3.

Figure 5 is a graph showing antisense inhibition of HPV luciferase expression in transiently transfected CHO cells treated with various concentrations of PS HPV4, HPV5 and HPV6.

Figure 6 is a graph showing antisense inhibition of HPV luciferase expression in transiently transfected CHO cells treated with a combination of various concentrations of PS HPV1, HPV4 and HPV6.

Figure 7 is a graph showing the effect of various concentrations of HPV1 or a random oligonucleotide on the expression of HPV luciferase in keratinocytes, which were introduced into the cells by means of a lipid carrier.

Figure 8 is a graphic representation of the antisense activity of base mismatched oligonucleotides in a test with stably transfected CHO cells.

Figure 9 is a graph of the antisense activity of base mismatched oligonucleotides and oligonucleotides in which mismatches have been replaced by inosine in a stably transfected CHO cell assay.

Figure 10A is a graph showing antisense activity of HPV1, HPV32, HPV33, HPV30 and HPV34 in a stably transfected CHO cell assay.

Figure 10B is a graph showing antisense activity of HPV1, HPV31, HPV38 and HPV35 in a stably transfected CHO cell assay.

FIG. 11 Figure 3 is a graphic representation of the effects of length and chemical modifications on antisense activity in stably transfected cells, where HPVn = phosphorothioate (PS); 2 OMe 3 '= 3' end with 2'-O-methyl RNA-PS modification at 5 nucleotides; Methylphos 3 '= 3'-end with methylphosphonate modification at 5 nucleosides; 2 'OMe PO or PS = only 2'-O-methyl-RNA phosphodiester or phosphorothioate; 2 'OMe 5', 3 'PO or PS = 2'-O-methyl-RNA-PO / PS modification at the 5' and 3 'ends at 5 nucleotides.

Recent advances in HPV research have now made it possible to more effectively target antiviral compounds against HPV. Two proteins encoded by the virus, E1 and E2, were found to be essential for replication of the viral genome (Ustav et al., 1991, EMBO J. 10: 449-457; Chiang et al., 1992, Proc. Natl. Acad Sci. (USA) 89: 5799-5803). Most HPV types need both proteins for initiation of viral DNA replication. However, it has recently been shown that only E1 is required in certain in vitro experiments (Gopalakrishnan et al (1994), Proc Natl Acad Sci. (USA) 91: 9597-9601).

E1 is one of eight viral proteins encoded by the circular, double-stranded, 7900 base pair DNA genome of all HPV types (see Figure 1). The genome can be divided into three distinct functional domains: the upstream regulatory region (URR), which contains the origin for viral DNA replication and enhancers and promoters involved in transcription; the L region encoding the structural proteins L1 and L2; and the E region, which encodes genes necessary for autonomic functions. The eight viral proteins shown schematically in FIG. 1 are translated starting from complex families of alternatively-spliced ​​mRNAs.

E1 is an ATP-hydrolyzing DNA helicase believed to be involved in the unwinding of DNA at viral origin during the replication of the HPV genome by the DNA replication complex of the human host (Hughes et al., 1993, Nucleic Acids Res. 21: 5817-5823; Chow et al. (1994), Intervirol. 37: 150-158). Thus, E1 represents a virus-specific target with a defined biochemical function that can be measured in cells expressing that gene.

For the development of a therapeutic anti-sense compound against human papillomaviruses, the E1 gene was targeted by HPV types 6 (GenBank HPV6b, accession number M14119) and 11 (GenBank HPV11, accession number X00203). Types 6 and 11 are associated with more than 90% of all cases of non-malignant genital warts. A region of 46 nucleotides (-17 to +29 of the E1 open reading frame) located at the center of the protein translation initiation site has been studied in detail. This region is conserved in a number of clinical isolates of HPV types 6 and 11. The entire open reading frame of the gene (-17 to +1950) was also examined as an antisense target. This entire region shows high sequence identity between HPV type 6 and HPV type 11.

It has been found that specific oligonucleotides that are complementary to certain portions of the nucleic acid encoding the translational start site for the human papillomavirus E1 gene can inhibit HPV replication and expression. This result has been exploited in accordance with the invention to provide synthetic oligonucleotides that are complementary to regions that extend beyond or are adjacent to the translational start site of the HPV E1 protein-encoding mRNA.

A "synthetic oligonucleotide" here comprises chemically synthesized polymers of from about 5 up to about 50, preferably from about 15 to about 30, ribonucleotide and / or deoxyribonucleotide monomers attached to each other by at least one, preferably more than one 5 'to 3' Internucleotide binding are linked or linked.

The term "oligonucleotide sequence which is complementary to nucleic acid or mRNA" according to the invention means an oligonucleotide which is linked to the nucleic acid sequence under physiological conditions, eg. B. by Watson-Crick base pairing (interaction between the oligonucleotide and a single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between the oligonucleotide and a double-stranded nucleic acid) or in any other way, including in the case where an oligonucleotide attached to RNA binds, causing the formation of a pseudo knot. The binding by Watson-Crick or Hoogsteen base pairing under physiological conditions is conveniently determined by measuring the interference with the function of the nucleic acid sequence.

In a first aspect, the invention provides synthetic oligonucleotides that are complementary to a nucleic acid extending across the translational start site of the human papillomavirus E1 gene. The oligonucleotides comprise at least 15 nucleotides. In preferred embodiments, the oligonucleotides of the invention are about 15 to about 30 nucleotides in length.

The oligonucleotides according to the invention are modified. In one embodiment, the modifications comprise at least one internucleotide linkage selected from the group consisting of alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate or carboxymethyl ester, including combinations of such linkages, e.g. In a chimeric oligonucleotide. In a preferred embodiment, an oligonucleotide of the invention comprises at least one phosphorothioate internucleotide linkage. In another preferred embodiment, all internucleotide linkages in the oligonucleotide are phosphorothioate internucleotide linkages. In another preferred embodiment, the oligonucleotide comprises at least one methylphosphonate internucleotide linkage. In a further particular embodiment, the oligonucleotide comprises at least one n-butylphosphoramidate bond. In one embodiment, at least one methylphosphonate or n-butylphosphoramidate linkage is at the 3'-end. Better are about five such bonds at the 3 'end.

In further embodiments, the oligonucleotides of the invention may also comprise at least one deoxyribonucleotide, at least one ribonucleotide, or a combination thereof, e.g. In a hybrid oligonucleotide. In a particular embodiment, the oligonucleotide may consist only of deoxyribonucleotides. In one embodiment of the invention, the oligonucleotide contains at least one 2'-O-methylribonucleotide. In particularly preferred embodiments of the invention, the oligonucleotide has five 2'-O-methylribonucleotides at the 3'-end or at the 3'- and 5'-ends. Other embodiments include at least one or at least two inosine residues at any position in the oligonucleotide.

In particular, in one embodiment, the oligonucleotides according to the invention have one of the sequences shown in Table 1A or in the sequence listing as SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 32, 36, 37 and 38 shown sequence. In a further embodiment, the oligonucleotides of the invention have one in Table 1B as SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 , 108, 109, 110, 111, 116, 117, 118, 119, 120, 121, 122 and 130, nucleotide sequence. All of these oligonucleotides may be further modified as shown in the description.

In further aspects, a pharmaceutical composition is provided according to the invention. The pharmaceutical composition is a physical mixture of at least one and preferably two or more HPV-specific oligonucleotides having the same or different sequences, modification (s) and / or lengths. In some embodiments, this pharmaceutical formulation also includes a physiologically or pharmaceutically acceptable carrier. Specific embodiments include a therapeutic amount of a lipid carrier.

The oligonucleotides of the present invention are suitable for use as therapeutically active compounds, particularly for use in the control or prevention of human papillomavirus infection.

In this aspect of the invention, a therapeutic amount of a pharmaceutical composition containing HPV-specific synthetic oligonucleotides is administered to a cell to inhibit the replication of human papillomavirus. In a similar aspect, the oligonucleotides of the present invention may be used for the treatment of human papillomavirus infection, including, but not limited to, a therapeutic amount of a pharmaceutical composition containing at least one HPV-specific oligonucleotide and in some embodiments at least two HPV-specific oligonucleotides to an infected animal or a cell is administered. In some preferred embodiments, the method comprises administering at least one oligonucleotide or at least two oligonucleotides having one of in Table 1A or in the Sequence Listing as SEQ ID NO: 1-11, 16, 18-28, 21-32 and 36-38 or in Table 1B as SEQ ID NOS: 41-111, 116-122 and 130, including modifications thereof.

In all methods involving administration of oligonucleotide (s) of the invention, at least one and preferably two or more identical or different oligonucleotides may be administered simultaneously or sequentially in a single treatment in the form of separate pharmaceutical compositions.

In a further aspect, the invention provides a method for detecting HPV in a sample, such as a solution or biological sample. In this method, the sample is brought into contact with a synthetic oligonucleotide according to the invention or with an oligonucleotide having a sequence complementary thereto. Hybridization of the oligonucleotide to the HPV nucleic acid is then detected if HPV is in the sample.

Another aspect of the invention are kits for the detection of HPV in a sample. Such kits comprise at least one inventive synthetic oligonucleotide or an oligonucleotide with a sequence complementary thereto and means for detecting the hybridized to the nucleic acid oligonucleotide. In a kit with more than one oligonucleotide according to the invention, these oligonucleotides may have the same or different nucleotide sequences, length and / or modification (s).

Synthetic oligonucleotides of the invention specific for the E1 nucleic acid, in particular mRNA, consist of deoxyribonucleotides, ribonucleotides, 2'-O-methylribonucleotides or any combination thereof, wherein the 5'-end of one nucleotide and the 3'-end of another Nucleotides are covalently linked together. These oligonucleotides are at least 15 nucleotides in length, preferably 15 to 30 nucleotides, with 20 to 30mers being most common.

The oligonucleotides can be prepared by known methods. For example, the nucleotides may be covalently linked together by known methods such as phosphoramidite, H-phosphonate or methylphosphoramidite chemistries (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 5,149,798). These methods can be performed by manual or automated synthesis, and the oligonucleotides are then further processed (for a review, see Agrawal et al., 1992, Trends Biotechnol., 10: 152-158).

The oligonucleotides of the invention may also be modified in various ways without impairing their ability to hybridize to the HPV nucleic acid. For example, the oligonucleotides may contain internucleotide linkages other than phosphodiester internucleotide linkages between the 5-terminus of one nucleotide and the 3'-end of another nucleotide in which the 5'-nucleotide phosphate has been replaced by any chemical moiety such as phosphorothioate. Oligonucleotides with phosphorothioate bonds can be prepared in a conventional manner, such as. By phosphoramidite chemistry (see, e.g., Agrawal et al (1988), Proc Natl Acad Sci. (USA) 85: 7079-7083) or H-phosphonate chemistry (see, for example, U.S. Pat Froehler (1986), Tetrahedron Lett 27: 5575-5578). The Bergot et al. (1992, J. Chromatog. 559: 35-42) can also be used. Examples of other chemical groups capable of forming an internucleotide linkage include alkylphosphonates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, carbamates, acetamidates, carboxymethyl esters, carbonates, and phosphate triesters.

Exemplary of a combination of internucleotide linkages, U.S. Patent 5,149,797 describes conventional chimeric oligonucleotides having a phosphorothioate core region located between flanking methyl phosphonate or phosphoramidate regions. Other chimeras are "inverted" chimeric oligonucleotides having one or more non-ionic oligonucleotide regions (e.g. Alkylphosphonate and / or phosphoramidate and / or phosphotriester internucleoside linkage) flanked by one or more regions of oligonucleotide phosphorothioates. Chimeric and reverse chimeras can be synthesized as described in the Examples for methylphosphonate-containing oligonucleotides. These "chimeric" and "reverse chimeric" oligonucleotides are preferred modifications of the oligonucleotides of the present invention.

Various oligonucleotides with modified internucleotide linkages can be prepared in a manner known per se (see, for example, 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).

Self-stabilized oligonucleotides are also considered to be modified oligonucleotides and are useful in the methods of the invention (Tang et al., 1993, Nucleic Acids Res. 20: 2729-2735). These oligonucleotides comprise two regions: a target-hybridizing region and a self-complementary region having an oligonucleotide sequence that is complementary to a nucleic acid sequence within the self-stabilized oligonucleotide. These oligonucleotides form loop structures that are believed to stabilize the 3 'end from exonuclease attack, with hybridization to the target still possible.

On the other hand, sugar modifications include modifications at the 2-position of the ribose residue, including, but not limited to, 2'-O substitutions by an -O-lower alkyl group having 1-6 saturated or unsaturated carbon atoms or by an -O-aryl group. or allyl group having 2-6 carbon atoms, wherein the -O-alkyl, aryl or allyl group may not be substituted or substituted (e.g. by halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxy or amino groups) or wherein the 2-O group is substituted by an amino or halogen group. Neither of these substitutions is intended to exclude the native 2'-hydroxyl group in the case of ribose or the 2'-H group in the case of deoxyribose. WO 94/02498 describes conventional hybrid oligonucleotides having regions of 2'-O-substituted ribonucleotides flanking a DNA core region. Another form of hybrid is a "reverse" hybrid oligonucleotide, e.g. For example, an oligonucleotide comprising a 2'-O-substituted (or 2'-OH-unsubstituted) RNA region located between two oligodeoxyribonucleotide regions, a structure that inverted compared to "conventional" hybrid oligonucleotides is. Hybrid and reverse hybrid oligonucleotides can be synthesized as described in the Examples for oligonucleotides with 2'-O-methyl RNA. The hybrid and reverse hybrid oligonucleotides of the present invention are particularly preferred because of their increased stability and activity in the presence of serum. In another embodiment, the hybrid or reverse hybrid oligonucleotide may comprise at least one n-butyl phosphoramidate or methyl phosphonate linkage.

Preferably, the ribonucleotide is a 2'-O-methyl ribonucleotide. In another embodiment, the oligonucleotide comprises at least one, preferably one to five, 2'-O-methyl ribonucleotides at the 3'-end of the oligonucleotide. In addition, the oligonucleotide may further comprise at least one, preferably one to five, 2-O-methyl ribonucleotides at the 5'-end.

Other oligonucleotide structures according to the invention include the so-called dumbbell and open dumbbell structures (Table 1B). Ashly and Kushlan ((1991), Biochem 30: 2927-2933) describe the synthesis of oligonucleotide dumbbells, including open dumbbells. A dumbbell is a double-helical stem terminated by two hairpin loops. The antisense activity of open dumbbells (free-end dumbbell molecules) is described by Yamakawa et al. ((1996), Nucleosides and Nucleotides 15: 519-529). It is believed that these structures possess favorable properties, similar to those of the self-stabilized oligonucleotides described above.

Other modifications may be in or on the end (s) of the oligonucleotide molecule and include additions to the molecule's internucleoside-phosphate bonds, such as cholesteryl, cholesterol or diamine compounds having a different number of carbon moieties between the two amino groups, and Modifications of the terminal ribose, deoxyribose, and phosphate that bind opposite chains or associated enzymes or other proteins that bind to the viral genome, network or split off. Other linkers, including non-nucleoside linkers, include, but are not limited to, polyethylene glycol of a different length 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 non-basic linkers or commercially available asymmetric and symmetric linkers (Clone Tech, Palo Alto, California) (e.g., Glen, Research Product Catalog, Sterling, VA).

Further examples of modified oligonucleotides include those with a modified base and / or a modified sugar such as arabinose instead of ribose or a 3,5'-substituted oligonucleotide with a sugar at the 3 'or 5' position or at both positions a chemical group except a hydroxyl or phosphate group (at the 3 'or 5' position) is bound.

In addition, oligonucleotides having a ribose cap at the 3'-end may undergo NalO₄ oxidation / reductive amination. Examples of such species are given in Table 1B. Amination includes, but is not limited to, the following: spermine, spermidine, tris (2-aminoethyl) amine (TAEA), DOPE, long chain alkylamines, crown ethers, coenzyme A, NAD, sugars, peptides, dendrimers.

In another embodiment, at least one cytosine base may be modified by a known methylation, e.g. 5'-methylated deoxycytosine (5-Me-dC) (see Table 1B). Such methylation may, for. B. for reducing stimulation of the immune system by the oligonucleotide, if necessary, be desired.

Other modified oligonucleotides carry a cap with a bulky substituent at the 3 'and / or 5' ends conferring resistance to nuclease, or carry substitution on one or both non-bridging oxygens per nucleotide. Such modifications may be present at some or all of the internucleoside linkages as well as at one or both ends of the oligonucleotide and / or within the molecule (see review in Agrawal et al., 1992, Trends Biotechnol. 10: 152-158). Non-limiting examples of the capped species include 3'-O-methyl, 5'-O-methyl, 2'-O-methyl and combinations thereof as shown in Table 1B.

In a preferred embodiment, the oligonucleotide has a complementary nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 (HPV1), 11 (HPV19), 18 (HPV30), 19 (HPV31), 20 (HPV32), 21 (HPV33). and 26 (HPV38) as shown in Table 1A, including modifications thereof.

In a further embodiment, the oligonucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NOS: 54 (HPV56), 118 (HPV53), 119 (HPV52), and 121 (HPV50), as shown in Table 1B, including modifications thereof.

In a particular embodiment, the oligonucleotides of the aforementioned two embodiments are deoxyribonucleotides and have phosphorothioate internucleotide linkages.

In a further specific embodiment, the oligonucleotide is selected from the group of sequences having SEQ ID NOS: 1, 41-111, 116-122, and 130 as shown in Table 1B, wherein the oligonucleotide comprises the internucleotide linkage composition and further modifications as described in Table 1B has.

Most preferably, the oligonucleotide has a nucleotide sequence and further modifications as indicated for an oligonucleotide selected from the group consisting of SEQ ID NOS: 88 (HPV1 8-4-8 IH 2'-Ome PO), 88 (HPV1 8-4-8 IH 2'-OMe PS), 89 (7-6-7 IH 2'-OMe PO), 89 (7 6-7 IH 2'-OMe PS), 90 (HPV1 9-6-5 IH 2'-OMe PO), 90 (HPV1 9-6-5 IH 2'-OMe PS), 91 (5-6-9 IH 2'-OMe PO), 91 (5-6-9 IH 2'-OMe PS), 92 (10-6-4 IH 2'-OMe PO), 92 (10-6-4 IH 2'-OMe PS), 93 (HPV1 6-8-6 IH 2'-OMe PO), 93 (HPV1 6-8-6 IH 2'-OMe PS) and 96 (HPV1 0x5 hybrid), from SEQ ID NOS: 41 (SS1), 42 (SS2), 43 (SS3), 44 (SS4), 49 (SS9) and 51 (SS11), from SEQ ID NOS: 54 (HPV56 CAP), 57 (SS16), 59 (SS18), 65 (SS26), 67 (SS28) and out of 104 (HPV56 0 · 5 hybrid), and from SEQ ID NOS: 1 (HPV1 5-Me-dC) , 24 (HPV36 5-Me-dC) and 112 (HPV43 5-Me-dC).

20mer phosphorothioate oligonucleotides complementary to the E1 gene of HPV strains 6a and 6b (in vitro transcribed RNA = 2328 bases) were probed with a ribonuclease H (RNase H) assay with 100 nM synthetic oligonucleotide and in vitro transcribed RNA tested. The RNase H test identified regions of the target RNA that were accessible to the antisense oligonucleotide. Cleavage revealed that the oligonucleotide had hybridized to the target RNA such that the target was cleaved by RNase H. The results of RNase H-mediated cleavage are shown in Table 1A. Position +1 of the E1 target site is the first base of the start site for translation. TABLE 1A

a possible triplex-forming oligonucleotide

b the lowercase letters represent a base with a mismatch, cursive letters represent triplex forming bases. The internucleotide bond is PS unless otherwise stated.

These results suggest that the region closest to the translation start site (AUG) is accessible to antisense oligonucleotides and can be cleaved by RNase H. The data also defines a very active region for hybridization and cleavage in the range of -13 to +20. The best of these oligonucleotides were HPV1 (+1 to +20) (SEQ ID NO: 1), HPV3 (-9 to +11) (SEQ ID NO: 3), HPV4 (-13 to +7) (SEQ ID NO: 1). 4) and HPV5 (-5 to +15) (SEQ ID NO: 5).

Furthermore, four regions in the downstream coding region that appear to be amenable to hybridization with antisense oligonucleotides were identified by the Randomer RNase H test. The oligonucleotides bound to these regions are HPV20 (+203 to +222) (SEQ ID NO: 12), HPV21 (+231 to +250) (SEQ ID NO: 13), HPV22 (+282 to +301) (SEQ ID NO: 14) and HPV23 (+373 to +392) ​​(SEQ ID NO: 15). The results are shown in Table 1A. The data suggest that the region at +373 is the site most heavily cleaved by RNase H in the presence of the complementary DNA phosphorothioate sequence.

The oligonucleotides identified outside the E1-luciferase fusion target sequences can be tested by assaying the expression of the complete E1 gene product (see Example 6 below).

These and other antisense oligonucleotides directed against the translation start site were tested in mammalian cells by reporter gene assays using firefly luciferase. The 46 nucleotide region of the HPV E1 gene from nucleotides -17 to +29 relative to the translation start site was cloned 5 'to and in frame with the entire open reading frame of the firefly luciferase gene in the plasmid pGLori to produce the plasmid pE1Luc6 , Transcription of this E1-luciferase gene fusion was placed under the control of the cytomegalovirus early gene promoter. Expression of E1-luciferase fusion in mammalian cells was quantified in a luminometer by adding luciferin substrate and ATP cofactor to cell lysates. The reduction of luciferase levels in antisense oligonucleotide-treated cells as compared to luciferase levels in negative control (random oligonucleotide; randomer) treated cells serves as a measure of the sequence-specific activity of the antisense oligonucleotides.

In all cellular antisense assays, a 20-mer phosphorothioate oligonucleotide with a random sequence was used as the negative control. Further, a 20-mer phosphorothioate antisense oligonucleotide directed against the first 20 nucleotides of the firefly luciferase gene coding region was used as a positive control (Luc +1 - +20) (SEQ ID NO: 39). This goal is present in the E1 fusion and control luciferase constructs.

Chinese hamster ovary cells (CHO-K1) were stably transfected with the pE1Luc6 construct. The percentage of luciferase expression, measured relative to the effective concentration of control (EC50), was then measured for the oligonucleotide which gives an inhibition of 50% of the control (ie, cells treated with lipid only). The phosphorothioate (PS) 20mer oligonucleotides 1, 3, 4, 5, and 17 all displayed sequence-specific antisense activity against the E1 Luc6 target, as did the positive control Luc + 1-20 PS antisense oligonucleotide, which was directed against the first 20 nucleotides of the coding region of the luciferase gene. Two E1-specific 20mer oligonucleotides, 2 and 6, and the negative PS 20mer oligonucleotide of the negative control showed little or no activity (Figure 2). There was a clear correlation between in vitro RNase H cleavage of the target RNA and sequence-specific antisense activity in stably transfected cells (Figure 2). None of the oligonucleotides except the positive control Luc +1-20 + oligonucleotide exhibited sequence-specific antisense activity in CHO-K1 cells stably transfected with the parental pGLori construct carrying only the luciferase gene.

The other oligonucleotides listed in Table 1B below also showed activity.

Capital letters represent the antisense sequence

Lowercase letters represent the Nicbt antisense sequence

Outlined residues are base-paired

The underlined sequence is 2'-OMe RNA

The bold sequence is methylphosphonate

L = non-nucleosidic polyethylene glycol (PEG) linker

The internucleotide linkage is PS, unless otherwise stated

Antisense tests with the oligonucleotides according to the invention were also carried out in transiently transfected CHO cells. The cells were transfected using the lipid carrier Lipofectamin with either plasmid pE1 Luc6 or control plasmid pGLori in the presence of PS oligonucleotides (Figure 3). Two independent methods for analyzing antisense activity were performed. In the first method, the amount of reporter plasmid was titrated over a 1000-10000 fold range to determine the linear range of luciferase expression under these assay conditions. Antisense oligonucleotides were added at fixed concentrations to each of these plasmid dilution series and the luciferase activity measured. A decrease in the luciferase signal in a plasmid titration curve caused by the addition of oligonucleotide shows an antisense effect. This method was later improved by fixing the concentration of the reporter plasmid at an optimum to accurately titrate the amount of an oligonucleotide required for a specific antisense effect. This method was used to determine relative luciferase expression, measured in relative luciferase units (see Figures 4 and 5) for particular compounds, and to determine small differences in their activity.

Figures 4 and 5 show that phosphorothioate oligonucleotides tested in this region, including HPV1 (SEQ ID NO: 1), HPV2 (SEQ ID NO: 2), HPV3 (SEQ ID NO: 3), HPV4 (FIG. SEQ ID NO: 4), HPV5 (SEQ ID NO: 5) and HPV6 (SEQ ID NO: 6) are active antisense compounds. HPV17 (SEQ ID NO: 9) was also active in this assay. Randomer negative control caused minor effects on both plasmids up to 300 nM. The Luc +1 - +20 positive control, directed against both constructs, shows specific antisense activity against both constructs. HPV-specific antisense activity occurs at concentrations of less than 1 nM to more than 300 nM. HPV1 to 6 show similar specific activities against pE1Luc6 (Figures 4 and 5). At 100 nM, all compounds specifically reduce expression of E1 luciferase by more than 90% compared to the Randomer control. At concentrations greater than 100 nM, the random oligonucleotides have non-sequence specific inhibitory effects in the transiently transfected cell system. Accordingly, the data are not shown for oligonucleotide concentrations above 100 nM. Compared with gene expression from the control plasmid pGLori, these compounds show the same effect as the randomer, indicating that they are only specifically directed against the HPV E1 sequence.

HPV24 (SEQ ID NO: 29) is a 28mer variant of HPV17 (SEQ ID NO: 9) with a 3 'appendage created to refold to form a stabilizing triplex structure. In the transiently transfected CHO cell assay, this oligonucleotide retained antisense activity. Other similar oligonucleotides also showed antisense activity (see Table 1B).

The use of a mixture of different oligonucleotides directed against different conserved sites within a particular viral gene may be desirable. Such an oligonucleotide mixture may be in the form of a therapeutic composition comprising at least one, two or more oligonucleotides in a single therapeutic composition (ie, a composition comprising a physical mixture of at least two oligonucleotides). Alternatively, these oligonucleotides may have two different sequences. For example, various compounds have been mixed that are directed against distinct, separate or overlapping regions within the E1 luciferase transcript, with the absolute oligonucleotide concentration maintained at 100 nM. Figure 6 shows that E1-specific oligonucleotides were active in admixture with other E1-specific oligonucleotides, Randomer or Luc + 1-20. This demonstrates that lower concentrations of individual oligonucleotides can be combined to maintain strong specific antisense activity.

A relevant cell line for measuring antisense activity against HPV is the target cell of the virus, human keratinocytes. HPV-specific oligonucleotides of the present invention were tested in transient transfection assays similar to those above for CHO cells. Neonatal human epidermal foreskin keratinocytes (NHEK) were transiently transfected with pE1Luc6 or pGLori using the lipid carrier lipofectamine. PS oligonucleotides were added to the cells in the presence of a lipid carrier. The results shown in Figure 7 show that luciferase expression is strong in the presence of a random oligonucleotide or in the absence of oligonucleotide in the keratinocytes (between 10 & and 10 & Relative Radiation Units (RLU) in each well). The randomer does not cause measurable nonspecific effects in cells transfected with either of the two reporter plasmids pE1 Luc6 or pGLori. The HPV1 oligonucleotide added in the presence of lipofectamine to cells transfected with pE1 Luc6 reduced luciferase expression to 2 x 10 & sup4; RLU at a concentration of 100 nM, which shows a sequence-specific effect. A similar effect was observed if the oligonucleotides were added in the absence of a lipid carrier.

Thus, oligonucleotide-specific reductions in reporter plasmid expression in normal human keratinocytes could be demonstrated in these experiments if the oligonucleotides were administered to the cells with a lipid carrier.

The activity of the oligonucleotides according to the invention can be confirmed in in vitro cultured three-dimensional epithelia. This involves applying HPV-positive keratinocytes to a collagen membrane (collagen raft) and culturing the cells at the air-liquid interface. The keratinocytes used in these experiments may be derived from normal neonatal foreskin or may be obtained from condylomata acuminata biopsy material. These collagen-raft cultures (organotypic) allow the keratinocytes to differentiate and form a three-dimensional structure similar to that found in vivo. This directed process of normal cellular differentiation may allow vegetative replication of papillomavirus, a process that requires replication of the viral genome in the cell. Antisense oligonucleotides are added to the culture medium under the raft. As in vivo, the oligonucleotides must be taken up by the keratinocytes and must reach the cells where active viral DNA replication takes place to prevent this process. The effect of antisense oligonucleotides on the life cycle of HPV can be monitored by visualizing the amount of virus in each raft culture using in situ hybridization with probes for HPV DNA. Quantification can be done by image analysis. In addition, the expression of individual viral genes can be shown and the possible mechanism of action of the antisense oligonucleotide can be determined if specific ribo probes are used for individual viral open reading frames. A common immunohistochemical analysis of collagen raft cultures is also used to demonstrate expression (or lack) of viral proteins. In addition, classical histology, combined with immunohistochemistry, is also used to show an association between active papillomavirus infection, atypical cell histology, and aberrant cellular differentiation.

To determine whether oligonucleotides of the invention possessed sequence-specific antisense activity, an increasing number of mismatches were introduced into the HPV1 sequence: the G residues were sequentially mutated to A (see Table 1A, in which the lower case letters in HPV7-10 , 12-14 and 29 represent the position of mismatches relative to the target sequence). With CHO-K1 cells stably transfected with the E1Luc6 construct, it was demonstrated that mismatch did not appreciably affect the sequence-specific antisense activity, but that two or more mismatches affected the activity of HPV1 (SEQ ID NO: 1). eliminated (Figure 8). This was consistent with the effectiveness of RNase H cleavage of the oligonucleotides shown in Table 1A. HPV7 (SEQ ID NO: 31) with a base mismatch showed no effect on RNase H cleavage. However, two mismatches (HPV8, SEQ ID NO: 32) reduced RNase H cleavage by 50% and three mismatches (HPV9, SEQ ID NO: 33) substantially abolished RNase H activity. Similar results were obtained in the system with transiently transfected CHO cells.

The development of a compound effective against many clinical isolates of HPV requires the selection of a highly conserved region of E1. However, base mismatches may occur in the antisense oligonucleotides directed against more than one type of HPV, and two base mismatches can eliminate the antisense activity of HPV1 (see Figure 8). One solution to the problem of sequence variation is the creation of oligonucleotides that can bind to many sequences. An oligonucleotide was designed in which mismatches are replaced by inosine nucleosides (HPV12-14, Table 1A, Figure 9, where the "i" end in oligonucleotides HPV12-14 shows the positions where the mismatched bases are replaced by inosine residues were). Inosine forms hydrogen bonds with all normal bases to a different extent. In the test system with stable transfection, replacement of one or the other mismatch in HPV8 (SEQ ID NO: 32) with inosine partially restored antisense activity (Figure 9). However, replacement of both mismatches by inosine produced antisense activity almost of HPV1. This again agrees with the data of the RNase H cleavage shown in Table 1A. In the presence of two mismatches (HPV8, SEQ ID NO: 32), the cleavage efficiency decreased to 52% over that of HPV1. Replacement of the 5 'most mismatch (in the oligonucleotide) with inosine (HPV12, SEQ ID NO: 36) increased cleavage to 61% over HPV1. Replacement of only the most 3 'mismatch by inosine (HPV13, SEQ ID NO: 37) more effectively reduced the mismatch effect and increased cleavage to 76% over HPV1. Replacement of both mismatches by inosine (HPV14, SEQ ID NO: 38) further increased cleavage to 81% over HPV1. This shows that the positioning of inosine at the sites where there are differences between the strains allows oligonucleotide activity against multiple HPV strains. Similar results were obtained comparing HPV8 to HPV14 in transiently transfected CHO cells.

The relationship between oligonucleotide length and activity was also examined. Extension of the 20mer HPV1 at the 3 'end to a 24mer (HPV15, SEQ ID NO: 8) or 28mer (HPV11, SEQ ID NO: 7) had no effect on the antisense activity of the oligonucleotide as measured in the luciferase Tests with stable or transiently transfected CHO-K1 cells. Further, in the system of stably transfected CHO cells, gradual truncation of bases at the 5 'or 3' end of HPV1 (HPV30-39, Table 1A) showed that antisense activity was maintained even when four bases from the 5 th 'End (Figure 10A) and five bases from the 3-end (Figure 10B).

The effect of chemical modifications on antisense activity was also examined. Several different chemical modifications were tested: 5 nucleotides at the 3'-end with methylphosphonate or 2'-O-methyl-RNA chemical modifications; 5 nucleotides at the 5 'and 3' ends with 2'-O-methyl-RNA chemical modifications and complete 2'-O-methyl-PO and -PS oligonucleotides.

Figure 12 shows the data for the various chemical modifications tested in stably transfected CHO-K1 cells. The introduction of five 2'-O-methyl-RNA chemical modifications at the 3 'end or at the 3' and 5 'ends of the sequence appears to increase the activity of the 20mer PS HPV1, while similar methylphosphonate modifications decreased the activity of the 20mer PS HPV1. Longer oligonucleotides improved the activity of methylphosphonate modifications at the 3'-end. Oligonucleotides with an exclusive 2'-O-methyl RNA backbone with PO or PS bonds were inactive, suggesting a role of RNase H in antisense activity. Compounds with an n-butyl phosphoramidate backbone, 5 n-butyl phosphoramidates at the 3 'end or a mixed n-butyl phosphoramidate and 2'-O-methyl RNA structure should be active and have an activity which is between that of the phosphorothioate and methyl phosphonate compounds.

The 2'-O-methyl-RNA phosphorothioate hybrid oligonucleotides had even greater activity than deoxyribose phosphorothioates and, regardless of oligonucleotide length, each hybrid oligonucleotide was more active than its corresponding homogeneous phosphorothioate oligonucleotide. The 2'-O-methyl-RNA-phosphorothioate-mixed backbone version of HPV1 was more active than the phosphorothioate compound in similar assays with transiently transfected CHO cells and methylphosphonate-HPV1 retained antisense activity.

Experiments with a mixed backbone design were repeated with oligonucleotides of different lengths to determine if increasing the length could alter the activity of the compound. Therefore, two longer versions of HPV1 (a 20mer) in three backbone configurations (PS, M, and OMe) were examined in transiently transfected CHO cells. In the case of 20mers (HPV15), the PS compound showed good antisense activity. The 2'-O-methyl-RNA compound was similarly active. The compound with the methylphosphonate backbone was slightly less active. When incorporating these modifications into a 28mer oligonucleotide (HPV11), similar results were observed.

Since the results showed similar or better activity of chimeric and hybrid oligonucleotides after 24 hours of cellular incubation time, the antisense effects of these oligonucleotides were examined over extended periods of time. The modified oligonucleotides have a greater resistance to degradation in the serum, which could result in a higher activity in the cells. In the test with transiently transfected CHO cells, the phosphorothioate compound showed a loss of activity from day 1 to day 7. In contrast, the 2'-O-methyl-RNA-phosphorothioate hybrid retained high activity until day 7. Similar results were obtained with 24meres and 28meres.

In summary, a combination of a chimeric backbone and phosphorothioate linkages (which mediate cellular RNase H activity) and modifications at the 3 'and / or 5' ends revealed that the antisense effect against E1 expression one week after administration to the Cells was retained.

For the testing of the toxicity of the oligonucleotides according to the invention, a commercially available cytotoxicity test (CellTiter 96 Non-Radioactive Cell Proliferation / Cytotoxicity Assay, Promega, Madison, WI) was used. The toxicity of the compounds was measured in parallel to the antisense activity using the standard transient transfection test system of cells. Regardless of the backbone, the oligonucleotides of the invention were non-toxic to cells at concentrations where specific antisense activity is observed.

Another test designed to demonstrate antisense activity against the native biochemical function of the viral E1 gene is to measure the ability of this protein to stimulate DNA replication initiated at the HPV origin of replication. DNA replication of papillomavirus in mammalian cells requires only three viral components, the E1 and E2 gene products, and a DNA sequence containing the HPV origin of replication. For the measurement of antisense activity against E1 gene expression, two plasmids were constructed expressing E1 or E2 by a CMV promoter. These two plasmids can be driven by oligonucleotides that bind anywhere in the E1 or E2 transcripts. As a reporter for E1 activity, a plasmid was constructed which expresses luciferase and additionally contains the HPV type 6 origin of replication. When transfected into a mammalian cell, the copy number of this plasmid increases if the E1 and E2 proteins are present. Finally, the cellular luciferase expression increases. This increase in enzyme activity can be quantified with a luminometer and the overall effect on viral DNA replication determined. A similar luciferase expression plasmid without the HPV origin can be prepared which serves as a negative control for these experiments. This plasmid is not affected by the expression of the viral E1 and E2 genes and luciferase expression remains constant.

The synthetic antisense oligonucleotides of the present invention may be in the form of a therapeutic composition or formulation useful for inhibiting DNA replication in a cell and treating human papillomavirus infections and concomitant diseases in an animal such as cutaneous and genital warts, epidermodysplasia verruciformis, papillomatosis of the respiratory system or larynx or cervical carcinoma. They may be used as part of a pharmaceutical composition in combination with a physiologically and / or pharmaceutically acceptable carrier. The properties of the carrier will depend on the route of administration. Such a composition may contain, in addition to the synthetic oligonucleotide and carrier, diluents, fillers, salts, buffers, stabilizers, solvents and other known materials. The pharmaceutical composition of the invention may also contain other active factors and / or agents that enhance inhibition of HPV expression. For example, combinations of synthetic oligonucleotides, each directed against another region of the HPV nucleic acid, may be used in the pharmaceutical composition of the invention. The pharmaceutical compositions of the invention may further contain other chemotherapeutic agents for the treatment of cervical carcinomas. Such additional factors and / or agents may be incorporated into the pharmaceutical composition to produce a synergistic effect with the synthetic oligonucleotides of the present invention or to reduce side effects of the synthetic oligonucleotides of the present invention. On the other hand, the synthetic oligonucleotides of the present invention may be incorporated into formulations of a particular anti-HPV or anti-cancer factor and / or substance for reducing the side effects of the anti-HPV factor and / or substance.

The pharmaceutical composition of the present invention may be in the form of a liposome in which the synthetic oligonucleotides of the present invention are present in an aqueous solution in addition to other pharmaceutically acceptable carriers with amphipathic agents such as lipids in an aggregate form as micelles, insoluble monolayers, liquid crystals or lamellar layers , be combined. Suitable lipids for a liposomal formulation include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. A preparation of such liposomal formulations is carried out in a conventional manner and is z. In U.S. Patents 4,235,871, 4,501,728, 4,837,028 and 4,737,323. The pharmaceutical composition of the present invention may further contain other lipid carriers such as lipofectamine or cyclodextrins and the like, thereby enhancing the delivery of the oligonucleotides to the cells, or may contain sustained release polymers.

As used herein, the term "therapeutically effective amount" means the total amount of each active ingredient of the pharmaceutical composition or method sufficient to effect a marked improvement in the patient, ie, a reduction in the number or size of cutaneous and genital warts Reduction of epidermodysplasia verruciformis, papillomatosis of the respiratory system or larynx or regression of cervical carcinoma. When used in conjunction with a single active ingredient administered alone, the term refers to that ingredient alone. When used in relation to a combination, the term refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, series or concurrently.

In carrying out the method of treatment or use of the invention, a therapeutically effective amount of one or more synthetic oligonucleotides of the invention is administered to a subject suffering from a HPV-related disease. The synthetic oligonucleotide of the invention may be administered in accordance with the method of the invention, either alone or in combination with other known therapies for HPV-related disease. When coadministered with one or more other therapies, the synthetic oligonucleotide of the present invention may be administered either simultaneously or in sequence with the other treatment (s). In a sequential administration, the attending physician will establish a suitable administration sequence of the synthetic oligonucleotide of the present invention in combination with the other therapy.

The use of a mixture of different oligonucleotides directed against different conserved sites in a particular viral gene may be desired. Such a mixture of oligonucleotides may be in the form of a therapeutic composition comprising at least one, two or more oligonucleotides in a single therapeutic composition (ie, a composition comprising a physical mixture of at least two oligonucleotides). Alternatively, these oligonucleotides may have two different sequences. At least one, preferably two or more, oligonucleotides may be administered simultaneously or in sequence in a single treatment in the form of separate pharmaceutical compositions.

The administration of the synthetic oligonucleotide of the present invention used in the pharmaceutical composition or the method of treating an animal may be by various conventional routes, such as intraocular, oral, inhalation or cutaneous, subcutaneous, intramuscular or intravenous injection.

If a therapeutically effective amount of the synthetic oligonucleotide of the invention is administered orally, the synthetic oligonucleotide will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as gelatin or an adjuvant. The tablet, capsule and powder contain about 5 to 95% synthetic oligonucleotide and preferably about 25 to 90% synthetic oligonucleotide. When administered in a liquid form, a liquid carrier such as water, petroleum, animal or vegetable oils such as peanut oil, mineral oil, soybean oil, sesame oil or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain a physiological saline solution, a solution of dextrose or other saccharide or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in a liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of the synthetic oligonucleotide, and preferably about 1 to 50% of the synthetic oligonucleotide.

If a therapeutically effective amount of the synthetic oligonucleotide of the present invention is administered intravenously, cutaneously or subcutaneously by injection, the synthetic oligonucleotide will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions having a suitable pH, isotonicity, stability and the like is known. A preferred pharmaceutical composition for intravenous, cutaneous or subcutaneous injection should preferably contain, in addition to the synthetic oligonucleotide, an isotonic carrier such as sodium chloride solution, Ringer's solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution or another contain known carrier. The pharmaceutical composition of the invention may also contain stabilizers, preservatives, buffers, antioxidants or other known additives.

The amount of synthetic oligonucleotide contained in the pharmaceutical composition of the present invention will depend on the type and severity of the disease being treated and on the nature of previous treatments the patient had undergone. Finally, the attending physician will determine the amount of synthetic oligonucleotide that will be used to treat the individual patient. Initially, the attending physician will administer low levels of the synthetic oligonucleotide and review the patient's responses. Larger amounts of the synthetic oligonucleotide may be administered until the optimal therapeutic effect for the patient is obtained. At this point, the dose is not increased any further. It is contemplated that the various pharmaceutical compositions used in the method of the invention will contain about 1.0 ng to about 2.5 mg of synthetic oligonucleotide per kg of body weight.

The duration of intravenous therapy with the composition of the invention will vary, depending on the severity of the disease being treated and the condition and possible idiosyncratic response of each individual patient. It is contemplated that each administration of the synthetic oligonucleotide will be for 12 to 24 hours by continuous intravenous administration. Finally, the attending physician will determine the appropriate duration of intravenous therapy with the pharmaceutical composition of the invention.

The oligonucleotides of the present invention may also be part of kits for the inhibition of human papillomavirus replication and infection in a cell. Such a kit comprises a synthetic oligonucleotide specific for an HPV nucleic acid, such as those described herein. For example, the kit may contain at least one synthetic, continuous oligonucleotide of the invention, e.g. For example, those with SEQ ID NOS: 1-39. These oligonucleotides may have a modified backbone, such as those described above, and may be RNA / DNA hybrids, e.g. B. at least one 2'-O-methyl. The kit of the invention may optionally contain buffers, cell or tissue treatment reagents, cell or tissue treatment agents, vessels and the like.

Further kits according to the invention serve to detect HPV in a sample such as a solution or a biological sample such as liquid, tissue, tissue homogenate and the like. These kits contain at least one synthetic oligonucleotide complementary to a nucleic acid extending across the translational start site of the human papillomavirus E1 gene, and means for detecting the oligonucleotide hybridized to the nucleic acid in the case of HPV in the sample ,

The following examples illustrate the invention and are not intended to be limiting.

EXAMPLES

1. RNase H test

A. Linearization of the DNA template

The E1 gene from plasmid pE16B1 was subcloned by polymerase chain reaction into the vector PCR-Script (Stratagene, La Jolla, CA). Plasmid PCR-pE16B1 (20 μg) was linearized with the restriction enzyme NotI (New England Biolabs, Beverly, MA, 60 E) for 4 hours at 37 ° C, for 1 hour at 37 ° C with proteinase K (Stratagene, La Jolla, CA) (0, 1 μg / μl) and extracted twice with phenol / chloroform. The linearized plasmid was precipitated with ethanol and isolated from the supernatant by centrifugation. The dried precipitate was dissolved in diethylpyrocarbonate (Aldrich, Milwaukee, WI) -treated water at a concentration of 0.5 μg / μl.

In vitro transcription and32 P labeling of HPV RNA

HPV E1 mRNA was transcribed in vitro using the Stratagene mRNA transcription kit (La Jolla, CA) and the T7 RNA polymerase supplied with the kit. Transcription was in the presence of 7.5 mM CTP, 7.5 mM ATP, 7.5 mM UTP, 6 mM GTP and 6 mM guanosine hydrate. The lower GTP concentration allowed initiation of a large portion of the transcripts with guanosine to allow end-labeling of the RNA without pretreatment with alkaline phosphatase. After 3 hours of transcription at 37 ° C, the reaction was treated with RNase-free DNase (Stratagene, La Jolla, CA, or Ambion, Austin, TX), extracted twice with phenol / chloroform, and purified on a G-50 Sephadex spin column (Boehringer's column). Mannheim, Indianapolis, IN or Pharmacia, Uppsala, Sweden) to eliminate unreacted nucleotides and nucleosides. The recovered RNA was quantified by measuring the UV absorbance at 260 nm using an extinction coefficient of 10,000 M-1 cm-1 base-1 of the RNA.

The RNA (5 μg) was end-labeled with 20-25 units of T4 polynucleotide kinase (Pharmacia, Uppsala, Sweden) and 50 μl of γ-32P-ATP (Amersham, Arlington Heights, IL, 6000 Ci / mmol). The labeled RNA was purified by chromatography on a G-50 Sephadex spin column (Boehringer-Mannheim, Indianapolis, IN or Pharmacia, Uppsala, Sweden).

C. RNase H cleavage with a random 20mer library

End-labeled RNA (20-100 nM) was incubated with a 20-base (50-100 μM) random DNA library (synthesized on a Pharmacia Gene Assembler, as described below) previously used to dissociate aggregates at 37 ° C for 90 minutes in FIG. 9 1 x buffer (40 mM Tris-HCl, pH 7.4, 4 mM MgCl 2, 1 mM DTT). RNase H (Boehringer-Mannheim, Indianapolis, IN) (1 μl, 1 unit / μl) was then added. The reaction was incubated at 37 ° C for 10 minutes, quenched by addition of 10 μl of 90% formamide with 0.1% phenol red / 0.1% xylene cyanol and frozen on dry ice. The stopped reactions were boiled for 2.5 to 3 minutes, chilled on ice and loaded 5 to 7 μl onto a 4% denaturing polyacrylamide gel which had been prewarmed to 50-55 ° C. Typically, the phenol red ran to the bottom of the gel, which was then dried at 80 ° C under reduced pressure. The gel was autoradiographed using a XOMAT film (Kodak, Rochester, NY) or was analyzed with a Phosphorimager from Molecular Dynamics (Sunnyvale, CA) or Bio Rad (Hercules, CA).

D. Cleavage of HPV RNA with Specific Antisense Oligonucleotides

20-100 nM [5'-32 P] -labeled RNA and 100 nM oligonucleotides (ODN) were dissolved in 9 μl of 1 × RNase H buffer (40 mM Tris-HCl, pH 7.4, 4 mM MgCl 2, 1 mM DTT ) Preincubated at 37 ° C for 15 minutes. 1 μl of RNase H (1 U / μl) was added and the reaction was incubated at 37 ° C for 10 minutes. The reactions were stopped and analyzed as described above. Quantification of the cleavage products was accomplished using software supplied with the Phosphorimager (Molecular Dynamics, Sunnyvale, CA or Bio Rad Laboratories, Hercules, CA). "Counts" were determined by drawing a box around the band of interest and subtracting the background determined by a box drawn near it. Counts in a product band were compared to the total counts in the trace above to determine the percentage of cleavage.

E. Cleavage of HPV mRNA by semi-random oligonucleotides

Semi-random oligonucleotides (100 μM in water) are boiled for 1 minute for the dissociation of aggregates that form between complementary sequences in the mixture, and 1 μl (final concentration 10 μM) to 8 μl of 1 × RNase H buffer (40 mM Tris-HCl, pH 7.4, 4mM MgCl 2, 1mM DTT) with labeled mRNA (20-100 nM). After preincubation at 37 ° C for 15 minutes, RNase H (1E) is added and incubated at 37 ° C for 10 minutes. The reactions are stopped and analyzed as described above. Cleavage sites are determined with DNA and / or RNA size markers.

Second Synthesis of oligonucleotides

Oligonucleotides were prepared by standard phosphoramidite chemistry (Beaucage (1993), Meth. Mol. Biol. 20: 33-61) on 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) according to the manufacturer's standard procedures and proprietary procedures. The proprietary procedures served to increase the coupling time from 1.5 minutes to 12 minutes for the 2'-O-methyl-RNA amidites. The Pharmacia synthesizers required additional drying of the amidites, activating reagent and acetonitrile. This was done by adding 3 Å molecular sieves (EM Science, Gibbstown, NJ) before insertion into the machine.

DNA β-cyanoethyl phosphoramidites were purchased from Cruachem (Glasgow, Scotland). The DNA support was 500 A pore size controlled pore glass (CPG) (PerSeptive Biosystems, Cambridge, MA), which was derivatized at a loading of between 30 and 40 mmol per gram with the appropriate 3'-base. 2'-O-methyl-RNA-β-cyanoethyl phosphoramidites and CPG (500 Å) supports were purchased from Glen Research (Sterling, VA). For the synthesis of random sequences, the DNA phosphoramidites were mixed from the synthesizer according to the manufacturer's instructions (Pharmacia, Uppsala, Sweden).

All 2'-O-methyl RNA-containing oligonucleotides were synthesized with ethylthiotetrazole (American International Chemical (AIC), Natick, MA) as an activator which was dissolved at 0.25M in low water acetonitrile (Aldrich, Milwaukee, WI) has been. Some DNA syntheses were carried out with 0.25 M ethylthiotetrazole, but most with 0.5 M 1-H-tetrazole (AIC). The thiosulfurizing reagent used for all PS oligonucleotides was 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage Reagent, RI Chemical, Orange, CA or AIC, Natick, MA) as 2 % solution in low water content acetonitrile (w / v).

Cholesteryl CPG (chol) and polyethylene glycol (PEG), 5'-amino modifier [C &NH₂], and cholesteryl (chol) phosphoramidites for the synthesis of oligonucleotides having linkers as described in Table 1B were prepared according to the manufacturer's instructions used (Glen Research, Sterling, VA).

The 3'-NH 2 cap is a 3 '- (3-amino-2-propanol) conjugate (Table 1B) prepared with 3'-amino modifier C3 CPG according to the manufacturer's instructions (Glen Research , Sterling, VA).

For an oxidation, redox reaction or amination of oligonucleotide phosphorothioates with a ribonucleotide at the 3'-terminus (Table 1B), the synthesis was as follows. An oligonucleotide phosphorothioate (1 mM) with a ribonucleotide at the 3'-terminus was ligated with NalO & sub4; (1.2 mM) on ice in 0.1 M sodium acetate, pH 4.75, for 30 minutes to give the 3'-dialdehyde product (Ox.). For the addition of amines, 6 equivalents of amine in 0.2 M sodium phosphate buffer (pH 8) was added to the oxidized oligonucleotide at room temperature for 30 minutes, followed by the addition of 30 equivalents of NaCNBH 3. The solution was allowed to stand at room temperature overnight. The product was purified by preparative polyacrylamide gel electrophoresis on a 20% denaturing gel. The same procedure was used in the absence of the amine to obtain the 3'-diol product (Ox./Red.).

After complete synthesis, the CPG was air dried and transferred to a 2 ml microcentrifuge tube with a screw cap. The oligonucleotide was deprotected and cleaved from the CPG with 2 ml of ammonium hydroxide (25-30%). The tube was sealed, incubated for 20 minutes at room temperature and then incubated at 55 ° C for 7 hours. After completion of the deprotection, the tubes were removed from the heating block and cooled to room temperature. The lids were removed and the tubes centrifuged at 10,000 rpm for 30 minutes to remove most of the ammonium hydroxide. The liquid was then transferred to a new 2 ml microcentrifuge tube with a screw cap and lyophilized in a Speed ​​Vac concentrator (Savant, Farmingdale, NY). After drying, the residue was dissolved in 400 μl of 0.3 M NaCl and the DNA precipitated with 1.6 ml of absolute EtOH. The DNA was precipitated by centrifugation at 14,000 rpm for 15 minutes, the supernatant decanted and the precipitate dried. The DNA was again precipitated from 0.1 M NaCl as described above. The precipitate was dissolved in 500 μl H 2 O and centrifuged at 14,000 rpm for 10 minutes to remove all solid material. The supernatant was transferred to another microcentrifuge tube and the amount of DNA determined spectrophotometrically. The concentration was determined by measuring the optical density at 260 nm. The E & sub2; & sub6; & sub0; for the DNA portion of the oligonucleotide was calculated using OLIGSOL (Lautenberger (1991), Biotechniques 10: 778-780). The E & sub2; & sub6; & sub0; of the 2'-O-methyl portion was calculated using the OLIGO 4.0 Primer Extension Software (NBI, Plymouth, MN).

The purity of the oligonucleotides was checked by polyacrylamide gel electrophoresis (PAGE) and UV shading. 0.2 OD₂ & & units were loaded with 95% formamide / H₂O and orange G dye onto a 20% denaturing polyacrylamide gel (20 x 20 cm). The gel ran until the orange G dye was within one inch from the bottom of the gel. The band was prepared by short wavelength UV shading on a thin layer chromatography plate (Kieselgel 60 F254, EM Separations, Gibbstown, NJ).

Some oligonucleotides were synthesized without removal of the 5'-trityl group (with trityl) to facilitate purification by reverse phase HPLC. Trityl oligonucleotides were dissolved in 3 ml of 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 analyzed on a 1.5 x 30 cm glass liquid chromatography column (Spectrum, Houston, TX.) Using 600E HPLC (Waters, Franklin, Mass.) ), which had been packed with C-18 uBondapak Chromatography Matrix (Waters, Franklin, MA). The oligonucleotide was passed through a 40 minute gradient of 14-32% acetonitrile (Baxter, Burdick and Jackson Division, Muskegon, MI) in 0.1 M ammonium acetate (JT Baker, Phillisburg, MJ) at 5 ml / min followed by 32 % Acetonitrile eluted for 12 minutes. Peak detection was at 260 nm with a Dynamax UV-C absorption detector (Rainin, Emeryville, CA).

The HPLC-purified oligonucleotide with trityl was dried by evaporation and the trityl group 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 precipitated by ethanol. The precipitate was isolated by centrifugation and precipitated again with ethanol from 3 ml of 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 used for the synthesis of methylphosphonate-linked oligonucleotides using two Pharmacia Gene Assembler Special DNA Synthesizers. A synthesizer was used for the synthesis of the phosphorothioate portions of the oligonucleotides using the β-cyanoethyl phosphoramidite method described above. The other synthesizer was used to introduce methylphosphonate moieties. Reagents and synthetic cycles that were found to be beneficial for methylphosphonate synthesis were used (Hogrefe et al., Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs (Agrawal, Ed.) (1993), Humana Press Inc., Totowa, NJ). For example, 0.1M methyl phosphonamidites (Glen Research, Sterling, VA) were activated with 0.25M ethylthiotetrazole. A coupling time of 12 minutes was used and oxidized immediately after the coupling step with iodine (0.1 M) in tetrahydrofuran / 2, 6-lutidine / water (74, 75/25 / 0.25). Dimethylaminopyridine (DMAP) was used for capping to replace standard N-methylimidazole (NMI). The chemicals were from Aldrich (Milwaukee, WI).

The processing procedure was based on the method described by Hogrefe et al., (1993, Nucleic Acids Research, 21: 2031-2038). The product was cleaved from the resin by incubation with 1 ml of ethanol / acetonitrile / ammonium hydroxide (45/45/10) for 30 minutes at room temperature. Ethylenediamine (1.0 ml) was then added to the mixture to deprotect for 4.5 hours at room temperature. The resulting solution and two washes of the resin with 1 ml of 50/50 acetonitrile / 0.1M triethylammonium hydrogencarbonate (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) (pH 7). The mixture was then concentrated to dryness with a Speed ​​Vac concentrator. The resulting solid residue was dissolved in 20 ml of water and the sample was desalted using a Sep-Pak cartridge. After passing the aqueous solution through the cartridge twice at 2 ml per minute, the cartridge was washed with 20 ml of 0.1 M TEAB and the product was eluted with 4 ml of 50% acetonitrile in 0.1 M TEAB at 2 ml per minute. The eluate was evaporated to dryness in a Speed ​​Vac. The crude product was purified by polyacrylamide gel electrophoresis (PAGE) and desalted with a Sep-Pak cartridge. The oligonucleotide was precipitated with ethanol from 0.3 M NaCl and then from 0.1 M NaCl. The product was dissolved in 400 μl of water and quantified by UV absorption at 260 nm.

Third E1-luciferase Genfusionstest

A. Use of stably transfected cells

The E1 luciferase fusion construct pE1Luc6 (Roche, Welwyn Garden City, England) contains 46 nucleotides extending across the translation start site of the HPV-6b E1 gene and between the immediate early gene promoter of cytomegalovirus and the luciferase reporter gene in the Plasmid pGLori (Hoffman-La Roche, Nutley, NJ) are inserted. The E1 target and the luciferase gene were subcloned by polymerase chain reaction from this plasmid and the parental plasmid pGLori into the vector pCR-Script (Stratagene, La Jolla, CA) and further into the vector pcDNA3 (Invitrogen, San Diego, CA ) subcloned. These constructs (pcDNA3GLori and pcDNA3E1 Luc6) were stably transfected with lipofectamine (GIBCO-BRL, Gaithersburg, MD) into CHO-K1 cells (American Type Culture Collection (ATCC CCL60), Rockville, MD). Several geniticin-resistant, luciferase-expressing clones were randomly selected for each construct.

Stably transfected CHO cells were spotted on 96-well plates. Cellfectin (GIBCO-BRL, Gaithersburg, MD) was diluted to a concentration of 4 μg / ml in Optimem Serum Free Medium (GIBCO-BRL, Gaithersburg, MD) and 100 μl distributed in each well of the 96-well plate. Oligonucleotides were diluted to 5 μM or 25 μM in 4 μg / ml Cellfectin in Optimem and 25 μl distributed on 3 wells of the 96-well plate. The oligonucleotide was serially diluted at a fivefold increase along the plate. Four hours after addition of the oligonucleotide, the holes were aspirated and 100 μl CCM5 medium (Hyclone, Logan, Utah) was distributed into each well. The plates were incubated overnight at 37 ° C. The cells were washed twice with Dulbecco's phosphate buffered saline (PBS) and lysed in 50 μl of cell lysis buffer (Analytical Luminescence Laboratory, San Diego, CA). Luciferase activity was measured in 20 μl of lysate with Analytical Luminescence Laboratory substrates in a MicroLumat LB 96 P luminometer (EG & G Berthold, Nashua, NH).

B. Using transiently transfected CHO cells

CHO cells were propagated in complete DMEM medium (PMEM + 10% fetal calf serum + nonessential amino acids + sodium pyruvate + L-glutamine + penicillin / streptomycin). 10 4 CHO cells per well were seeded on white 96-well Luminometer plates about 15 hours before transfection. The medium was removed and the cells washed twice with DMEM half full medium (no fetal calf serum or penicillin / streptomycin sulfate).

100 μl of transfection mixture with E1-luciferase fusion or luciferase reporter plasmids (pE1Luc6 or pGLori, 0.01 to 20 ng / 100 μl), oligonucleotide (0.1 nM to 1000 nM) and 8 to 12 μg / ml lipofectamine (GIBCO -BRL, Gaithersburg, MD) in DMEM half full medium were added. The mixture was incubated at 37 ° C for 6 hours. 100 μl of DMEM + 20% fetal calf serum + 2 × penicillin / streptomycin sulfate were then added and the cells were incubated for 1 to 7 days.

The cells were washed twice with 100 μl of phosphate buffered saline (PBS). Cells were lysed by a freeze (-80 ° C) / thawing cycle in 20 μl of reporter lysis buffer (Promega, Madison, WI). Quantities of the luciferase enzyme were measured by the addition of 100 μl luciferin assay reagent (Promega, Madison, WI) and by means of a luminometer (EG & G Berthold Microlumat LB96P, St. Albans, Herts, UK). Each hole was counted 40 s.

The activity data of the luciferase enzyme can be plotted against the plasmid concentration or oligonucleotide concentration. The specific activity of the antisense oligonucleotides is defined as the percent activity of the oligonucleotide compared to a randomer against the E1-luciferase target.

C. Use of transiently transfected human keratinocytes

Neonatal human foreskin keratinocytes (NHEK cells) were transiently transfected with the E1-luciferase fusion plasmid pE1Luc6 or control plasmid pGLori (described above) using lipofectamine. Antisense oligonucleotides were added to the cells with the plasmid or after transfection without lipid carrier or before and after transfection without a lipid carrier.

If oligonucleotides of the invention were added to the plasmid, the following procedure was used. NHEK cells at the second passage (strain 2718, Clonetics Corp., San Diego, CA) were placed in each well of a 96-well Luminometer plate (Dynatech, Billingshurst, West Sussex, UK) at a concentration of 10 & sup4; Cells / well in 100 μl of keratinocyte growth medium (KGM) (Clonetics Corp., San Diego, CA). The cells were cultured overnight at 37 ° C in a humidified CO 2 atmosphere. The following transfection mixtures were prepared for each well in 100 μl of keratinocyte basal medium (KBM, Clonetics Corp., San Diego, CA): 1% lipofectamine (GIBCO-BRL, Gaithersburg, MD), 50 ng of plasmid DNA and 0, 0, 1, 1, 10 or 100 nM antisense oligonucleotide. Immediately prior to transfection, the cells were washed with KBM. The transfection mixture was added to the cells for 6 hours at 37 ° C. This mixture was then removed from the cells. Full KGM was added and the culture increased for an additional 48 hours. For counting in a luminometer, the cells were harvested by removal of the medium, washed once with PBS, and then 50 μl of cell lysis buffer (Promega, Madison, WI) was added to each well of the plate and frozen at -80 ° C. Prior to reading the plate in the luminometer (Berthold Microlumat L96P, St. Albans, Herts, UK), it was thawed for 30 minutes at room temperature and then 100 μl of luciferase substrate buffer (Promega, Madison, WI) added to each well. After 3 seconds, the luciferase activity in each well was measured for 40 seconds.

If oligonucleotides according to the invention were added after transfection, the following procedure was used. NHEK cultures were seeded in 96-well plates as described above. For these experiments, the transfection mixture contained 50 ng of plasmid and 1% of lipofectamine in KBM. The transfections were as described above. After 6 hours of incubation, the transfection mixture was removed, replaced with KBM and then incubated overnight in KGM. The following day, the KGM was replaced by KGM with 0, 0.2, 1.0, 5.0, or 10.0 μM antisense oligonucleotide. The cultures were maintained in this medium for 48 hours before further treatment for the luminometer count made as described above. In some cases, cells were treated with KGM-diluted antisense oligonucleotides (0-10 μM) prior to transient transfection. They were then transiently transfected and post-treated with oligonucleotide as described above.

4th cytotoxicity

The transfection mixture with reporter plasmid, oligonucleotide and lipofectamine in DMEM half full medium was prepared as in 3B, supra. Portions of these were applied in duplicate to two microtiter plates: one to assay for luciferase expression and one to measure viability of the cells. The viability of the cells was measured by the Celltiter 96 non-radioactive cell proliferation / cytotoxicity test (Promega, Madison, WI). The luciferase activity in plate 1 was measured as described in B, supra. For plate 2, 15 μl of MTT staining solution was added to the CHO cells in 100 μl of DMEM medium. The plates were incubated at 37 ° C in humidified 5% CO 2 atmosphere for 4 hours. 100 μl solubilization / stop solution (all reagents are included in the Promega kit) was added and the mixture was incubated for 1 hour. The optical density of each hole was measured at 570 nm (versus controls).

5th In vivo test of HPV-specific oligonucleotides

The in vivo method of Kreider et al. (U.S. Patent 4,814,268) is used to determine whether the oligonucleotides of the present invention are capable of inhibiting expression of HPV-specific genes. In particular, mice were treated in the Kreiss-nude mouse xenograft model of HPV type 11 infection in human foreskins [MK Howett, JW Kreider and KD Cockley (1990), Human xenografts. A model system for human papillomavirus infection. Intervirology 31: 109-115]. Human foreskin grafts were rinsed with Minimum Essential Medium with 800 μg / ml gentamycin (GlBCO-BRL, Gaithersburg, MD) and then incubated for 1 hour at 37 ° C in 1 ml of Condyloma acuminatum (HPV-containing) extract. The extract is prepared from a vulvar condyloma which is minced and destroyed in 50 ml of PBS at 4 ° C with a tissue homogenizer at 25,000 rpm for 30 minutes. The cell debris are removed by centrifugation. Athymic mice (nu / nu in a BALB / c background) (Harlan Sprague Dawley, Inc., Madison, Wi) are amnesticated with Nembutal and the kidneys are separately delivered through dorsal, bilateral, paravertebral, subcostal incisions. The kidney capsule is incised and the foreskin graft is placed in each kidney with toothless forceps. The skin incisions are closed with wound clips and the mice are offered drinking water with trimethoprine (0.01 mg / ml) and sulfamethoxazole (0.05 mg / ml) for the duration of the experiment.

In the experiment, 10 mice, each containing two grafts (one per kidney), 25 mg kg-1 day-1 of the antisense oligonucleotide HPV1 0x5 hybrid (SEQ ID NO: 96, Table 1B), which was five 2'-OMe ribonucleotides at the 3 'end, administered subcutaneously for 34 days and then 5 mg kg-1 day-1 for the remaining 56 days of the experiment for a total exposure of 90 days. As controls, 10 mice were treated with two grafts each with saline. The mice were sacrificed by cervical dislocation, the kidneys with the cysts removed and their size measured. The standard cyst size metric used by Kreider is the "Large Mean Diameter" (GMD) or the Average Dimension [ie (I + w + h) / 3]. The calculated GMD was 2.89 ± 0.23 mm (Table 2) for 10 control animals given subcutaneous saline and 1.62 ± 0.14 mm for 9 animals given HPV1 0 .5 OMe ( Table 3). The statistical measurement accuracy for the drug effect was calculated to be p <0.001 according to Student's t-test (T = 4.59, n = 18). Although the GMD was used to measure size, a more representative comparison of the differences between the two groups is the ratio of cyst volumes, ie, the third power of the two GMDs or 1.62³ / 2.89 = = the tumor volume in HPV1 0 · 5 OMe compound-treated mice are 82% lower than control mice. This is a conservative estimate since it is believed that the original foreskin chip used has no volume at implantation and does not grow in the absence of a viral infection. None of these assumptions is correct. Foreskin chips are large at implantation ~ 1 x 1 mm in skin thickness and grow easily even in the uninfected state as determined in a previous experiment (GMD = 1.20 ± 0.363 mm). Therefore, the effect on subtracting this baseline of uninfected implants is calculated to be (1.62-1.20) 3 / (2.89-1.20) 3 = a 65-fold (> 98%) decrease in cyst size for the Antisense oligonucleotide compared to the salt control. Table 2 Control (saline) Table 3 HPV1 0x5 OMe (administered as described above)

In addition, after determination of cyst size, the kidneys are fixed in neutral-buffered formalin, embedded in paraffin, cut at 6 microns and stained with hematoxylin and eosin. Cohort sections are paraffin-free and incubated with antibody to disrupted bovine papillomavirus (Dakopatts, Accurate Chemica. & Scientific Corp., Westbury, NY) to display the group-specific antigen (GSA) by the immunoperoxidase method (see Jensen et al., (1980) J. Natl. Cancer Inst. 64: 495-500 and Kurman et al. (1983), Am. J. Surg. Path 7: 39-52)). GSA is a capsid antigen that is common to most papillomaviruses. Positive controls consist of rabbit papilloma or human vulvar condyloma. Negative controls are normal human skin.

6th Studies of CHO-K1 cells stably transfected with the complete HPV E1 gene

The complete E1 gene is subcloned from the plasmid pE16B1 (Roche Welwyn, Garden City, UK) (SEQ ID NO: 40) by polymerase chain reaction into the vector pcDNA3 (Invitrogen, San Diego, CA). This is transfected into CHO-K1 cells and isolated for geneticin-resistant (GIBCO-BRL, Gaithersburg, MD) clones. These clones are tested by western blot for expression of E1 protein. Positive clones are used for antisense oligonucleotide assays, with efficacy measured by Western blots for translation inhibition and by Northern blots and ribonuclease protection assays for RNA loss and RNase H cleavage products. In addition, E1-expressing cells are transiently transfected with pHPVE2 and pGLori to measure the inhibition of HPV DNA replication.

7th E1 RNA dot blot test

To confirm the accuracy of the E1-luciferase enzyme assay, which measured E1-luciferase expression as a surrogate marker for the expression of the actual E1 viral target, levels of E1 mRNA in CHO cells were compared to the E by Plumpton et al. ((1995), Biotechnol. 31: 1210-1214).

CHO cells were transfected with pE1681 (SEQ ID NO: 40), a plasmid expressing the entire open reading frame of E1 together with 103 nt of 5 'untranslated region. The cells were then treated with a placebo or 100 nM HPV1, HPV9 (with three mismatches) or Randomer phosphorothioate compounds. Another set of CHO cells was treated with the same antisense compounds but not transfected with the expression plasmid. Finally, RNA was isolated from all eight CHO samples. Total RNA was hybridized with labeled oligonucleotide probes for the E1 messenger or an actin control, and the amounts of each transcript were measured by quantification of marker intensity on a phosphorimager.

Cells transfected with the E1 construct, but treated only with placebo, expressed high levels of the E1 messenger. Cells treated with the control randomer oligonucleotide expressed the same large amounts of E1. However, cells treated with the mismatched HPV29 showed 40% less E1 expression. Cells treated with HPV1 that exactly matched the targeted viral gene showed 80% lower levels of E1 mRNA. In contrast, control CHO cells that had not been transfected with the E1 construct did not show effects due to treatment with the antisense oligonucleotides. Furthermore, all eight CHO RNA samples showed similar levels of actin RNA. This shows that the antisense effects were specific for E1 gene expression. This work suggests that oligonucleotides directed against the E1 gene of human papillomavirus directly reduce the amount of mRNA in the cell and confirm that the antisense activity in the E1 luciferase surrogate test used for routine screening coincides with the direct measurements of E1 RNA levels.

Äguivalente

One skilled in the art will recognize many equivalents to the specific substances and methods described herein or may find these through routine experimentation. Such equivalents are within the scope of the invention and are covered by the following claims.

SEQUENCE LISTING

(1. GENERAL INFORMATION:

(i) REGISTERS:

(A) NAME: F. HOFFMANN-LA ROCHE AG

(B) ROAD: Grenzacherstrasse 124

(C) LOCATION: Basel

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(E) COUNTRY: Switzerland

(F) POSTCODE: CH-4070

(G) TELEPHONE: 061-688 39 43

(H) TELEFAX: 061-688 13 95

(I) TELEX: 962292/965542 hIr ch

(A) NAME: Hybridon, Inc

(B) ROAD: One Innovation Drive

(C) LOCATION: Worcester

(D) BUNDESLAND: MA

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP): 01605

(G) TELEPHONE: 508 / 752-7000

(H) TELEFAX: 508 / 752-7001

(ii) TITLE OF INVENTION: Oligonucleotides specific for human papillomaviruses

(iii) NUMBER OF SEQUENCES: 40

(iv) CORRESPONDENZADDRESS:

(A) ADDRESSAT: F. HOFFMANN-LA ROCHE AG

(B) ROAD: Grenzacherstrasse 124

(C) LOCATION: Basel

(D) BUNDESLAND: BS

(E) COUNTRY: Switzerland

(F) POSTCODE: CH-4070

(v) COMPUTER READABLE VERSION:

(A) DATA CARRIER: floppy disk

(B) COMPUTER: Apple Macintosh

(C) OPERATING SYSTEM: System 7.1 (Macintosh)

(D) SOFTWARE: Word 5.1

(vi) DATA OF THE PRE-REGISTRATION:

(A) REGISTRATION NUMBER: US 08 / 471,974

(B) REGISTRATION DATE: 06.06.1995

(C) CLASSIFICATION:

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(A) LENGTH: 15 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GTACCTGAAT CGTCCGCC 18

(2) PARTICULARS TO SEQ ID NO: 27:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 15 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

TGAATCGTCC GCCAT 15

(2) PARTICULARS TO SEQ ID NO: 28:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 30 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(iv) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GTACCTGAAT CGTCCGCCAT CGTTGTTAGG 30

(2) PARTICULARS TO SEQ ID NO: 29:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 30 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

TCTTTTTTT TTTTCTGTAC CTGAATCGTC 30

(2) PARTICULARS TO SEQ ID NO: 30:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 30 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

ACCCAGACCC CTCATTTTCT TTTTTCTTTT 30

(2) PARTICULARS TO SEQ ID NO: 31:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

GTACCTAAAT CGTCCGCCAT 20

(2) PARTICULARS TO SEQ ID NO: 32:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

GTACCTAAAT CATCCGCCAT 20

(2) PARTICULARS TO SEQ ID NO: 33:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART; nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

GTACCTAAAT CATCCACCAT 20

(2) PARTICULARS TO SEQ ID NO: 34:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

ATACCTAATE CATCCACCAT 20

(2) PARTICULARS TO SEQ ID NO: 35:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

GTGCCAGAGT CGTCCGCCAT 20

(2) PARTICULARS TO SEQ ID NO: 36:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

GTACCTNAAT CATCCGCCAT 20

(2) PARTICULARS TO SEQ ID NO: 37:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

GTACCTAAAT CNTCCGCCAT 20

(2) PARTICULARS TO SEQ ID NO: 38:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

GTACCTNAAT CNTCCGCCAT 20

(2) PARTICULARS TO SEQ ID NO: 39:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 20 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

ATGTTTTTGG CGTCTTCCAT 20

(2) PARTICULARS TO SEQ ID NO: 40:

(i) SEQUENCE MARKINGS:

(A) LENGTH: 107 base pairs

(B) ART: nucleic acid

(C) STRING FORM: single strand

(D) TOPOLOGY: linear

(ii) ART OF MOLECULAR: DNA

(iii) HYPOTHETIC: no

(iv) ANTISENSE: yes

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

TCGAAGCTCA GATCCGAAGA CCTAACAACG ATGGCGGACG ATTCAGGTAC AGAAAATGAG 60

GGGTCTGGGT GTACAGGATG GTTTATGGTA GAAGCTATAG TGCAACA 107