MXPA96005514A - Horqui medianteribozima human papilloma virus inhibition - Google Patents

Abstract

The present invention provides synthetic ribozymes, i.e., ribozymes including a hairpin portion, binding sites for binding to a human papilloma virus (HPV) after the viral base 419 to 434, respectively, and dissociation sites to dissociate to the HPV virus. The invention also includes a diagnostic assay to determine the presence of HPV-16 in tissues, based on P amplification.

Description

"INHIBITION OF HUMAN PAPILOMA VIRUS THROUGH RIBOZIMA DE HORQUILLA" FIELD OF THE INVENTION The present invention relates to an RNA catalyst, i.e., ribozyme, which cleaves Human Papilloma virus into a fragment having a 5 'hydroxyl and a fragment having a 2' 3 'cyclic phosphate. The products of the reaction described herein resemble those resulting from the natural hydrolysis of RNA.

BACKGROUND OF THE INVENTION Papilloma viruses are small DNA viruses that induce hyperproliferation of epithelial cells. Approximately 70 different genotypes have been isolated from humans. Some types (1, 2, 4 and 7) are associated with benign squamous papillomas (wrinkles, condylomas) in humans, while at least two types (16 and 18) have been associated with neoplastic and preneoplastic human lesions (DiPaolo and others, 1993, Critical Reviews in Oncogenesis 4: 337-360).

In the United States, cervical cancer affects approximately 8.6 women per 100,000 each year. In women, HPV-16 is frequently associated with latent infections, benign and premalignant cervical lesions (dysplasias / CIN), and half of invasive cervical carcinomas. In men, HPV-16 is associated with subclinical or papular clinical macular lesions. Bowenoid palulosis of the penis resembles carcinoma in si tu. Cervical cancer, which kills at least 500,000 women worldwide each year, progresses through progressive cell changes from benign condylomata to dysplasias / high quality CIN before developing into invasive cancer. More than five trillion dollars for health care are spent in the United States each year for the detection and treatment of these injuries. Evidence from epidemiology indicates that up to 89 percent of human and oral tumors harbor types of HPV that are capable of immortalizing primary human keratinocytes and transforming rodent cells. The oncogenic potential of HPV seems to be associated with products of two viral genes, E6 and E7. These products are required for the acquisition and maintenance of the transformed phenotype. The proteins encoded by these genes are ligated, with high affinity in neoplastic-associated types, and neutralize the products of tumor suppressor cells Rb and p53 (Nasseri, 1991, Virol 194: 136, Sedman et al., 1991, J. Virol 65: 4860-4866, S otkin et ettstein, 1989, J. Virol 63: 1441-1447, Smotkin and Wettstein, 1986, J. Virol 63: 1441-1447, Steele et al., 1993, Cancer Res. 53: 2330; Storey et al., 1991, Nuc Acids, Res. 19 (15): 4109). The current policy in genitourinary clinics is surgery for superior quality injuries due to the lack of superior alternatives. Long-term cervical laser ablation therapy does not influence the natural history of diseases associated with cervical human papillomavirus in women. Interferons, per se, have been disappointing in relation to acute viral infection, usually because the treatment can not be started in time. Therefore, it has been assumed that any benefit with interferons is due to the anti-proliferative effect and not due to the antiviral. Chemotherapy in combination is also used in cancer therapy and cisplatin is one of the drugs that is selected for cervical cancer, alone or in combination with other chemotherapy agents. However, the current success obtained with a chemotherapy treatment is unsatisfactory. The response regimen for cisplatin in combination with 5FU treatment in phase II studies in cervical cancer patients is only effective in 22 percent of patients, while the same combination produced an 88 percent response in carcinoma of scaly cell on the head and neck. The use of cytotoxic agents for cancer therapy have limitations due to the toxic side effects and the development of multiple resistance to the drug. Thus, there must be a consideration of a therapy shift that does not involve the direct toxic reaction, but may modify the growth of the tumor cells. Current new therapeutic suggestions for treatment in HPV infections have focused on the use of antisense oligonucleotides to interrupt the use of viral mRNA (DiPaolo et al., 1993, Critical Reviews in Oncogenesis 4: 337-360; Steele et al., 1993, Cancer Res. 53: 2330, Storey et al., 1991, Nuc. Acids Res. 19 (15): 4109). However, antisense therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pages 305 to 325). Ribozymes are RNA molecules that possess catalytic RNA capacity (see Cech et al., US Pat. Number 4,987,071) that segment a specific site in a 7? RN of white. The number of 5 RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stenochemistry (Hampel and Tritz, 1989, Biochem 28: 4929-4933, Uhlenbeck, 1987, Nature 328: 596-600). This provides an advantage in relation to antisense technology. 10 Antisense therapy has two disadvantages when compared to ribozymes: (1) by its nature, the antisense molecule is not catalytic; and (2) the antisense molecules are usually longer than the reference recognition sequence of ribozyme. This increases the possibility of antisense molecules that have a detrimental effect on the sequences of similar mRNAs found in the same gene family. Ribozymes have been designated in the motif of "hammer head" (Sedman et al., 1991, J. Virol. 65: 4860-4866). However, catalytic RNAs, such as those that were designed based on the "hammerhead" model, have several limitations that restrict their use in vitro and can predict their use in vivo. For example, The optimum temperatures for the reaction is 50 ° C to 55 ° C (Haseloff and Gerlach, 1988, Nature 334: 585, Uhlenbeck, 1987, Nature 328: 596-600). In addition, the Km is 0.6 micrometers (Uhlenbeck, 1987, Nature 328: 596: 600), implying that the reaction requires high concentrations of the substrate, which makes it difficult if not impossible for the catalytic 7? RN to target low levels. of the target RNA substrate, as it will be found in vi vo. A "hairpin" motif has been found to be more efficient than the "hammerhead" motif (Hampel and Tritz, 1989, Biochem. 28: 4929-4933; Hampel et al., 1990; Nuc. Acids Res. 18: 299-304). In addition, hairpin ribozymes have been used to target targets in HIV (Ojwang et al., 1992, Proc. Nati. Acad. Sci.

United States of America 89, 10802-10806; Yu et al., 1993, Proc. Nati Acad. Sci. United States of America 90: 6340-6344). However, ribozymes for a virus will generally not segregate other virus species. Not only do ribozymes require specific reference sequences for segmentation, but they require modifications to the ribozyme structure itself to be able to efficiently segment a specific target. At present there is no hairpin ribozyme that has been shown to segment the HPV RNA and no site has been identified in HPV that is able to be cleaved by the hairpin ribozyme.

Certain molecular components have been used in the past to deliver ribozymes. For example, retroviral vectors have been used to deliver ribozymes. PoIIII promoters and vectors retrovirals with PoIIII promoters have also been used.

COMPENDIUM OF THE INVENTION AND ADVANTAGES • * - In accordance with the present invention, the Synthetic catalytic RNAs, ie ribozymes including a hairpin portion, have been constructed as a binding site to bind to a human papilloma virus, either after the viral base 434 or after the base 419 and a site of segmentation to segment the virus binding site. In one aspect, the present invention comprises a synthetic ribozyme comprising a hairpin portion, a binding site for ligating to a nucleotide sequence of a human papilloma virus and a site of segmentation to segment the sequence. The cleavage site is either a site after base 434 of the viral sequence or a site after base 419 in the viral sequence. The binding site of this synthetic ribozoma is preferably linked to one of the following sequences: 430-ACUG U * GUC CUGAAGA-4444 (SEQ ID NO: 2), 430-ACUG U * GUCCUGAAGAA-445 (SEQ ID N0: 3), 430-ACUG U * GUCCUGAAGAAA-446 (SEQ ID NO:) 415-UAAC U * GUCAAAAGC-428 (SEQ ID NO: 7), 415-UAAC U * GUC AAAAGCC-429 (SEQ ID NO: 8), 415-UAAC U * GUCAAAAGCCA-430 (SEQ ID NO: 9), and 415-UAAC U * GUC AAAAGCCAC-431 (SEQ ID NO: 10). "*" Indicates the targeting site. The hairpin portion of the ribozyme, also preferably comprises the sequence of SEQ ID NO: 5. "" 'In a preferred embodiment, the synthetic ribozyme .0 is linked to a nucleotide sequence of HPV-16. In this embodiment, the ribozyme preferably comprises the sequence of SEQ ID NO: 6 or the sequence of SEQ ID NO: 11. In a further embodiment, the synthetic ribozyme may have the two-dimensional configuration shown in FIG.

Figure 2. Alternatively, the synthetic ribozyme may have the two-dimensional configuration shown in Figure 6. In another aspect of the present invention, the invention includes a vector comprising a coding of the DNA sequence for the synthetic ribozyme described above. In this regard, the DNA sequence is preferably operably linked to the expression control sequences. The vector can be, for example, a plasmid. A further aspect of the invention comprises a host cell transformed with the vector described above, wherein the host cell is capable of expression for ribozyme from the vector. In still another aspect of the present invention, the invention comprises a method for cleaving a human papillomavirus by means of a ribozyme. This method includes the steps of: identifying a segmentation site in the virus genome; determine the sequence on either side of the segmetation site; constructing a synthetic hairpin ribozyme, wherein a binding site in the synthetic ribozyme includes a sequence not complementary to the cleavage site and a complementary binding region with respect to the sequences on either side of the cleavage site; and provide the synthetic ribozyme of hairpin to the viral genome, thereby allowing the ribozyme to segment the viral genome. In this aspect of the invention, the step of providing can be carried out in vi tro.

Alternatively, the step of providing can be carried out in vivo. The step to construct the synthetic hairpin ribozyme may also further include incorporating the tetralazo sequence of SEQ ID NO: 5 into the ribozyme.

In a further aspect of the present invention, the invention comprises a method for detecting a human papilloma virus-16 (HPV-16) in human tissue. This method comprises the steps of: obtaining a sample of the human tissue containing RNA, exposing the RNA in the tissue to a ribozyme that is linked to a nucleotide sequence of the HPV-16 RNA, such as the HPV-16 RNA present in the sample it is segmented by the ribozyme; amplify the cDNA using primers complementary to: the 5 'end of a full length HPV-16 transcript, a 5' fragment of the ribozyme cleavage site of the full length transcript HPV-16, and a 3 'fragment of the ribozyme cleavage of the full-length transcript VPH-16; and identify the amplified DNA fragments. In the above method, a larger DNA fragment represents a full-length HPV transcript and a smaller DNA fragment represents the fragment resulting from the cleavage of the ribozyme of the full-length HPV transcript. If the HPV-16 RNA is present in the sample, a preponderance of the smallest fragment is identified relative to the largest fragment. In one embodiment, the HPV-16 RNA cleaved by the ribozyme is an E6 transcript of HPV-16. The ribozyme used in that method of preference is either RHPV434 or RHPV419, and the human tissue is a sample of the cervical tissue. This method may further comprise the step of providing the cDNA of the RNA present in the sample after the exposure step and before the amplification step. Another aspect of the present invention comprises a method of treating cervical cancer comprising the steps of: constructing a synthetic ribozyme comprising a hairpin portion, a binding site for ligating to a nucleotide sequence of a human papilloma virus, and a segmentation site for segmenting the sequence, wherein the targeting site is selected from the group consisting of a site after the base 434 of the sequence and a site after the base 419 of the sequence; and delivering an effective amount of the synthetic ribozyme to the cervical tissue. In this method, the delivery step can provide for suspending the synthetic ribozyme in a lipofection-based liposomal delivery system. In addition, this method may further comprise the step of administering additional agents in combination with the synthetic ribozyme, such as immunological agents or chemotherapeutic agents. Preferably, the immunological agents are LAK cells and the chemotherapeutic agent used is cisplatin.

BRIEF DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as it is better understood by reference to the following detailed description when considered in connection with the accompanying drawings. Figure 1 is a diagram of the HPV target sites, showing the location of the target sites selected for cleavage by the fork ribozyme, the overlaps at the target sites of the mRNA for both the E6 and E7 region of the HPV 16 and the point of dissociation of this target site by the hairpin ribozyme occurring in "*" after nucleotide 434 and nucleotide 419. Figure 2 is a diagram of the hairpin ribozyme with a helix 1 driven at optimal comprising 8 bp, designed to segment HPV-16 after position 434, which shows the sequences of the optimal ribozyme (VPHR), and the substrate (VHPS), the base regions remaining paired between the substrate of white and the ribozyme (labeled Helix 1 and Helix 2) and 5 the base pairing regions required in the "hairpin" portion of the catalyst (labeled Propellers 3 and 4). Figure 3 is an autoradiography of the '' '' - results of the segmetation of the HPV substrates after site 434 by the present invention, with helix 1 having lengths of 7bp, 8bp and 9bp, as shown; the reference controls were ribozyme R53 and substrate S17 (lanes 1 and 2), the reaction was at 37 ° C with 25 nM of ribozoma and 50 nM of substrate for 60 minutes, and the reference reaction was (-) sTRSV seguence S17 / R53 at 10 nM and 100 nM during the times shown (Hampel and Tritz, 1989, Biochem 28: 4929-4933). Figure 4 illustrates the results of a time course of segmentation using RHPV434 showing: (Figure 4A) an autoradiograph of the segmentation at each time point, and (Figure 4B) a graph of the results of 4A, where the segmentation conditions were the same as in Figure 3 using [RJ-25 nM and [ S] -100 nM for the times shown.

Figure 5 illustrates the kinetic analysis of the cleavage by RHPV434, showing: (Figure 5A) a graph of the results of Figure 4B, and (Figure 5B) an autoradiograph of the segmentation results after 10 minutes at each concentration of [ S], with segmentation conditions as in Figure 3 using [R] = 20 nM and [S] of 400 nM (lane 1), 200 nM (lane 2), 150 nM (lane 3), 100 nM (lane 4), 75 nM (lane 5), 50 nM (track 6) and 25 nM (track 7). Figure 6 is a diagram of the hairpin ribozyme with an optimized helix 1 comprising 7 bp, designated to segment HPV-16 after position 419, showing the sequences of the optimized ribozyme (VPHR) and the substrate (VPHS) ), pairing the base regions between the target substrate and the ribozyme (labeled Helix 1 and Helix 2), the base pairing regions required in the "hairpin" portion of the catalyst (labeled Propellers 3 and 4). Figure 7 is an autoradiograph of the results of cleavage of HPV substrates after site 419 by the present invention with helix 1 having lengths of 6 bp, 7 bp, 8 bp, 8bp and 9bp as shown; the reference controls were the R53 ribozymes and the S17 substrate (lanes 1 and 2), the reaction was at 37 ° C with 25 nM ribozyme and 50 nM of the substrate for 60 minutes, and the reference reaction was the sequence (- ) s native TRSV S17 / R53 at 10 nM and 100 nM during the periods shown (Hampel and Tritz, 1989, Biochem 28: 4929-4933). Figure 8 illustrates the results of a time course of segmentation using VPHR419 that shows: (Figure 8A) an autoradiograph of the segmentation results at each time point, and (Figure 8B) a graph of the result of Figure 8A, where the segmentation conditions were the same as in Figure 3 using [R] = 25 nM and [S] = 100 nM for the times shown. Figure 9 illustrates the kinetic analysis of segmentation by VPHR 419 which shows: "(Figure 9A) a graph of the results of Figure 9B, and (Figure 9B) an autoradiograph of the segmentation results after 10 minutes at each concentration of [S], with segmentation conditions as in Figure 3 using [R] = 20 nM and [S] of 200 nM (track 1, 150 nM (lane 2), 100 nM track 3), 75 nM (lane 4), 50 nM (lane 5), and 25 nM (lane 6), and controls to complete the reaction were zero minute (lane 7) and one hour (track 8) for [S] of 20 nM. Figure 10 shows diagrammatically the region of the polylinker that was cloned into pBluescript KS to provide plasmid pBKS LNKR.

Figure 11 is a diagram of the plasmid ptVl containing the RNAval promoter, its upstream region, the tetralazo variant and the polyT termination sequence. This was prepared by cloning the ARNtva-L promoter and its upstream region to pBKS LNKR. Figure 12 shows the DNA sequence (SEQ ID NO: 19) of plasmid ptVl including the polylinker, the promoter of ttva -'-, the upstream region, the tetralazo, the termination region; the new ribozymes can be cloned in the Xhol / Mlul sites. Figure 13 is a diagram of the plasmid pBtVl-434 containing the ribozyme VPHR 434 in the plasmid ptVl. Figure 14 shows the DNA sequence (SEQ ID NO: 20) of the polylinker and the ribozyme in the plasmid pBtVl-434. Figure 15 is a digram of the plasmid pZIPVl-434 (without) containing the ribozyme VPHR434 in. the "without" orientation where the transcription is the same direction as that of the retroviral genome in the retroviral vector. Figure 16 shows the DNA sequence (SEQ ID NO: 21) of the delivery cassette and the ribozyme in the plasmid pZIP Vl-434 (without). Figure 17 is a diagram of the plasmid pZIPVl-43 (anti) containing the ribozyme VPHR 434 in the "anti" orientation where the transcript is in the opposite direction to that of the retroviral genome in the retroviral vector. Figure 18 shows the DNA sequence (SEQ ID NO: 22) of the polylinker and the ribozyme in the plasmid zZIP V1434 (anti). Figure 19 is a diagram of the plasmid pBlVl-434 (i) which contains the clone of the catalytically inactive mutant ribozyme VPHR434 in ptVl. Figure 20 shows the DNA sequence (SEQ ID NO: 23) of the delivery cassette and ribozyme in the plasmid pBtVl-434 (i). Figure 21 shows the expression of the HPV specific ribozyme in transformed human CXTl carcinoma cells as tested by RNase protection of the transcripts; the arrow indicates the expected size of the protected ribozyme transcript of the poIIII promoter, 160 nt. Figure 22 shows the expression of the HPV E6 transcript in CXTl cells containing the hairpin ribozyme as tested by the RT and PCR method.

DETAILED DESCRIPTION OF THE INVENTION A hairpin ribozyme containing a tetralazo modification was designed, tested and displayed to segment a specific sequence in the primary transcript of type 16 human papillomavirus. The cleavage sites immediately followed nucleotide 434 and 419, respectively, in the sequence of this virus. Optimization of the ribozyme was carried out showing that a helix 1, 8 nt was optimal for site 434 and that a helix 1, 7 nt was optimal for site 419. The time course of the reaction showed almost complete segmentation of the substrate. The kinetic parameters for site 434 were measured using symmetric analysis of normal Michaelis enzyme. The Km for the reaction was 21 nM which shows a very tight bonding of the ribozyme and the substrate. The Kcat or investment number was 0.083 min-1 to provide a total catalytic efficiency (kcat / Km) of 4 micrometers-1 min-1. The kinetic parameters for site 419 were also measured using the kinetic analysis of normal Michaelis enzyme. The Km for the reaction of 98 nM and the kcat or inversion number was 0.18 min-1 to provide a total catalytic efficiency (kcat / Km) of 1.8 micrometers-1 min-1. The optimized target sites are shown in Figure 1. Segmentation occurred after washing 434 and after washing 419, respectively and before the GUC sequence shown as indicated in the diagram. All the white sequence is part of the primary transcript (SEQ ID N0: 1) for the E6 and E7 regions of HPV 16 (Nasseri 1991, Virol. 194: 136; Smotkin and Wettstein, 1989, J. Virol. 63: 1441-1447). The cap for this mRNA is nt 97. A splice donor exists in nt 226 and two splice acceptors exist in nt 409 and nt 526. As a result, three different E6 E7 mRNAs can be produced: E6E7, E6: (I) E7 and E6 (II) E7, E6E7 is the result of the full-length E6E7 transcript where the splicing tamer in nt 226 is not used. The E6E7, the E6 transfer termination occurs at nt 557. E6 (I) E7, the main transcript, is the result of using the splice donor on nt 226 and the splice acceptor on nt 409 and su. transfer completion signal at E6 (I) is at nt 415. This provides a truncated E6 coding region and a full-length E7 region. E6 (II) E7, the small transcript is the result of the use of the emplame donor at nt 226 and the splice acceptor at nt 526 and its transfer completion signal is E6 (II) is at nt 541 to provide the region of truncated E6 coding and a full-length E7 (Nasseri, 1991, Virol. 194: 136). An RNA catalyst (ribozyme) has been identified comprising an RNA sequence that can accurately segment HPV. The target sequences for cleavage by ribozymes are present in the primary transcript E6E7 and E6 (I) E7, the main transcript. Segmentation of these transcripts would have the effect of decreasing the production of full-length E6 and E7 proteins, both of which appear to play a key role in the transformation of keratinocytes (Sedman et al., 1991, J. Virol. : 4860-4866). The hairpin ribozyme (Hampel et al., 1990, Nuc Acids Res. 18: 299-304) designed to be cleaved after site 434 in HPV is shown in Figure 2 and designated VPHR434. In the preferred embodiment, this hairpin ribozyme has the tetralazo modification as shown (Anderson et al., 1994, Nuc Acids Res. 22: 1096-1100). The GUU sequence of Loop 3 of the basic structure has been replaced by a tetralogy sequence GGAC (UUCG) GUCC which in the present invention is shown to generate a very stable structure with high catalytic efficiency. In particular, the invention comprises certain synthetic RNA catalysts capable of cleaving an RNA substrate containing the target sequences: 430-ACUG U * GUC CUGAAGA-444 (SEQ IN NO: 2) 430-ACUG U * GUC CUGAAGAA-445 (SEQ ID NO: 3) 430-ACUG U * GUC CUGAAGAAA-446 (SEQ ID NO: 4) The hairpin ribozyme designated to be segmented after site 419 in HPV is shown in the Figure 6 and designated VPHR419. In the preferred embodiment this hairpin ribozyme also has the modification of tetralazo as shown. The GUU sequence of Loop 3 of the basic structure has been replaced by a tetralogy sequence GGAC (UUCG) GUCC which in the present invention has been shown to generate a very stable structure with '* "" high catalytic efficiency. In particular, the invention comprises certain synthetic RNA catalysts capable of cleaving an RNA substrate containing the target sequences: 415-UAAC U * GUC AAAAGC-428 (SEQ ID NO: 7) 415-UAAC U * GUC AAAAGCC-429 (SEQ ID NO. : 8) 15 415-UAAC U * GUC AAAAGCCA-430 (SEQ ID NO: 9) 415-UAAC U * GUC AAAAGCCAC-431 (SEQ ID NO: 10) The term "synthetic RNA catalyst" as used herein means a catalyst (ribozyme) that is not an RNA catalyst that occurs naturally, even when the term "synthetic catalysts" can be the truncated or altered versions of the catalysts that occur naturally. The term "Synthetic catalyst" includes catalysts synthesized in vi tro and catalysts synthesized in vivo. In In particular, the term "synthetic catalysts" may include catalysts produced by hosts transformed by a vector comprising a sequence coding for the catalyst. RNA of any length and type can be used as the substrate as long as it contains the target sequence represented by the formula 5 '-F? -CS-F2 ~ 3 In this formula, CS is the segmentation sequence, ie, a sequence of bases containing the site in which the catalyst segments the substrate. CS is a short sequence of bases that is not a base paired with the ribozyme and in the present invention CS preferably has the sequence 5'-NGUC-3A where N is any base and the substrate is cleaved by the ribozyme between N and G to produce a fragment having an OH at the 5 'end or terminus and the fragment having a 2, 3' cyclic phosphate at the 3 'end. CS is flanked by two short base sequences Fi and F2 which constitutes the base pairing with the RNA catalyst. F] _ is preferably at least 3 bases in length and more preferably 4 bases in length. F2 is also preferably at least 3 bases in length, more preferably 6 to 12 bases in length. Ribozymes, in accordance with the present invention also include a substrate binding portion and a "hairpin" portion. A binding portion of the catalyst substrate is represented by the following formula: 3'F4-L1-F3-5 'wherein F3 is a sequence of bases which are selected such that F3 is essentially a base paired with F2 (Helix 1 , Figures 2 and 6) when the catalyst has been bound to the substrate; F4 is a sequence of bases that are selected so that F4 essentially performs the base pairing with F_, when the catalyst is bound to the substrate (Helix 2, Figures 2 and 6); The sequences of F3 and F4 are selected such that each contains a base frame number to achieve sufficient binding of the RNA substrate in the RNA catalyst so that substrate cleavage can be effected; and L] _ is a sequence of bases that are selected such that 1 > Do not perform base pairing with CS when the catalyst is bound to the substrate (Loop 1, Figures 2 and 6). As used herein, the term "essentially base-paired" means that more than 65 percent of the bases of the two RNA sequences in question are paired with base and preferably more than 75 percent of the bases are paired with base. The term "essentially unpaired" means that more than 65 percent of the bases of the two sequences in question are not paired with base and preferably more than 75 percent of the bases are unpaired. F3 is preferably, at least, 3 bases long, and more preferably, 6 to 12 bases long. F4 is preferably 3 to 5 bases in length, particularly preferably 4 bases in length. 1_ is a short distance from the bases that preferably has the sequence 5? GAA-3 'when CS has the sequence 5'-NGUC-3 In addition, when L] _ is 5'-AGAA-3' and CS is 5 ' -NGUC-3A when the first base pair between F and F4 adjacent to CS and L ^ is preferably G: C or C: G (Figures 2 and 6). Accordingly, in the present invention, a preferred target sequence in a selected substrate contains the sequence 5'-BNGUC-3 'wherein B is G, C or U (Anderson et al., 1994, Nuc Acids Res. 22: 1096-1100). The "fork" portion is a portion of the catalyst that bends in a fork-like configuration when the substrate-catalyst complex is modeled in two dimensions for minimum energy fold.

This is shown in Figures 2 and 6. The "fork" portion is not an absolute fork in the sense that not all the bases of the "fork" portion have been paired in base. Of course, it is necessary that the "fork" portion have at least one essentially unpaired region so that the catalyst can assume a tertiary structure that permits better or optimal catalytic activity. The "fork" portion of the catalyst preferably has the sequence: P3-S2-P4"3 'L2 ^ \ P2-S! .- Pi-5' where, Pi and P4 are base sequences which are selected from so that Pi and P4 essentially undergo base pairing (Helix 3, Figures 2 and 6) Pl is covalently fixed to F4, Si and S2 are sequences that are selected so that Si (Loop 2) and S2 (Loop 4) they have essentially not been paired, p2 and p3 are base sequences that are selected such that P2 and P3 have essentially undergone base pairing (Helix 4, Figures 2 and 6), and L2 is a sequence of unpaired bases ( Loop 3) The terms "essentially subject to base pairing" and "essentially unpaired" have the same meanings as discussed above, P and P each preferably being from 3 to 6 bases in length and in length. greater preference P] _ has the sequence '-ACCAG-3 'And P4 has the sequence 5' -CUGGUA-3 '. It has been found that A at the end or 5 'end of 5'-ACCAG-3' (underlined) has not undergone base pairing with the U at the end or 3 'terminal of 5'-CUGGUA-3' (underlined) and unpaired A can act as a joint (Figures 2 and 6). If and S2 each preferably is from 4 to 9 bases in length and more preferably, If it has the sequence 5? GAAACA-3 'and S2 has the sequence 51- GUAUAUUAC-3'. Unexpectedly, it was found that the hairpin ribozyme as constructed for the HIV target sequence (Ojwang et al, 1992, Proc Nati Acad Sci USA 89, 10802-10806) was not as efficient as a hairpin ribozyme built with a "tetralazo" modification.

In the prior art, the preferred P2 sequence has the sequence 5'-CAC-3 ', P3 has the sequence 'GUG-3 'and L2 has the sequence 5'-GUU-3' (Ojwang et al., 1992, Proc. Nati, Acad. Sci. USA 1989, 10802-10806). In the preferred embodiment of the present invention, L2, P2. P3 (Figures 2 and 3, Loop 3, Helix 4) are constructed to include the stable RNA hairpin sequence. 5'-GGAC UUCG GUCC-3 '(SEQ ID NO: 5) results in the modification of "tetralazo". as a result, Helix 4 is extended by four base pairs through the prior art sequence mentioned above. In addition, the GUU sequence of Loop 3 is replaced with the UUCG sequence. The resulting ribozyme is more active and more thermally stable than the unmodified ribozyme. The structure of the present invention, as shown in Figure 2 for VPHR434 and which is described above, can be represented diagrammatically by the formula: P3 - S2 - P4"3 '• L / L2 - Si - Pi - F4 - Li - F3 - 5 'The complete ribozyme sequence of the preferred embodiment of the present invention is 5' -UUCUUCAGAGAACAGU ACCAGAGAAACACACGGUCUUCGUCCGUGGUAUAUUACCUGGUA-3 '(SEQ ID NO: 6) The structure of the present invention, as shown in the Figure 6 for VPRH419 and as described above can be represented diagrammatically by the formula: PX - S2 - P4"3 'L2 ^ P2 - Si - Pi - F4 - Li - F3 - 5' The complete ribozyme sequence of the modality Preferred of the present invention is 5'-GGUUUUAGAAGUUAACCAGAGAAACACACGGACUUCGUCCGUGGUAUAUUACCUGGUA-3 '(SEQ ID No.11). The ribozyme of the present invention that cleaves HPV RNA can be used as a therapeutic agent in the treatment of HPV infections that are associated with genital warts and genital neoplasms. In the preferred embodiment, there are two methods for administering the therapeutic agent: gene therapy and a modification of the antisense methodology. The therapeutic agent used in the present invention is administered in combination with other drugs or individually in a manner compatible with good medical practice. The composition is administered and dosed in accordance with good medical practice taking into account the clinical condition of the individual patient, the site and the method of administration, the programming of the administration and other factors known to those who practice medicine. The term "effective amount" for purposes of the present is therefore determined by considerations, such as those known in the art.

HUMAN GEN THERAPY The coding sequence for the HPV16 specific ribozyme is cloned into a vector as described herein and used in human gene therapy (Mulligan, 1993, Science 260: 926-932). In one embodiment, a U6 promoter was cloned into the vector and placed immediately before the coding region of the ribozyme. The U6 promoter is a pol III eukaryotic promoter capable of driving the transcription of the ribozyme using the RNA-II polymerase of the host cell (Das, 1988, EMBO J. 7 (2): 503-512). The use of a retroviral vector to carry the encoded ribozyme aids in the integration of the ribozyme coding sequence into the genomic DNA of the cell thereby providing long-term production of the anti-HPV 16 ribozyme within the cell (Chatterjee and Wong, 1993, Methods: Companion to Methods in Enzymology 5 (l): 51-59). To supply the ribozyme coding vector to the target cells, a liposomal delivery system based on Lipofectin is used. The use of liposome auxiliaries to obtain vector-ribozyme DNA to the cell without degradation, since the liposome acts as a protective barrier of nucleases (Sullivan, 1993, Methods: Companion to Methods in Enzymology, 5 (1): 61 -66). The cells absorb the liposomes that contain the vector through the process of endocytosis that occurs naturally. The advantage of using the Lipofectin reagent is that it allows the liposome, once it has been taken into the cell, to divert degradation by liposomal enzymes which is the usual destination of an endocytic material (Felgner et al., 1993, Methods: Companion to Methods in Enzymology 5 (l): 67-75 In a preferred embodiment, ribozymes directed against one or both of E6 and E7 are administered in combination with immunological agents, such as LAK cells or chemotherapeutic agents, such as cisplatin, which has use in cervical cancer The supply of a ribozyme to the cervical area can be done either by painting or injection.

In addition to the vector described above, other vectors in the delivery of specific HPV ribozymes can be used in gene therapy. These vectors are described in greater detail below. The methods used with and the utility of the present invention can be further demonstrated by the following examples.

MATERIALS AND METHODS Enzymes and chemical compositions. All restriction enzymes used were either from Bethesada Research Laboratories (BRL) or Boehringer Mannheim Biochemicals. The stabilization agents for restriction enzymes were supplied by the manufacturer. The DNA ligand T4 and the kit sequences were obtained from Pharmacia. The transcription kit in vi tro and the related enzymes were obtained from Promega. Bovine calf serum, antibiotics (penicillin and streptomycins), L-glutamine, sodium pyruvate, phosphate-stabilized saline (PBS) and modified Dulbecco's Eagle medium (DMEM) were purchased from GIBCO. The T7 RNA polymerase used was manufactured by US Biochemicals (USB). With the exception of RNA T7 polymerase, the stabilization agents for enzymes used were supplied by the manufacturer. The RNA T7 polymerase transcription stabilization agent consisted of the following: 40mM Tris, pH 8.0, 6mM MgCl2, 5mM DTT, 1mM Spermidine, 1 percent Triton X 100. Synthetic DNA templates used for in vitro prescriptions and cloning were produced using an Applied Biosystems 392 DNA synthesizer. Recombinant DNA techniques. Unless otherwise stated, the techniques were carried out as described in the Sambrook et al. Article (1989, Molecular Cloning: A Laboratory Manual (second edition), Sections 1.25 to 1.28, 1.60 to 1.61, 1.68 a 1.69, 1.82 to 1.84, 6.9 to 6.13, 6.46 to 6.48) the entire disclosure of which is incorporated herein by reference. Segmentation of HPV substrates. Segmentation was carried out in 12 mM MgCl2, 2mM spermidine and 40mM Tris, pH 7.5, using previously published methods (Hampel and Tritz, 1989, Biochem.28: 4929-4933). All reactions were carried out at 37 ° C with 25 nM of ribozyme and 50 nM of the substrate for 60 minutes unless otherwise indicated. The reference reaction was the native STRV sequence (-) S17 / R53 at 10 nM and 100 nM during the times shown (Hampel and Tritz, 1989, Biochem 28: 4929-4939).

Irradiation of P32. The substrate and the ribozymes were radiated with p32-cPT by transcription of the synthetic DNA templates using RNA T7 polymerase as described previously (Hampel and Tritz, 1989, Biochem, 28: 4929-4933) and the separated reaction products. in 15 to 18 percent polyacrylamide gels in 7M urea. Construction of the ribozyme. The ribozyme was constructed by T7 transcription of complementary synthetic DNA templates. This was carried out as described above (Hampel and Tritiz, 1989, Biochem 28: 4929-4939). Construction of Master Plasmids Containing VPHR. The coding and non-coding chains for VPHR were synthesized and purified by HPLC. The chains included an EcoRI site, the ribozyme coding region, a poly-T termination signal for Polymerase III RNA and a BamHl site. The two chains were then annealed by adding an equimolar amount of each, covered in H2O at 90 ° C for 5 minutes and then allowed to cool slowly to room temperature over a period of 30 minutes. The resulting double strand fragment was digested with EcoRI and BamHI. The digestion products were carried out on an agarose gel and the ribozyme coding fragment was isolated and purified. Plasmid pHC (Altschuler, 1992, Gene, 122: 85-90) was digested with EcoRI and BamHI and the fragment was isolated and purified as above. The VPHR434 or PVHR419 fragment was then ligated into pHC, and the ligation mixture was used to transform the competent bacterial DH5alpha cells. Individual colonies were selected and grown in CircleGrow bacterial media and the plasmids were extracted and purified using a normal miniprep protocol (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (second edition) Sections 1.25 to 1.28, 1.60 a 1.61, 1.68 to 1.69, 1.82 to 1.84, 6.9 to 6.13, 6.46 to 6.48). Plasmids were selected for incorporation of the VPHR434 or PVHR419 insert. A colony that incorporated the insertion was then subjected to sequence using the enzymes of Sequence Version 2.0 and the protocol to verify the appropriate DNA sequence. The resulting plasmid was named pHC-4343 or pHC-419, respectively. The ribozymes were cloned into a Moloney retrovirus expression vector for in vivo testing in human cells transformed with HVP-16. The cloning project is as follows. The ribozyme oligos were synthesized with the Pol III termination signal and the EcoRl / BamHl terms. These were then cloned in pHC (Altschuler, 1992, Gene, 125: 85-90), the normal bacterial expression vector used in a preferred embodiment. The ribozyme is cut with EcoRl HindIII and cloned into pU6 which is a Bluescript vector containing a mouse U6 promoter (Das, 1988, EMBO J. 7 (2): 503-512). The insert containing the U6 promoter is then cloned into the BamH1 site of pZIP-NeoSV (X) (Cepko et al., 1984, Cell 37: 1053-1062). pHC-434 and pMU6, a plasmid containing the promoter region of RNA polymerase III (Das, 1988, EMBO J. 7 (2): 503-512) were digested with Eco RI and Hind III. The VPHR434 fragment that retained the hairpin cassette region and the pMU6 fragment were isolated and purified as described above. The ligation and bacterial transformation of the two fragments was carried out as described above. The colonies were selected and subjected to sequence as described above. The resulting plasmid was named pMU6-434. Selection of the HPV sequence (SEQ ID No: l) for segmentation site. The data of the VPH16 sequence was obtained through Gen Bank. The E6 and E7 regions of HPV16 were inspected for potential target sequences as disclosed above.

All potential sites containing potential target sequence were tested and ribozymes that showed significant catalytic activity were further developed. VPHR434 and VPHR419 are examples of ribozymes and showed significant catalytic activity. The general principles of the present invention can be more fully appreciated by reference to the following non-limiting examples.

EXAMPLE 1 In the preferred embodiment of the present invention, as shown in Figure 2, they constructed Loop 3 and Helix 4 to include the stable RNA hairpin sequence. 5'-GGAC UUCG GUCC -3 '(SEQ ID No: 5) resulting in the "tetralazo" modification (Cheong et al., 1990, Nature, 346: 680-682; Varani et al., 1991, Biochem. -89). As a result, Helix 4 was extended by four base pairs through the unmodified sequence. In addition, the GUU sequence of Loop 3 was replaced by the UUCG sequence. To determine the activity of the ribozyme, RNA was added to a substrate at a ratio of 1:30 and the time course of segmentation was studied as the parameters varied. The reaction was carried out in 12 mM MgCl 2, 40 mM Tris, pH 7.5 and 2 mM spermidine through 150 minutes. For temperature dependence, the cleavage regimen of a ribozyme containing the tetralazo modification is tested through a temperature scale and compared to the control reaction at 37 ° C. The reaction products are analyzed on polyacrylamide / urea gels. The bands are cut and counted in a liquid scintillation counter. In the control reaction only 2 percent of the substrate remains after 150 minutes indicating that the ribozyme must imtreat with multiple substrates during the course of the reaction since it speaks 30 times more substrate than the ribozyme. In addition, the amount of ribozyme remains unchanged as expected from a catalyst. In the temperature-dependent study of the tetralazo modification compared to the prior art, the activity of the ribozyme was measured at 20 ° C, 27 ° C, 33 ° C, 37 ° C, 41 ° C and 45 ° C . The reaction showed a temperature dependence similar to that which was to be expected in a reaction involving RNA molecules subjected to base pairing. The Arrhenius trace of the data provides an optimum temperature of 37 ° C for the reaction. Higher temperatures reduce the reaction rate with a very fast rate reduction of about 41 ° C compatible with the fusion of the catalytic RNA structure. At 50 ° C no reaction was detectable. The reaction regime at temperatures below 37 ° C showed a linear reciprocal temperature dependence consistent with the classical decrease in activation energy for the reaction. The inclination of the line in the Arrhenius trace provided an activation energy of 19 Kcal / mol which is close to that found for the catalysts that conform to a hammer head segmentation mechanism (13.1 Kcal / mol) (Uhlenbeck, 1987, Nature 328: 596-600). The example shows that a ribozyme with the tetralazo modification is more active and more thermally stable than that of the prior art. This form of ribozyme remains active at 45 ° C, while the unmodified ribozyme loses most of its activity at this temperature. It was concluded from this experiment that Loop 3 does not have a conserved or invariable base sequence and that helix 4 can extend into Loop 3 by at least four base pairs without loss of activity. The four additional base pairs in Helix 4 provide helical stabilization of this region. The secondary bending energy of helix 4 and Loop 3 in the structure of the prior art is +0.6 Kcal / mol, while that of the ribozyme having Helix 4 extended and Loop 3 of the sequence UUCG (tetralazo) ) of the present invention was determined as being -11.1 Kcal / mol. Therefore, the presence of the tetralazo sequence increases bending energy by 11.7 Kcal / mol.

EXAMPLE 2 The segmentation reaction and the optimization of the length of the helix 1 for PVHR434. A segmentation study was carried out to optimize the length of the helix 1. Figure 3 shows bands in a denaturing polyacrylamide gel that identifies the ribozyme, the substrate and the segmentation products, three substrates were segmented by the ribozyme, each with a helix 1 of different length. The substrates were the following: SUBSTRATE LENGTH OF PERCENTAGE HELIX 1 SEGMENTED 430-ACUG U * GU9 * C CUGAAGA-444 7 5.4 (SEQ ID No: 2) 430-ACUG U * GUC CUGAAGAA-445 8 6.7 (SEQ ID No: 3) 430-ACUG U * GUC CUGAAGAAA-4 6 9 6.5 (SEQ ID No: 5) The most efficiently segmented substrate was one that had a helix 1 of 8bp (SEQ ID No: 3) and was used for all additional studies. It is referred to as VPHS and the corresponding ribozyme is referred to as VPHR-434 (Figure 2). Segementasión time course. The time course for VPHS segmentation using VPHR-434 was carried out over a period of 180 minutes (Figure 4). The ribozyme efficiently segmented the substrate up to 88 percent completion.

Kinetic segmentation parameters. The Michaelis kinetic analysis of the reaction was carried out using a ribozyme limit and an excess of the substrate for constant concentration of the ribozyme and variable concentrations of the substrate in order to measure the initial rates (Figure 5). The Km for the reaction was 21 nM and the kcat: or the investment number was 0.083 min "1.

This provides a total catalytic efficiency of (kcat / Km) of 4 micrometers-1 min-1, which is about 7 percent of that of the original native hairpin sequence (Hampel and Tritz, 1989, Biochem.28: 4929-4933).

EXAMPLE 3 The segmentation reaction and optimization of the length of Helix 1 for PVHR419. A segmentation study was carried out to optimize the length of Helix 1. Figure 7 shows bands in a denaturing polyacrylamide gel that identifies the ribozyme, the substrate and the cleavage products. Four substrates were segmented and the substrates were the following: SUBSTRATE LENGTH OF PERCENTAGE HELIX 1 SEGMENTED 415-UAAC U * GUC AAAAGC-428 7.5 (SEQ ID NO: 7; 415-UAAC U * GUC AAAAGCC-429 62.8 (SEQ ID NO: 8) 415-UAAC U * GUC AAAAGCCA-430 12.1 (SEQ ID NO: 9) 415-UAAC U * GUC AAAAGCCAC-431 28.9 (SEQ ID NO: 10) The most efficiently segmented substrate was one that had a helix 1 of 7bp (SEQ ID No: 8) and was used for all additional studies. The same is referred to as VPHS and the corresponding ribozyme is referred to as VPHR-419 (Figure 6). Segmentation time course. The timepo course for VPHS segmentation using VPHR-19 was carried out over a period of 180 minutes (Figure 8). The ribozyme efficiently segmented the substrate up to 88 percent completion. Kinetic segmentation parameters. A Michaelis-Menton kinetic analysis of the reaction was carried out using a ribozyme limit and an excess of the substrate for constant ribozyme and excess substrate for a constant concentration of ribozyme and varying concentrations of the substrate in order to measure the initial rates ( Figure 9). The Km for the reaction was 98 nM and the Kcat or inversion number was 0.18 min-1. This provides a total catalytic efficiency (kcat / Km) of 1.8 micrometers-1 min-1, which is approximately 3 percent of that of the original native hairpin sequence (Hampel and Tritz, 1989, Biochem.28: 4929-4933). The preferred embodiments of our improved vector are described in detail in the following non-limiting examples. Construction of vector ptVl containing a supply cassette. Vector ptVl (Figures 11 and 12) was prepared by cloning a delivery cassette consisting of a new polylinker, a human ANRtval poIIII promoter with a human upstream region (Arnold et al., 1986, Gene 44: 287-297). , a variant of the tetralazo sequence and a poly-T sequence to the pBluescript KS vector (from Estratagene). The human upstream region v.gr. is shown in Figure 12 (see also Arnold and Gross, 1987, Gene 51: 237-246). The steps to build the vector are as follows. Both chains of a new linker region of 119 nt (Figure 10) was prepared by chemical oligonucleotide synthesis. This linker region contained different restriction sites, the tetralazo sequence described above and a poly-T pathway to complete transcription of poIIII. This was cloned to the Asp718 (Kpnl) / Sacl sites of pBluescript KS (Stratagene) to provide pBKSLNKR (not shown). Plasmid pHTVl (see Arnold et al., 1986, Gene 44: 287-297) contains the human RNAva promoter. The ttvalval gel plus an additional 50 nt of the upstream sequence was simplified by PCR with the EcoRl / Xhol terminals. This was cloned into the corresponding sides of the linker region of pBKSLNKR to provide the final ptVl construct (Figures 11 and 12). The ribozyme coding sequences can be cloned at the XhoI / MLl sites of ptVl. All the chemical syntheses of the DNA oligonucleotides were carried out using normal methods in an ABI 392 DNA / RNA synthesizer (using manufacturer's recommendations for procedures). All plasmid constructs were carried out using normal procedures (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (second edition) .The tetralazo variant at the 3 'end of the ribozyme has the sequence CCUG (UUCG) CAGG ( SEQ ID NO: 16) This sequence is a variant of the highly stable tetralazo sequence GGAC (UUCG) GUCC (SEQ ID NO: 5) which is found in several native RNA sequences (Cheong et al., 1990, Nature 346: 680) In contrast to the native tetralazo, the variant inverts the two sequences forming the helix in relation to the original, this difference in relation to the native tetralazo and other synthetic fork ribozyme constructions (Anderson et al., 1994, Nuc Acids). Res. 22: 1096-1100), decreases the likelihood of in vivo recombination between the construct and the native or other synthetic sequences.This vector construct also has the advantage that the tetralazo variant can remove is replaced by another tetralazo sequence by simply digesting the vector with MIul / Bstxl and inserting another tetralazo sequence with appropriate ends. Plasmid ptVl has additional advantages based on its components and its disposition. The poIIII promoter provides very high transcription of the ribozyme (Yamada et al., 1994, Gene Therapy 1: 38-45). The upstream region improves the levels of transcription in vi vo of the ribozyme (Arnold and Gross, 1987, Gene 51: 237-246). The tetralazo variant provides enhanced stability to the ribozyme by protecting the 3 'terminal of degradation by 3' exonuclease. The poly-T sequence allows the efficient termination of the transcript originating in the pollin promoter. Restriction sites (XhoI / MIuI or BGIII / MIuI) after the tRNA coding region are suitable for inserting ribozyme coding sequences. For example, an overlay Bglll can be linked to an overlapping BamHl. Transcription is initiated in the tRNA coding region and terminates efficiently after the poly-T pathway and is transcribed from the poly-U portion of the ribozyme. The entire delivery cassette can be easily digested from ptVl as a unit and cloned towards the corresponding restriction sites of the viral expression vectors including vectors based on Moloney retroviruses, adenoviruses, adeno-associated viruses and the like. This approach has been used to construct a series of constructs for the delivery of HPV-specific ribozymes to living cells. Construction of the vector for the supply of specific ribozymes of human papillomavirus to human cells. Our constructions containing the delivery cassette, a hairpin ribozyme sequence and a retroviral vector have been designed to specifically segment the HPV sequences. Ribozyme VPHR434 was designed to segment site 434 in the human papillomavirus HPV-16 sequence (access GenBank # K02718). Site 434 is in the E6 / E7 sequence of human papilloma virus (HPV) that has been found in human carcinoma (DiPaolo et al., 1993, Critical Reviews in Oncogenesis 4.337-360). The retroviral vector used was pZIP-NeosSV (X) 1 (Cepko et al., 1984, Cell 37: 1053-1062) which is a vector based on Moloney retroviruses used in human genetic engineering (see, for example, the 1994 RAC Application of Wong-Stall). DNA corresponding to the HPV-16 specific hairpin ribozyme for site 434 in the HPV-16 sequence was synthesized and cloned into the XhoI / MLl sites of the ptV1 vector as described above to provide the pBtVl plasmid -434 (Figures 13 and 14). The pBtVl-434 vector was digested with Sau3Al and an * 226 bp insert containing the delivery cassette with its inserted ribozyme was isolated after separation by agarose gel electrophoresis using standard methods. Digestion of this plasmid with Sau3ala produces a number of fragments. The 226 bp fragment (cut between the BamHl and Bell sites) was identified by running a pBluescript KS control digester side by side by the pBtVl-434 digester; the 226 bp fragment is only seen in the digester pBtVl-434. This 226 bp fragment was cloned into the BamH1 site of pZIP-Neo (SV (X) I in both orientations to provide plasmids pZIPVl-434 (syn) (Figures 15 and 16) and pZIPVl-434 (anti) (Figures 17 and 18), respectively The "sin" orientation refers to constructs in which the poIIII promoter inserted is in the same orientation as the poIIII promoter of the retrovirus.The "anti" constructs have the poIIII promoter inserted which is in the orientation In these constructions, the ribozyme coding region VPHR434 is downstream and, therefore, transcribed from a very strong poIIII promoter into a vector based on Molonesy retroviruses. POIIII promoter provides transcription of the effective ribozyme in vi vo (Yam, ada et al, 1994, Gene Therapy 1: 38-45) .The upstream region improves the levels of in vivo transcription of the ribozyme (Arnold and Gross, 1987). , Ge ne 51-237-256). Tetralazo provides enhanced stability by protecting the 3 'end of the ribozyme against nuclease degradation. The pol-T sequences complete the transcription from the poIIII promoter. A corresponding inactive construction was also developed. The inactive construct had a mutation change from an AAA to a CGU in loop 2 of the hairpin ribozyme cloned in ptVl (as described above). The catalytically inactive ribozyme was encoded by the plasmid pBtVl-434 (i) (Figures 19 and 20). To verify that these and similar constructs have activity in vitro, the constructs were transfected into tissue cultured cells and the ribozyme production was assayed.

EXAMPLE 4 Test in vivo. The in vivo test was carried out using the specific constructions of HPV-16. Vectors pZIPvl-434 (sin) and pZIPVl-434 (anti) were stably transfected with human CXTl cells using normal methods (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, second edition). The CXT1 cell line derived from a spontaneous human cervical cancer tumor has been shown to express HPV E6 and E7 proteins 16.

The ribozyme is expressed in vivo by means of delivery in the retroviral vector. The expression of the ribozyme was determined by the RNase protection assays (Figure 21, using the method of Lee and Costlow, 1987, Methods Enzymol 152: 633-648). CXTl cells were transfected with the pZIP-Neo-SV (X), pZIP-V1434 (anti) and PZIP-V1434 (without) constructs and the transformants were selected as resistance to the drug G418. The RNA was isolated from the infected cells using the acid phenol method (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, second edition). The test probe used was transcribed with the T7 RNA polymerase of the plasmid pBtVl-434 (Figures 13 and 14) which was previously cut with BamHI; the 265 nt RNA transcript produced in this manner was used as the test probe. The RNase protection was carried out and the products were separated by gel electrophoresis using normal procedures (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, second edition). Referring to Figure 21, the arrow indicates the 160 nt size of the protected ribozyme transcript of the poIIII promoter. The ribozyme is seen in the pZIPVl-434 (sin) track while none is seen in the tracks corresponding to the RNA of cells transfected with pZIP-Neo-SV (X) and pZIP-V1434 (anti). Similarly, no 160 nt fragment is seen in the control track for cells that were not transfected ("NT" track). The other clues are the controls that show the 32p test probe that is intact (track "test probe") and that the RNase is active for digestion of the cellular RNA and the test probe ("RNase +" track). Note that only a band of 160 nt is visible. If a band longer than 230 nt had been seen, it would have indicated the transmission originating from the vector poIIII promoter. The presence of only the 160 nt fragment shows that the ribozyme transcript was transferred from the poIIII promoter of the delivery cassette and not from the vector promoter. The hairpin ribozyme appeared as decreasing the expression of E6 mRNA in vivo. To determine whether ribozyme expression affects HPV expression, an assay based on reverse transcription (RT) and amplification by polymerase chain reaction (PCR) was used. CXTl cells were stably transfected with the pZIP-Neo-SV (X), pZIP-V143 (anti) and pZIP-V1434 (without) constructs and were selected by resistance to G418 and used as a source of the HPV mRNA. The mRNA was isolated by the acid phenol method and further purified using poly-A binding using standard methods (Sambrook, 1989, Molecular Cloning: A Laboratiry Manual, second edition). The mRNA was transferred in reverse to produce the cDNA that was amplified by PCR with the E6-specific primers to detect a loss of the E6 mRNA. The primer used for reverse transcription (called "16E7R") complementary to the 3 'end of E6 mRNA was: 5' TTATGGTTTCTGAG 3 '(SEQ ID NO: 12). Once the PCR primers were in the probe probe anchored specific to the E6 cDNA that amplifies the lower strand of the E6 cDNA that has or has not been cleaved by the ribozyme. The other two PCR primers were specific for amplification of the upper strand of E6 cDNA. One of these upper chain primers is 5 'from the ribozyme cut site at nt 434 and the other is 3' from the ribozyme cut site. These three primers were: the anchored primer called E7X having the sequence 5 * CCCTCTAGAGGCACACAATTCCTAGTG 3 '(SEQ ID NO: 13); the ribozyme cut site primer 31 called E6-U2 having the sequence 5 'CACGTAGAGAAACCCAGC3' (SEQ ID NO: 14); and the 5 'primer of the ribozyme cut site named E6-16U, which has the sequence 5' CAGCAATACAACAAACCG 3 '(SEQ ID NO: 15). PCR amplification was carried out using normal conditions: 25 cycles at 9 ° C for one minute for fusion and then 65 ° C for 45 seconds for annealing and 72 ° C for one minute for polymerization using an automated thermal cycler (Perkin Elmer model) 480). Five units of the Amplitaq polymerase (Perkin Elmer), 100 microns of each NTP, 10 ng of the E6-16U primer (SEQ ID NO: 15), 10 ng of the E6-U2 primer (SEQ ID NO: 14) and 20 ng of the E7X primer (SEQ ID NO: 13) were used, of course. This combination of three primers provided two PCR products of approximately 300 nt and 500 nt respectively in cells in which the E6 mRNA was intact. However, in cells where the ribozyme cut the E6 mRNA, the smaller of the two 300 nt PCR products was more predominant due to the molar ratio of the ribozyme cut of the E6 mRNA to the full length of the E6 mRNA. The products were separated by gel electrophoresis. Figure 22 shows that RT and the PCR assay detects the presence of HPV of the E6 mRNA in the infected cells. The Figure also shows that the ribozyme cleaves E6 mRNA in the transfected cells expressing the ribozyme. The arrows indicate the positions where the two amplified PCR products migrate in a gel, with the arrow to the left indicating the position of the 300 bp fragment and the arrow to the right indicating the position of the 500 bp fragment as determined from the positions in the gel of molecular weight markers (track "MWM"). The positive control track ("HPV-16") showed bands at both positions indicating that the RNA in the tiny E6 space is present in the CXT1 cells and that the assay successfully amplified both fragments. Cells transfected with the plasmid pZIP-Neo-SV (X) also serve as a control because the plasmid does not encode a specific HPV ribozyme and the bands are seen in both positions. Amplification using the three primers can be used to identify the presence of HPV-16 in human tissue. A sample containing RNA from human tissue can be subjected to the ribozyme and subsequently amplified using the three primers. The ribozyme will only cut the RNA that has the HPV-16 binding sequence. Therefore, samples containing the HPV-16 RNA will be cut by the ribozyme and will result in the preferential amplification of the shorter product produced by the 5 'primer of the ribozyme cutting site.

EXAMPLE 5 Selection of the transformed cell line that is not HPV-16 with specific ribozymes of HPV-16 and transfection in an expression vector of HPV-16 / E7. HeLa cells are stably transfected with constructs capable of expressing a specific HPV ribozyme for site 434 in HPV. These constructs express the ribozyme using a poIIII promoter from either mouse U6 (MU6) or human ANRtval (tV1) origin. Verification of ribozyme expression is determined using RNA test probes in RNase protection assays. The ribozyme specific for HPV site 434 is cloned into vectors with the transcribed ribozyme ending from either a mouse poIIII promoter U6 (MU6) or human ARNtval (tVl). Both constructions of the bacterial plasmid and the constructions based on Moloney retroviruses are carried out. The bacterial plasmid constructs have the human tRNAva promoter cloned in the plasmid pBluescript (KS) (which can be obtained from stratagem). The specific ribozyme 434 is cloned into pBluescripts with the tRNAval promoter at the multiple cloning site to provide the plasmid pBTVl-434. The insert is removed from this plasmid by cutting with the restriction enzyme Sau3Al and the appropriate fragment size containing the insert is isolated using the normal gel purification method. The insert is then subcloned into the BamHl site of pZIP-neo which is a plasmid based on Moloney murine leukemia virus to provide plasmid pZIP-434 with the insertion in either the "sin" or "anti" configuration. HeLa cells were transfected with the ribozyme constructs and stable transfectants were selected using the G418 resistance selection. The cells are grown and the RNA is isolated from the cells for use in an RNase protease assay to identify the expressed ribozyme. The test probe to be used for the identification of the ribozyme is a plasmid with the inserted ribozyme inserted between the T3 promoter and a T7 promoter. These two plasmids are called pMU6-434A for the construction of the ribozyme U6 promoter and pBTVl-434 for the construction of the ribozyme RNArval promoter. The test probe for the construction of the MU6 promoter is produced by linearizing the plasmid containing MU6, pMU6-434A, with EcoRI and transcribing the linearized plasmid with the T3 RNA polymerase using normal procedures. This test probe, which is 110 nt long, binds to the ribozymes produced from both the long terminal repeat (LTR) of plasmid pZIP-434 (without) and the poIIII promoter of plasmids pZIP-434 (without) and pZIP-43 (anti). No ribozoma can be produced by LTR of the pZIP434 (anti) configuration. After annealing and digestion of RNase, the protected fragment produced from LTR is 77 nt in length, while the protected fragment produced from the poIIII promoter is 64 nt long. The test probe used with the construction of the tV1 promoter is produced by linearizing the plasmid pBtVl-434 with BamHI and transcribing the DNA plasmid with T7 RNA polymerase using normal polymerization conditions. This test probe, which is 265 nt long, is fixed to the ribozymes produced by both LTR of pZIPVl-434 (without) and the poIIII promoter of the plasmids pZIPVl-434 (without) and pZIPVl-434 (anti), but the sizes of the protected fragments differ. After digestion of RNase, the protected fragment of LTR is 230 nt long, while the protected fragment produced from the poIIII promoter is 160 nt long. The cells are infected with a retroviral vector capable of expressing the E6 / E7 genes. The comparison levels for the three mRNA species for these two proteins (called E6E7, E6 (I) E7 and E6 (II) E7, the first two being the main species and the third being the small species) are measured using a transcriptase Reverse and a polymerase chain reaction (RT / PCR assay) as indicated in the article by Cornelissen et al. (J. Gen. Virol. 71: 1243-1246, 1990). The primers used in the RT / PCR assay are as follows. Primer 1 is 5'NNNAAGCTTCTGCAATGTTTCAGGACCC 3 '(SEQ ID: 16) and primer 2 is 5'NNNGGATCCCCATTGGTACCTGCAGGATC3' (SEQ ID NO: 18). Primer 2 is the primer used in the RT reaction; both primers 1 and 2 are used in the PCR reaction. The RP / PCR reaction yields fragments of the following sizes: 791 bp corresponding to the product E6E7; 608 bp for the product E6 (I) E7 and 491 bp for the product E6 (II) E7. Because ribozyme 434 cleaves the two main species of mRNA (E6E7 and E6 (I) E7) and not the secondary species (E6 (II) E7), the secondary species serves as an internal reference standard in the assay. The levels of the two main species (the products of 791 bp and 608 bp) relative to the secondary species (the product of 491 bp) are measured to determine the in vivo efficacy of ribozyme 434. The main species shows a reduction of 10 percent to 50 percent relative to the product of the internal secondary species when the ribozyme is expressed. As controls, cells transfected only with the ribozyme vector that does not contain the ribozyme insert and cells transfected with the inactive ribozyme construct are also used. The RT / PCR assay shows no decrease in the two main species in relation to the secondary species when these plasmids are processed.

It will be understood by those skilled in the art that similar constructions and assays can be carried out using the ribozyme specific for site 419. When these constructs are used and the 419-specific ribozyme is produced in the transfected cells, a similar decrease is seen. in the main species of HPV mRNA in relation to the secondary internal reference species that is not segmented by the specific ribozyme 419.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Hampel, Arnold DiPaolo, Joseph Siwkowski, Andrew M. (ii) TITLE OF THE INVENTION: INHIBITION OF HUMAN PAPILOMA VIRUS BY A FORK RIBOZYMA (Üi) SEQUENCE NUMBER : (iv) ADDRESS FOR CORRESPONDENCE (A) CONSIGNEES: Knobbe, Martens, Olson & Bear (B) STREET: 620 Newport Center Drive (C) CITY: Newport Beach (D) STATE: California (E) COUNTRY: United States of America (F) POSTAL CODE: 92660 (v) COMPUTER LEADABLE FORM: (A) MEDIUM TYPE: 8.89 cm disc (B) COMPUTER: Compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) ) DATE OF PRESENTATION (C) CLASSIFICATION: (vii) INFORMATION ABOUT THE LAWYER / AGENT: (A) NAME: Altman, Daniel E. (B) REGISTRATION NUMBER: (C) ATTORNEY NUMBER / TOCA: NIH113.001QPC (viii) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (714) 760-0404 (B) TELEFAX: (714) 760-9502 (2) INFORMATION FOR SEQ ID NO: l (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7904 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: circular (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) SOURCE ORIG INAL: (A) ORGANISM: human papilloma virus (B) CEPA: HPV16 (vi) DESCRIPTION OF SEQUENCE: SEQ ID NO: l ACTACAATAA TTCATGTATA AAACTAAGGG CGTAACCGAA ATCGGTTGAA CCGAAACCGG TTAGTATAAA AGCAGACATT TTATGCACCA AAAGAGAACT GCAATGTTTC AGGACCCACA GGAGCGACCC AGAAAGTTAC CACAGTTATG CACAGAGCTG CAAACAACTA TACATGATAT AATATTAGAA TGTGTGTACT GCAAGCAACA GTTACTGCGA CGTGAGGTAT ATGACTTTGC TTTTCGGGAT TTATGCATAG TATATAGAGA TGGGAATCCA TATGCTGTAT GTGÁTAAATG TTTAAAGTTT TATTCTAAAA TTAGTGAGTA TAGACATTAT TGTTATAGTT TGTATGGAAC AACATTAGAA CAGCAATACA ACAAACCGTT GTGTGATTTG TTAATTAGGT GTATTAACTG TCAAAAGCCA GTGTGTCCTG AAGAAAAGCA AAGACATCTG GACAAAAAGC AAAGATTCCA TAATATAAGG GGTCGGTGGA CCGGTCGATG TATGTCTTGT TGCAGATCAT CAAGAACACG TAGAGAAACC CAGCTGTAAT CATGCATGGA GATACACCTA CATTGCATGA ATATATGTTA GATTTGCAAC CAGAGACAÁC TGATCTCTAC TGTTATGAGC AATTAAATGA CAGCTCAGAG GAGGAGGATG AAATAGATGG TCCAGCTGGA CAAGCAGAAC CGGACAGAGC CCATTACAAT ATTGTAACCT TTTGTTGCAA GTGTACTCT ACGCTTCGGT TGTGCGTACA AAGCACACAC GTAGACATTC GTACTTTGGA AGACCTGTTA 'ATGGGCACAC TAGGAATTGT GTGCCCCATC TGTTCTCAGA AACCATAATC TACCATGGCT GATCCTGCAG GTACCAATGG GGAAGAGGGT ACGGGATGTA ' ATGGATGGTT TTATGTAGAG GCTGTAGTGG AAAAAAAAAA AGGGGATGCT ATATCAGATG ACGAGAACGA AAATGACAGT GATACAGGTG AAGATTTGGT AGATTTTATA GTAAATGATA ATGATTATTT AACACAGGCA GAAACAGAGA CAGCACATGC GTTGTTTACT GCACAGGAAG CAAAACAACA TAGAGATGCA GTACAGGTTC TAAAACGAAA GTATTTGGTA GTCCACTTAG TGATATTAGT GGATGTGTAG ACAATAATAT TAGTCCTAGA TTAAAAGCTA TATGTATAGA AAAACAAAGT AGAGCTGCAA AAAGGAGATT ATTTGAAAGC GAAGACAGCG GGTATGGCAA TACTGAAGTG GAAACTCAGC AGATGTTACA GGTAGAAGGG CGCCATGAGA CTGAAACACC ATGTAGTCAG TATAGTGGTG GAAGTGGGGG TGGTTGCAGT CAGTACAGTA GTGGAAGTGG GGGAGAGGGT GTTAGTGAAA GACACACTAT ATGCCAAACA CCACTTACAA ATATTTTAAA TGTACTAAAA ACTAGTAATG CAAAGGCAGC AATGTTAGCA AAATTTAAAG AGTTATACGG GGTGAGTTTT TCAGAATTAG TAAGACCATT TAAAAGTAAT AAATCAACGT GTTGCGATTG GTGTATTGCT GCATTTGGAC TTACACCCAG TATAGCTGAC AGTATAAAAA CACTATTACA ACAATATTGT TTATATTTAC ACATTCAAAG TTTAGCATGT TCATGGGGAA TGGTTGTGTT ACTATTAGTA AGATATAAAT GTGGAAAAAA TAGAGAAACA ATTGAAAAAT TGCTGTCTAA ACTATTATGT GTGTCTCCAA TGTGTATGAT GATAGAGCCT CCAAAATTGC GTAGTACAGC AGCAGCATTA TATTGGTATA AAACAGGTAT ATCAAATATT AGTGAAGTGT ATGGAGACAC GCCAGAATGG ATACAAAGAC AAACAGTATT ACAACATAGT TTTAATGATT GTACATTTGA ATTATCACAG ATGGTACAAT GGGCCTACGA TAATGACATA GTAGACGATA GTTGAATTGC ATATAAATAT GCACAATTGG CAGACACTAA TAGTAATGCA AGTGCCTTTC TAAAAAGTAA TTCACAGGCA AAAATTGTAA AGGATTGTGC AACAATGTGT AGACATTATA AACGAGCAGA AAAAAAAACA ATGAGTATGA GTCAATGGAT AAAATATAGA TGTGATAGGG TAGATGATGG AGGTGATTGG AAGCAAATTG TTATGTTTTT AAGGTATCAA GGTGTAGAGT TTATGTCATT TTTAACTGCA TTAAAAAGAT TTTTGCAAGG CATACCTAAA AAAAATTGCA TATTACTATA TGGTGCAGCT AACACAGGTA AATCATTATT TGGTATGAGT TTAATGAAAT TTCTGCAAGG GTCTGTAATA TGTTTTTGTAA ATTCTAAAAG CCATTTTTGG 'TTACAACCAT TAGCAQATGC CAAAATAGGT ATGTTAGATG ATGCTACAGT GCCCTGTTGG AACTACATAG ATGACAATTT AAGAAATGCA TTGGATGGAA ATTTAGTTTC TATGGATGTA AAGCATAGAC CATTGGTACA ACTAAAATGC CCTCCATTAT TAATTACATC TAACATTAAT GCTGGTACAG ATTCTAGGTG GCCTTATTTA CATAATAGAT TGGTGGTGTT TACATTTCCT AATGAGTTTC CATTTGACGA AAACGGAAT CCAGTGTATG AGCTTAATGA TAAGAACTGG AAATCCTTTT TCTCAAGGAC GTGGTCCAGA TTAAGTTTGC ACGAGGACGA GGACAAGGAA AACGATGGAG ACTCTTTGCC AACGTTTAAA TGTGTGTCAG GACAAAATAC TAACACATTA TGAAAATGAT AGTACAGACC TACGTGACCA TATAGACTAT TGGAAACACA TGCGCCTAGA ATGTGCTATT TATTACAAGG CCAGAGAAAT GGGATTTAAA CATATTAACC ACCAAGTGGT GCCAACACTG GCTGTATCAA AGAATAAAGC ATTACAAGCA ATTGAACTGC AACTAACGTT AGAAACAATA TATAACTCAC AATATAGTAA TGAAAAGTGG ACATTACAAG ACGTTAGCCT TGAAGTGTAT TTAACTGCAC CAACAGGATG TATAAAAAAA CATGGATATA CAGTGGAAGT GCAGTTTGAT GGAGACATAT GCAATACAAT GCATTATACA AACTGGACAC ATATATATAT TTGTGAAGAA GCATCAGTAA CTGTGGTAGA GGGTCAAGTT GACTATTATG GTTTATATTA TGTTCATGAA GGAATACGAA CATATTTTGT GCAGTTTAAA GATGATGCAG AAAAATATAG TAAAAATAAA GTATGGGAAG TTCATGCGGG TGGTCAGGTA ATATTATGTC CTACATCTGT GTTTAGCAGC AACGAAGTAT CCTCTCCTGA AATTATTAGG CAGCACTTGG CCAACCACCC CGCCGCGACC CATACCAAAG CCGTCGCCTT GGGCACAGAA GAAÁCACAGA CGACTATCCA GCGACCAAGA TCAGAGCCAG ACACCGGAAA CCCCTGCCAC ACCACTAAGT TGTTGCACAG AGACTCAGTG GACAGTGCTC CAATCCTCAC TGCATTTAAC AGCTCACACA AAGGACGGAT TAACTGTAAT AGTAACACTA CACCCAÍAGT ACATTTAAAA GGTGATGCTA ATACTTTAAA ATQTTTAAGA TATAGATTTA AAAAGCATTG TACATTGTAT ACTGCAGTGT CGTCTACATG GCATTGGACA GGACATAATG TAAAACATAA AAGTGCAATG GTTACACTTA CATATGATAG TGAATGGCAA CGTGACCAAT TTTTGTCTCA AGTTAAAATA CCAAAAACTA TTACAGTGTC TACTGGATTT ATGTCTATAT GACAAATCTT GATACTGCAT CCACAACATT ACTGGCGTGC TTTTTGCTTT GCTTTGTGTG CTTTTGTGTG TCTGCCTATT AATACGTCCG CTGCTTTTGT CTGTGTCTAC ATACACATCA TTAATAATAT TGGTATTACT ATTGTGGATA ACAGCAGCCT CTGCGTTTAG GTGTTTTATT GTATATATTA TATTTGTTTA TATACCATTA TTTTTAATAC ATACACATGC ACGCTTTTTA ATTACATAAT GTATATGTAC ATAATGTAAT TGTTACATAT AATTGTTGTA TACCATAACT TACTATTTTT TCTTTTTTAT TTTCATATAT AATTTTTTTT TTTGTTTGTT TGTTTGTTTT TTAATAAACT GTTATTACTT AACAATGCGA CACAAACGTT CTGCAAAACG CACAAAACGT GCATCGGCTA CCCAACTTTA TAAAACATGC AAACAGGCAG GTACATGTCC ACCTGACATT ATACCTAAGG TTGAAGGCAA AACTATTGCT GAACAAATAT TACAATATGG AAGTATGGGT GTATTTTTTG GTGGGTTAGG AATTGGAACA GGGTCGGGTA CAGGCGGACG CACTGGGTAT ATTCCATTGG GAACAAGGCC TCCCACAG CT ACAGATACAC TTGCTCCTGT AAGACCCCCT TTAACAGTAG ATCCTGTGGG CCCTTCTGAT CCTTCTATAG TTTCTTTAGT GGAAGAAACT AGTTTTATTG ATGCTGGTGC ACCAACATCT GTACCTTCCA TTCCCCCAGA "TGTATCAGGA TTTAGTATTA CTACTTCAAC TGATACCACA CCTGCTATAT TAGATATTAA TAÁTACTGTT ACTACTGTTA CTACACATAA TAATCCCACT TTCACTGACC CATCTGTATT GCAGCCTCCA ACACCTGCAG AAACTGGAGG GCATTTTACA CTTTCATCAT CCACTATTAG TACACATAAT TATGAAGAAA TTCCTATGGA TACATTTATT GTTAGCACAA ACCCTAACAC AGTAACTAGT AGCACACCCA TACCAGGGTC TCGCCCAGTG GCACGCCTAG GATTATATAG TCGCACAACA CAACAGGTTA AAGTTGTAGA CCCTGCTTTT CTAACCACTC CCACTAAACT TATTACATAT GATAATCCTG CATATGAAGG TATAGATGTG GATAATACAT TATATTTTTC TAGTAATGAT AATAGTATTA ATATAGCTCC AGATCCTGAC TTTTTGGATA TAGTTGCTTT ACATAGGCCA GCATTAACCT CTAGGCGTAC TGGCATTAGG TACAGTAGAA TTGGTAATAA ACAAACACTA CGTACTCGTA GTGGAAAATC TATAGGTGCT AAGGTACATT ATTATTATGA TTTAAGTACT ATTGATCCTG CAGAAGAAAT AGAATTACAA ACTATAACAC CTTCTACATA TACTACCACT TCACATGCAG CCTCACCTAC TTCTATTAAT AATGGATTAT ATGATATTTA TGCAGATGAC TTTATTACAG ATACTTCTAC AACCCCGGTA CCATC TGTAC CCTCTACATC TTTATCAGGT TATATTCCTG CAAATACAAC AATTCCTTTT GGTGGTGCAT ACAATATTCC TTTAGTATCA GGTCCTGATA TACCCATTAA TATAACTGAC CAAGCTCCTT CATTAATTCC TATAGTTCCA GGGTCTCCAC AATATACAAT TATTGCTGAT GCAGGTGACT TTTATTTACA TCCTAGTTAT TACATGTTAC GAAAACGACG TAAACGTTTA CCATATTTTT TTTCAGATGT CTCTTTGGCT GCCTAGTGAG GCCACTGTCT ACTTGCCTCC TGTCCCAGTA TCTAAGGTTG TAAGCACGGA TGAATATGTT GCACGCACAA ACATATATTA TCATGCAGGA ACATCCAGAC TACTTGCAGT TGGACATCCC TATTTTCCTA TTAAAAAACC TAACAATAAC AAAATATTAG TTCCTAAAGT ATCAGGATTA CAATACAGGG TATTTAGAAT ACATTTACCT GACCCCAATA AGTTTGGTTT TCCTGACACC TCATTTTATA ATCCAGATAC ACAGCGGCTG GTTTGGGCCT GTGTAGGTGT TGAGGTAGGT CGTGGTCAGC CATTAGGTGT GGGCATTAGT GGCCATCCTT TATTAAATAA ATTGGATGAC ACAGAAAATG CTAGTGCTTA TGCAGCAAAT GCAGGTGTGG ATAATAGAGA ATGTATATCT ATGGATTACA AACAAACACA ATTGTGTTTA ATTGGTTGCA AACCACCTAT AGGGGAACAC TGGGGCAAAG GATCCCCATG TACCAATGTT GCAGTAAATC CAGGTGATTG TCCACCATTA GAGTTAATAA ACACAGTTAT TCAGGATGGT GATATGGTTC ATACTGGCTT TGGTGCTATG GACTTTACTA CATTACAGGC TAACAAAAGT GAAGTTCCAC TGGATATTTG TACATÍTGATT TGCAAATATC CAGATTATAT TAAAATGGTG TCAGAACCAT ATGGCGACAG CTTATTTTTT TATTTACGAA GGGAACAAAT GTTTGTTAGA CATTTATTTA ATAGGGCTGG TACTGTTGGT GAAAATGTAC CAGACGATTT ATACATTAAA GGCTCTGGGT CTACTGCAAA TTTAGCCAGT TCAAATTATT TTCCTACACC TAGTGGTTCT ATGGTTACCT CTGATGCCCA AATATTCAAT AAACCTTATT GGTTACAACG AGCACAGGGC CACAATAATG GCATTTGTTG GGGTAACCAA CTATTTGTTA CTGTTGTTGA TACTACACGC AGTACAAATA TGTCATTATG TGCTGCCATA TCTACTTCAG AAACTACATA TAAAAATACT AACTTTAAGG AGTACCTACG ACATGGGGAG GAATATGATT TACAGTTTAT TTTTCAACTG TGCAAAATAA CCTTAACTGC AGACGTTATG ACATACATAC ATTCTATGAA TTCCACTATT TTGGAGGACT GGAATTTTGG TCTACAACCT CCCCCAGGAG GCACACTAGA AGATACTTAT AGGTTTGTAA CCCAGGCAAT TGCTTGTCAA AAACATACAC CTCCAGCACC TAAAGAAGAT GATCCCCTTA AAAAATACAC TTTTTGGGAA GTAAATTTAA AGGAAAAGTT TTCTGCAGAC CTAGATCAGT TTCCTTTAGG ACGCAAATTT TTACTACAAG CAGGATTGAA GGCCAAACCA AAATTTACAT TAGGAAAACG AAAAGCTACA CCCACCACCT CATCTACCTC TACAACTGCT AAACGCAAAA AACGTAAGCT GTAAGTATTG TATGTATGTT GAATTAGTGT TGTTTTTTTGT GTATATGTTT GTATGTGCTT GTATGTGCTT GTAAATATTA AGTTGTATGT GTGTTTGTAT GTATGGTATA ATAAACACGT GTGTATGTGT TTTTAAATGC TTGTGTAACT ATTGTGTCAT GCAACATAAA TAAACTTATT GTTTCAACAC CTACTAATTG TGTTGTGGTT ATTCATTGTA TATAAACTAT ATTTGCTACA TCCTGTTTTT GTTTTATATA TACTATATTT TGTAGCGCCA GGCCCATTTT GTAGCTTCAA CCGAATTCGG TTGCATGCTT TTTGGCACAA AATGTGTTTT TTTAAATAGT TCTATGTCAG CAACTATGGT - - TTAAACTTGT ACGTTTCCTG CTTGCCATGC GTGCCAAATC CCTGTTTTCC TGACCTGCAC TGCTTGCCAA CCATTCCATT GTTTTTTTACA CTGCACTATG TGCAACTACT GAATCACTAT GTACATTGTG TCATATAAAA TAAATCACTA TGCGCCAACG CCTTACATAC CGCTGTTAGG CACATATTTT TGGCTTGTTT TAACTAACCT AATTGCATAT TTGGCATAAG GTTTAAACTT CTAAGGCCAA CTAAATGTCA CCCTAGTTCA TACATGAACT GTGTAAAGGT TAGTCATACA TTGTTCATTT GTAAAACTGC ACATGGGTGT GTGCAAACCG ATTTTGGGTT ACACATTTAC AAGCAACTTA TATAATAATA CTAA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: ACUGUGUCCU GAAGA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: ACUGUGUCCU GAAGAA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO:: ACUGUGUCCU GAAGAAA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GGACUUCGGU CC (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: UUCUUCAGAG AACAGUACCA GAGAAACACA CGGACUUCGU CCGUGGUAUA UUACCUGGUA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: UAACUGUCAA AAGC (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: UAACUGUCAA AAGCC (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: UAACUGUCAA AAGCCA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: UAACUGUCAA AAGCCAC (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11: GGCUUUUAGA AGUUAACCAG AGAAACACAC GGACUUCGUC CGUGGUAUAU UACCUGGUA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: 5 'TTATGGTTTC TGAG 3' (xi) SEQ ID NO: 12 5 'TTATGGTTTC TGAG 3' (xi) SEQ ID NO: 13 5 'CCCTCTAGAGG CACACAATTCCT GTG 3' (xi) SEQ ID NO: 14 5 'CACGTAGAGA AACCCAGC 3 '(xi) SEQ ID NO: 15 5' CAGCAATAC AACAAACCG 3 '(xi) SEQ ID NO: 16 5' CCUGUUCGCA GG '(xi) SEQ ID NO: 17 5' NNNAAGCTTCTGCAATGTTTCAGGACCC 3 '(xi) SEQ ID NO: 18 51 NNNGGATCCCCATTGGTACCTGCAGGATC 3 '(xi) SEQ ID NO: 19 (See Figure 12) (xi) SEQ ID NO: 20 (See Figure 14) (xi) SEQ ID N0: 21 (See Figure 16) (xi) SEQ ID NO: 22 (See Figure 18) (xi) SEQ ID NO: 23 (See Figure 20) »-

Claims (14)

1. R E I V I N D I C A C I O N E S: 1. A synthetic ribozyme comprising a hairpin portion, a binding site for ligating to a nucleotide sequence of a human papilloma virus, a segmentation site for segregating the sequence, wherein the cleavage site is selected from the group consisting of a site after the base 434 of the sequence and a site after the base 419 of the sequence.
2. 2. The synthetic ribozyme according to claim 1, wherein the binding site is linked to a sequence that is selected from the group consisting of: 430-ACUG U * GUC CUGAAGA-444 (SEQ ID NO: 2), 430 -ACUG U * GUC CUGAAGAA-445 (SEQ ID NO: 3), 430-ACUG U * GUC CUGAAGAAA-446 (SEQ ID N0: 4), 415-UAAC U * GUC AAAAGC-428 (SEQ ID NO: 7), 415-UAAC U * GUC AAAAGCC-429 (SEQ ID NO: 8), 415-UAAC U * GUC AAAAGCCA-430 (SEQ ID NO: 9), and 415-UAAC U * GUC AAAAGCCAC-431 (SEQ ID NO: 10 ), where "*" indicates the segmentation site.
3. 3. The synthetic ribozyme according to claim 1, wherein the hairpin portion comprises the sequence of SEQ ID NO: 5. The synthetic ribozyme according to claim 1, wherein the ribozyme is linked to a nucleotide sequence of HPV-16. 5. The synthetic ribozyme according to claim 4, comprising the sequence of SEQ ID NO: 6. 6. The synthetic ribozyme according to claim 4, comprising the sequence of SEQ ID NO: 11. 7. The ribozyme The synthetic ribozyme according to claim 1, having the two dimensional configurations as shown in Figure 6. 9. A vector comprising a DNA sequence coding for the ribozyme according to claim 1, the DNA sequence is operably linked to the expression control sequences. 10. The vector according to claim 9, wherein the vector is a plasmid. 11. A host cell transformed with a vector according to claim 9, wherein the host cell is capable of expressing the ribozyme from the vector. 12. A method for cleaving the human papillomavirus by a ribozyme, including the steps of: identifying a cleavage site in the viral genome; determine the sequence on either side of the segmentation site; constructing a synthetic hairpin ribozyme wherein a binding site in the synthetic ribozyme includes a non-complementary sequence to the cleavage site and a binding region complementary to the sequences on either side of the cleavage site; and provide the synthetic ribozyme of hairpin to the viral genome thus allowing the ribozyme to segment the viral genome. 13. The method according to claim 12, wherein the step of providing is in vi tro. The method according to claim 12, wherein the step of providing is in vivo. - ll ¬ lb The method according to claim 12, wherein the step of constructing a synthetic hairpin ribozyme includes incorporating a tetralazo sequence of SEQ ID NO: 5. 16. A method for detecting a human papillomavirus-16 (VHP- 16) in human tissue, comprising: obtaining a sample of human tissue containing RNA; exposing the RNA in the tissue to a ribozyme that binds to a nucleotide sequence of the HPV-16 RNA such that the HPV-16 RNA present in the sample is cleaved by the ribozyme; amplify the cDNA using primers complementary to: a 5 'end of a full-length HPV-16 transcript, a 5' fragment of the ribozyme cleavage site of the full-length HPV-16 transcript and a 3 'fragment of the ribozyme cleavage of a full-length HPV-16 transcript; and identifying the amplified DNA fragments such that the larger DNA fragment represents a full length HPV transcript and a smaller DNA fragment represents the fragment resulting from the cleavage of the full length HPV transcript ribozyme, where if the HPV-16 RNA is present in the sample, a preponderance of the smallest fragment is defined relative to the largest fragment. 17. The method according to claim 16, wherein the HPV-16 RNA cleaved by the ribozyme is an E6 transcript of HPV-16. 18. The method according to claim 16, wherein the ribozome is VPHR434. 19. The method according to claim 16, wherein the ribozome is VPHR419. 20. The method according to claim 16, wherein the human tissue is a cervical tissue. The method according to claim 16, further comprising the step of producing cDNA of the RNA present in the sample after the exposure step and before the amplification step. 22. A method for treating cervical cancer comprising the steps of: constructing a synthetic ribozyme comprising a hairpin portion, a binding site for ligating to a nucleotide sequence of a human papilloma virus, and a targeting site for segment the sequence, where the segmentation site is selected from the group consisting of a site after the base 434 of the sequence and a site after the base 419 of the sequence; and delivering an effective amount of the synthetic ribozyme to the cervical tissue. 23. The method according to claim 22, wherein the step of delivering comprises suspending the synthetic ribozyme in a liposomal delivery system based on lipofection. 24. The method according to claim 22, further comprising administering additional agents in combination with the synthetic ribozyme, the additional agents are selected from the group consisting of immunological agents and chemotherapeutic agents. 25. The method according to claim 24, wherein the immunological agents are LAK cells. 26. The method according to claim 24, wherein the chemotherapeutic agent is cisplatin.