WO1995024477A1

WO1995024477A1 - Human papillomavirus targeted ribozymes

Info

Publication number: WO1995024477A1
Application number: PCT/US1994/002559
Authority: WO
Inventors: Kamehameha Kay-Min Wong, Jr.; Saswati Chatterjee
Original assignee: City Of Hope
Priority date: 1994-03-08
Filing date: 1994-03-08
Publication date: 1995-09-14

Abstract

Human papillomaviruses (HPV) have been strongly implicated as important cofactors in the development of several human malignancies, particularly anogenital carcinomas. Products arising from the E6 and E7 open reading frames (ORFs) from PHV-16, a serotype commonly associated with human cervical carcinoma, are essential for viral transformation. To develop a novel treatment for this disease, ribozymes were designed to cleave HPV-16 E6 and E7 ORF transcripts in proximity to their translational start sites ('AUG'). Cleavage sites occur immediately 3' to nucleotides 110 and 558 of the viral genomic DNA, respectively. Oligonucleotides corresponding to these ribozymes were synthesized and inserted into a eucaryotic viral vector derived from the nonpathogenic parvovirus, adeno-associated virus (AAV). Ribozyme transcription from this vector, termed CWRT7:SVN, is under control of both the highly active Rous sarcoma virus LTR and bacteriophage T7 promoters. T7 transcripts of the E6 and E7 ribozymes efficiently cleaved their cognate targets in vitro under a variety of conditions, including physiological temperature.

Description

Human Papiliomavirus Targeted Ribozymes
FIELD OF THE INVENTION
This invention relates to ribozymes which cleave the major transforming genes of human papillomavirus (HPV) and to adeno-associated virus (AAV) based vectors encoding such ribozymes.

BACKGROUND OF THE INVENTION
Human papillomaviruses are serious human pathogens. For example, HPV 16 and HPV 18 have been found in over 85% of the invasive squamous cell carcinomas of the uterine cervix and are considered important prognostic indicators for malignant transformation in histopathologically benign lesions.

Molecular biologic studies have defined the
HPV 16 early region 6 (E6) and early region 7 (E7)
ORFs as essential for viral transformation.

The use of antisense oligonucleotides to inhibit the E6 and E7 HPV-16 [and HPV-18] major transforming genes has been reported. No highly effective treatment is available, however.

SUMMARY OF THE INVENTION
Pursuant to this invention"hammerhead"ribozymes were designed to cleave HPV-16 E6 and E7 ORF transcripts in proximity to the translational start sites ("AUG") of the respective proteins. Cleavage was designed to occur immediately 3'to the"WC"and "GUA"corresponding to nucleotides 110 and 558 of the viral genomic DNA, respectively. Oligonucleotides corresponding to these ribozymes were synthesized and inserted into an AAV-derived vector. Ribozyme transcription from this vector, termed CWRT7: SVN, is under control of both the highly active Rous sarcoma virus LTR and bacteriophage T7 promoters. E6 and E7 ribozymes were transcribed in vitro, and cleavage of cognate targets were evaluated under a variety of conditions. Target cleavage occurs efficiently under normal physiologic conditions.

Oligonucleotides were synthesized and inserted into an eucaryotic viral vector derived from the non-pathogenic parvovirus, AAV for transcription under the control of Rous sarcoma virus long terminal repeat (LTR) and bacteriophage T-7 promoters. T-7 transcripts of the E6 and E7 ribozymes effectively cleave their cognate targets in vitro at physiological temperature. Recombinant AAV based vectors encoding these ribozymes are encapsidated for administration to disrupt cognate oncogene expression intracellularly and thereby abrogate tumorigenicity.

DESCRIPTION OF THE FIGURES
Figure 1 is a schematic depiction of HPV-16 open reading frames, and the E6 and E7 transcriptional map. E6 and E7 transcripts originate from an identical site at map position 97. Translation"AUG" start sites, E6 splice donor, acceptor, alternative acceptor sites, E6Rz110 and E7Rz558 cleavage sites, and CWR: T7; SVN are'depicted.

Figure 2 depicts construction of the pGEM: E6/E7
RNA target expression system. Sequences corresponding from 97 to 865 of the HPV-16 genome (Seedorf, et. al., 1985) were amplified from pl321 by thermal cycling polymerase chain reaction and subcloned into pGEM4 as an HindIII-NheI fragment.

Figure 3 depicts target cleavage by E7Rz558 at varying molar ratios. In vitro 32P-labelled transcripts were gel purified, mixed at various molar ratios with E7Rz558, incubated at 52 C for three hours, and resolved by denaturing PAGE. The uncleaved 793 base target sequence, and 480 and 313 base cleavage products are depicted.

Figure 4 depicts target cleavage by E6Rz110 at 37 C. To better resolve cleavage products by polyacrylamide gel electrophoresis (PAGE), pGEM: E6/E7 was first cleaved with Ndei. In vitro target transcripts were generated as in Figure 3, incubated with varying molar ratios of E6Rz110, and analyzed by denaturing PAGE. The uncleaved 202 base target, and the 171 base cleavage product are clearly shown.

The 31 base cleavage product is too small to be visualized in this system.

DETAILED DESCRIPTION OF THE INVENTION
Papillomaviruses are small DNA viruses that are responsible for a wide variety of proliferative lesions in many animals, including humans (zur
Hausen, 1977; McCance, 1986). Human papillomaviruses (HPV) have become increasingly recognized as serious human pathogens. Their importance as a threat to public health arises from their high prevalence rates (as many as 10% of the population in the United
States between the'ages of 15 and 49 may have been infected (6)), and increasing incidence (the number of cases reported by general practitioners in the
United States rose from 169,000 per year in 1966 to 1.15 million in 1984 (2)). Although acute infections are generally not life threatening, infection with er certain serotypes of HPV has been implicated as an important cofactor in the development of cancer, particularly anogenital carcinomas.

For example, HPV-16 and HPV-18 DNA have been found in over 85% of invasive squamous cell carcinomas of the uterine cervix, and are considered important prognostic indicators for malignant transformation in histopathologically benign lesions. Importantly, protective vaccines are unavailable, and current therapy, including topical podophyllin, and systemic or intralesional interferon are of uncertain value in the prevention of malignant transformation. Thus, novel approaches to the control of HPV infection are continually being sought.

Analysis of the papillomavirus biology has been severely hampered by the inability to replicate the virus in tissue culture. Human papillomaviruses have been functionally divided into two major groups; infection with certain"high risk"HPV serotypes, such as HPV-16 and HPV-18, is more frequently associated with malignant transformation, whereas infection with"low. risk" serotypes such as HPV-6 or 11 is more often associated with benign proliferative lesions. The genomes of several HPV serotypes, including HPV-16 and 18, have been cloned, allowing for detailed mutational analyses. Genomic sequencing has revealed multiple open reading frames (ORFs) which have been functionally grouped into early (E) regulatory and late (L) structural genes (Figure 1).

Although the exact mechanism of human papillomavirus-related carcinogenesis is currently unknown, certain aspects of HPV-mediated transformation have recently been delineated. The physical form of HPV genomic DNA differs within premalignant and malignant cells. Whereas HPV DNA exists primarily in an episomal form in premalignant lesions, transformation to the malignant state is associated with viral genomic integration. while integration within cellular DNA appears random, integration with respect to the viral genome is less variant, and is often associated with disruption of the E2 ORF. The E2 ORF product normally represses expression from E6 and E7 ORFs, the major HPV-encoded transforming genes; thus, E2 ORF disruption results in unregulated expression of E6 and E7.

Elegant mutational studies utilizing cloned subgenomic DNA fragments have defined the HPV E6 and E7 ORFs as being essential for viral transformation (1,8). DNA encoding HPV-16 and 18 E6 and E7 transform epithelial cells in vitro, including human cervical epithelial cells (Kanda et al., 1988; Yutsudo et al. 1988;
Bedell et al. 1989). Similarly, lens specific expression of HPV-16 and E6 and E7 ORFs in transgenic mice resulted in bilateral microphthalmia and cataracts resulting from an impairment in lens fiber cell differentiation and induction of cellular proliferation, with the formation of lens tumors in adult mice with the highest levels of E6 and E7 expression.

Mechanistically, the E6 product from transforming HPV serotypes has been shown to physically associate with the cellular tumor suppressor gene product p53 (Werness et al., 1990;
Hubbert et al., 1992; Lechner et al., 1992), resulting in its rapid catabolism, possible via the protein catabolic ubiquitin pathway. Likewise, the
E7 ORF product binds to the cellular retinoblastoma tumor suppressor gene product (105RB), disrupting its function (Phelps et al., 1988; Dyson, et al., 1989;
Munger, et al., 1989b**). Furthermore, the ability to bind 105RB and transform cells resides within the amino terminal end of E7 from high risk (HPV-16) but is absent in low-risk (HPV-7) serotypes. Thus, disruption of endogenous cellular anti-transformation protective mechanisms by virus-encoded genes may play a major role in HPV-mediated transformation.

Recently, approaches that disrupt or inhibit essential targeted gene expression have been proposed as potential therapeutic modalities in the treatment of a variety of human disease. For example, ribozymes are a class of RNA molecules that cleave
RNA transcripts in a sequence specific fashion (Cech, 1987). Acting like enzymes, ribozymes catalytically cleave multiple copies of a given RNA substrate, and have been engineered to efficiently cleave their targets in trans (Uhlenbeck, 1987, Haseloff and
Gerlach, 1988; Perriman et al. 1992; Larson et al., 1993). Therefore, ribozymes designed to cleave essential genes provide a novel therapeutic approach to controlling viral infections and oncogenesis.

Recently, intracellular production of specific ribozymes has been used to inhibit human immunodeficiency virus gene expression and replication (Chang et al., 1990; Sarver et al., 1990;
Weerasighe et al., 1991; Yu et al., 1993), and bcr-abl (Snyder et al., 1993), and fos oncogene expression (Scanlon et al., 1991) in vitro without evidence of associated cellular toxicity.

In addition, AAV has recently been proposed for efficient delivery of a variety of transdominant inhibitory molecules including antisense transcripts and ribozymes. AAV-based vectors possess several advantages as vehicles for gene therapy. AAV vectors have high transduction frequencies in cells of diverse species, and lineages, including several
HPV-transformed cell lines, irrespective of proliferating status, and often integrate in tandem in multiple copies, thereby enhancing transgene expression. Additionally, wild type AAV has been reported to integrate site specifically within human chromosomal DNA, thereby minimizing the risk of insertional mutagenesis and variability of transgene expression.

Finally, latent wild type AAV infections have been stably maintained in tissue culture for over 100 serial passages in the absence of selective pressure, attesting to the stability of AAV genomic integration. Thus, AAV-based vectors are well suited for the stable introduction of transgenes into human and animal cells.

MATERIALS AND METHODS
1. Target Site Selection and Construction of an
HPV-16 E6 and E7 Ribozyme Expression System
A schematic depiction of the HPV-16 E6 and E7 transcriptional map is presented in Figure 1.

Both E6 and E7 transcripts arise from the same site at map position 97. The E6 transcript is multiply spliced, with a splice donor site at nucleotide 226, a splice acceptor site at 409, and an alternative splice acceptor site at 426. Ribozyme target sites were specifically chosen within exons common to both
E6 and E7 transcripts, with preference given to sites with high cleavage efficiencies according to the hierarchy determined by Perriman et al.

Oligonucleotides were produced utilizing an
Applied Biosystems Model 391 synthesizer (Foster
City, CA). Complementary oligonucleotides were synthesized incorporating a hammerhead ribozyme motif targeting the"WC"almost adjacent to the translational E6"AUG"start site (E6Rz110), corresponding to nucleotides 108-110 of the viral genomic DNA (upper strand 5'-TCGAGTGGGT CCTCTGATGA
GTCCGTGAGG ACGAAAAACA TTGCAG-3') (SEQ ID NO. 1) (Seedorf et al., 1985). These oligonucleotides were designed to produce 5'-SalI and 3'-NheI (XbaI-compatible) overhangs after annealing. The sequences flanking E6Rz110 were complementary to
HPV-16 genomic sequences 98-118.

Similar oligonucleotides incorporating a ribozyme targeting the"GUA"sequence adjacent to the"AUG"of the E7
ORF (E7Rz558) corresponding to nucleotides 556-558 of the genomic sequence (5'-TCGAATGCAT GATCTGATGA
GTCCGTGAGG ACGAAACAGC TGGGTT-3') (SEQ ID NO. 2) (Seedorf et al., 1985). The flanking sequences of the ribozyme were complementary to nucleotides 547-567 and covered the E7 ORF translational"AUG".

Oligonucleotides were annealed, and inserted into
SalI and XbaI digested pCWRT7: SVN, under control of the Rous sarcoma virus LTR and bacteriophage T7 promoters using standard methods (Sambrook et al., 1989) (Figure 1). The resulting constructs are termed pE6Rz110 and pE7Rz558. pCWRT7: SVN is a modification of pCWR; SVN, a previously described adeno-associated virus (AAV)-based vector containing the RSV promoter upstream of the AAV polyadenylation signal as well as a cassette conferring G418 resistance (Chatterjee et al., 1992). An oligonucleotide containing the T7 promoter and a multiple cloning site were inserted into pCWR; SVN downstream of the RSV promoter to yield pCWRT7; SVN.

All enzymes were utilized under conditions specified by the manufacturers.

An E6/E7 ribozyme substrate expression system was constructed as follows. Oligonucleotides were synthesized corresponding to nucleotides 97-109 (5'-ATCGATAAGC TTAACTGCAA TGTTT-3') (SEQ ID NO. 3) and nucleotides 854-865 (5'-ATCGATGCTA
GCTGGTAGATTATG-3') (SEQ ID NO. 4) of the genomic
HPV-16 sequence (Seedorf et al., 1985). A 5'-HindIII site was incorporated into the 5'-primer and a 5'-NheI recognition site was incorporated into the 3'-primer. These primers were used to amplify a 768 bp fragment from pl321, a plasmid containing nucleotides 50-865 of HPV-16. The amplified fragment was isolated, digested with HindIII and NheI, and inserted into HindIII and XbaI digested pGEM4 (Promega) to yield pGEM: E6/E7 (Figure 2).

All constructs were sequenced using a linear cycling/fluorochrome method (Applied Biosystems
Model 373), and were shown to be correct prior to subsequent studies.

2. Transcription Reactions
pE6Rz110 and pE7Rz558 were purified by anion exchange chromatography as per manufacturer's instructions Qiagen, and phenol/chloroform extracted to remove residual RNase. Ribozyme containing plasmids were linearized with XhoI and transcribed in vitro with T7 RNA polymerase as described in detail below. The resulting transcripts were 55 bases in length, including a 46 base ribozyme sequence and 9 bases at the 3'-end consisting of vector polylinker sequences.
pGEM: E6/E7 was purified by anion exchange chromatography, linearized with BamHI, and transcribed with T7 RNA polymerase as previously described. The resulting 793 base transcripts were used in E7 ribozyme cleavage reactions. To better demonstrate E6Rz110 cleavage products by PAGE, pGEM: E6/E7 was linearized with NdeI and transcribed with T7 RNA polymerase.

The resulting 202 base target transcripts were used in E6Rz110 ribozyme assays.

All in vitro transcription reactions were carried out using an Ambion Megascript kit. Reactions were performed in a total volume of 20 AL containing reaction buffer, 1 pg linearized template DNA, U T7
RNA polymerase, mM ATP, CTP, gTP, and UTP, and RNase inhibitor. Target transcripts were internally labeled with 10 pC a-32P UTP (Amersham) per reaction. The reaction mixture was incubated at 37 C for eight hours, then the DNA templates were digested with RNase-free DNase. T7 transcripts were separated in a denaturing 5% polyacrylamide-7M urea gel, full length transcripts were excised under UV shadowing, and eluted in a buffer containing 0.3 M NaOAc, pH 5.5,10 mM EDTA, and 0.1% SDS at room temperature overnight. The products were extracted with phenol and chloroform, ethanol precipitated, resuspended in
DEPC-treated water, and quantified spectrometrically.

3. Ribozyme Cleavage Assays
All cleavage reactions were carried out in a total volume of 20 p1 containing 20 mM Tris-Cl, pH 7.5, and 20 mM MgCl2. Approximate molar concentrations were determined spectrometrically.

Target transcripts and cognate ribozymes were mixed in tris buffer, denatured at 90 C for two minutes, cooled to room temperature, mixed with MgCl2 and incubated at specified temperatures ad times.

Cleavage reactions were terminated by addition of an equal volume of formamide gel loading buffer containing 20 mM EDTA. Samples were briefly denatured by heating to 90 C for two minutes prior to gel electrophoresis.

4. Electrophoresis and Autoradiograph
Specific RNA bands were resolved by electrophoresis in 5% polyacrylamide gels under denaturing conditions (7M urea). Gels were run at 450 volts for 80 to 90 minutes, harvested, and exposed to Kodak XAR-5 film at-70 C with intensifying screens.

5. Encapsidation of Vectors.

The vectors CWRT7: SVN and pGEM: E6/E7 were encapsidated in the following manner:
(a) Subconfluent 293 cells were infected with herpes simplex virus at a multiplicity of infection of 0.1 in 100 mm petri dishes one hour prior to transfection and incubated at 37 C in the presence of 5% C02.

(b) 10 ug/100 per dish of plasmid vector DNA was mixed with 3 ug per dish of pTAAV and brought to a total volume of 240 uL with sterile, distilled water.

(c) 240 UL of 0.5 M CaCl2 and 0.1 M Hepes were added, mixed and incubated for ten minutes at room temperature.

(d) 480 uL of 0.28 M NaCl, 0.05 M Hepes, 0.75 Mn
NaH2P04, and 0.75 Mm Na2GO94 were added, vortexed briefly and incubated at room temperature for 15 minutes.

(e) One hour post helper virus infection, the resultant slightly turbid calcium phosphate precipitate was carefully added to the infected cells.

(f) Thereafter, the cells were incubated for 48-72 hours at 37 C with observation for the development of helper virus cytopathic effects (CPE).

(g) The cells were harvested by scraping when the CPE affects 75% of the cells. The cells were then pelleted at 4 C and the pellets were separated from the supernatants.

(h) The cell pellets were resuspended in 1/10 volume of sterile 0.1 M Tris buffer at Ph 7.5.

(i) The resuspended cell pellets were freeze-thawed three times in dry ice/alcohol container.

(j) The freeze-thawed pellets were sonicated three times for 45 seconds.

(k) The sonicated cells were heated to inactivate the helper virus for four to six hours at 56 C with intermittent agitation.

(1) Aliquots were separated and. frozen.

RESULTS
1. E7Rz558 Cleaves its Target In vitro
32p-labeled E6/E7 target transcripts were generated in vitro and incubated with unlabeled
E7Rz558 under a variety of conditions. Reactions were first performed at different temperatures (from 25 C to 67 C) for one hour and at an approximate ribozyme to target molar ratios of 10: 1 or 100: 1. Both molar ratios produced effective cleavage. Three bands were observed in a 5% polyacrylamide gel (Figure 3), the uppermost band consisted of uncleaved target (793 bases), whereas the two lower bands represented predicted
E7Rz558-mediated cleavage products (480 and 313 bases, respectively). The most effective temperature for E7Rz558 cleavage was between 52 C and 57 C.

Additionally, increasing the ribozyme to substrate molar ratio increased cleavage efficiency within a given incubation temperature range (Figure 3).

Further experiments were performed to test a wider range of ribozyme-to-target (Rz: T) ratios from 2: 1 to 512: 1. At 52 C, the cleavage products were observed at a molar ratio of 8: 1, and the cleavage increased with an increasing molar ratio (Figure 4).

At a ribozyme to substrate molar ratio of 64: 1 or higher, E7Rz558 was able to cleave target transcripts at physiological temperature (37 C) (Figure 3).

Furthermore, increasing incubation time could increase cleavage (data not shown).

2. E6Rz110 cleaves its target in vitro
The designed cleavage site of the E6Rz110 target transcript was nucleotide 110 of the viral genomic
DNA, approximately 30 bases downstream of the 5'end of the transcript. To better resolve cleavage products utilizing polyacrylamide gel electrophoresis, the pE6/E7 target construct was digested with NdeI cleaving at nucleotide 279 of the viral genome. As a result, T7 transcription yielded a shortened 202 base target RNA. Incubation of
E6Rz110 with the truncated 32P-labeled target transcript for one hour at a Rz: T molar ratio of 100: 1 yielded a clearly detectable 171 base cleavage product at all incubation temperatures tested, from room temperature to 57 C. Approximately 60% of the target was cut at 37 C, and more than 90% of the target transcripts were cleaved at 52 C and 57 C (Figure 4).

3. E6Rz110 and E7Rz558 Cleave Their Substrates
In Vitro in the Presence of Total Cellular
RNA
To test whether normal cellular RNA could nonspecifically bind to E6Rz110 or E7Rz558 and interfere with cleavage, 2 pM of either E6Rz110 or
E7Rz558 were incubated with 20 nM of 32P-labelled substrate at 52 C for one hour in the presence of 50, 500, or 5,000 ng of total cellular RNA isolated form the 293 cell line, an Ad5-transformed human embryonic kidney cell line. Both ribozymes were able to cleave their appropriate target transcripts in the presence of total cellular RNA (data not shown).

4. A Preceeding Denaturation Step is Not
Essential for E6Rz110 and E7R558 Substrate
Cleavage In Vitro
As previously noted under Materials and Methods, ribozyme cleavage reactions were typically denatured at 90 C prior to incubation. However, a similar denaturation step is not feasible intracellularly.

To test whether denaturation was essential for substrate cleavage, 2 MM of E6Rz110 or E7Rz558 were incubated with 20 nM of substrate at 37 C for seven hours without prior heat-denaturation. Ribozyme cleavage efficiencies with and without prior denaturation were equivalent, indicating that denaturation was not essential (data not shown).

AAV-based vectors have been constructed which encode"hammerhead"ribozymes each of which was specifically designed to cleave both HPV-16 E6 and E7 transcripts immediately 3'-to sequences corresponding to nucleotides 110 (E6Rz110) and 558 (E7Rz558) of the genomic sequence. Both E6Rz110 and
E7Rz558 efficiently cleave their cognate target transcripts in a cell-free system under a variety of conditions, including physiological temperature.

E6Rz110 effectively cleaved its target transcript at a ribozyme: target ratio of 2: 1 at 37 C, the lowest ratio tested. In contrast, a high E7Rz558 ribozyme: target ratio appeared necessary for efficient cleavage, a requirement that could be overcome by increasing incubation time, a situation which can readily be achieved in vivo. In addition,
E7Rz558 cleaves the full length 793 base target transcript, a size which approximates the full length
E7 transcript in vivo, inferring that intracellular cleavage could also occur. Furthermore, cleavage occurs in the presence of an excess of total cellular
RNA without preceeding denaturation, also indicating the feasibility of future in vivo studies.

The relative inefficiency of E7Rz558 in comparison to
E6Rz110 target cleavage may result from the larger target transcript size for the former, with more complex target secondary structure and subsequent steric hindrance of ribozyme binding. It should be emphasized, however, that both ribozymes were designed to inhibit expression from both E6 and E7
ORFs. Thus, E6Rz110 not only removes the"AUG"from
E6 transcripts, but also removes the methyl-cap structure necessary from efficient translation from
E7 transcripts. Likewise, E7Rz558 cleaves upstream of the E7"AUG"and also removes the translational terminator ("UAA") from E6 transcripts.

The effectiveness of a gene therapeutic approach to HPV infection is dependent not only upon the efficacy of the inhibitory molecules, but also upon long term expression of the inhibitory species and the efficiency of gene transfer. Vectors based upon adeno-associated virus (AAV) are particularly attractive for this purpose. In addition to its previously mentioned attributes as a eucaryotic vector, epidemiologic studies indicate an inverse relationship between AAV-seropositivity and the development of cervical carcinoma, implying that prior infection with wild type AAV itself may be somewhat protective against the development of this disease. Infection with a variety of autonomous paroviruses has been shown to inhibit neoplastic transformation in vitro, resulting in actual attempts to harness this anti-oncogenic effect in vivo.

Similarly, infection with wild type AAV inhibits bovine papillomarvirus (BPV), HPV, and H-ras transformation in vitro, and also inhibits replication of adenovirus, which is a transforming virus in rodent cells. These anti-oncogenic effects of AAV have been mapped to the ITRs, which are present in CWR: T7; SVN, and the rep78 gene, which has been removed from this series of vectors. Thus,
AAV-based vectors may play a unique role by serving as both highly efficient delivery vehicles for anti-oncogenic transdominant genes, in addition to having innate anti-cancer properties.

SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT: Kamehameha Kay-Min Wong, Jr.

Saswati Chatterjee
(ii) TITLE OF INVENTION: Ribozymes Which
Cleave the Transforming Genes of Human
Papillomavirus
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: City of Hope
(B) STREET: 1500 East Duarte Road
(C) CITY: Duarte
(D) STATE: California
(E) COUNTRY: United States of America
(F) ZIP: 91010-0269
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3M Double Density 5
1/4"diskette
(B) COMPUTER: Wang PC
(C) OPERATING SYSTEM: MS-DOS (R) Version
3.30
(D) SOFTWARE: Microsoft (R)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 08 March 1994
(C) CLASSIFICATION: Unknown
(vii) PRIOR APPLICATION DATA: None
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Irons, Edward S.

(B) REGISTRATION NUMBER: 16,541
(C) REFERENCE/DOCKET NUMBER: None
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 783-6040
(B) TELEFAX: (202) 783-6031
(C) TELEX: None (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 1:
TCGAGTGGGT CCTCTGATGA GTCCGTGAGG ACGAAAAACA TTGCAG 46 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:
TCGAATGCAT GATCTGATGA GTCCGTGAGG ACGAAACAGC TGGGTT 46 (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION:

SEQ ID NO. 3:
ATCGATAAGC TTAACTGCAA TGTTT 25 (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4:
ATCGATGCTA GCTGGTAGAT TATG 24

Claims

CLAIMS: 1. The vector CWRT7: SVN.

2. The vector pGEM: E6/E7.

3. Nucleotide SEQ ID NO. 1.

4. Nucleotide SEQ ID NO. 2.

5. Nucleotide SEQ ID NO. 3.

6. Nucleotide SEQ ID NO. 4.

7. The construct pE6Rz110.

8. The construct pE7Rz558.

9. The encapsidated vector CWRT7: SVN.

10. The encapsidated vector pGEM: E6/E7.

AMENDED CLAIMS received by the International Bureau on 3 August 1994 (03.08.94); new claims 11-13 added; remaining claims unchanged (1 page)] 1. The vector CWRT7: SVN.