WO1997002841A1 - Genetic therapy employing the hhv-6a derived ts gene or polypeptide - Google Patents

Abstract

The HHV-6 transcription suppressor (ts) gene acts at the level of transcription to shut-off expression of the HIV LTR promoter and of the H-ras gene, a gene whose mutational activation is associated with numerous human cancers. The ts gene is delivered to cells by a pharmaceutically acceptable vehicle and acts as either a preventative or therapeutical agent against cancer or against viral infections.

Description

GENETIC THERAPY EMPLOYING THE HHV-6A DERIVED TS GENE OR POLYPEPTIDE

This application claims priority to Provisional U.S. Application Serial No. 60/001,010, filed July 10, 1995, the contents of which are incorporated by reference herein in their entirety.

Background of the Invention Trans-acting genetic elements that control gene expression are well documented in the literature. Some genetic elements control genes from the same genome while others act upon genes from heterologous organisms. Many examples of trans-inactivation and of trans-activation are known, particularly by phage and viral trans-acting elements. One family of trans-acting genetic elements that has been of particular interest in the past are the rep genes of the human adeno-associated virus type 2 (AAV-2) . AAV is a parvovirus, but unlike other parvoviruses, AAV grows efficiently only in cells co-infected by a helper virus. Helper viruses include adenoviruses, herpes virus and pox viruses. In the absence of helper virus, AAV is integrated into the host-cell chromosome and there is little or no gene expression from the integrated provirus. Carter, in Handbook of Parvoviruses , Vol . I, CRC Press, 1989, pp. 155-68 and pp. 255-82.

The rep gene is required for AAV-2 DNA replication. The gene is located on the left half of the AAV genome. It is expressed by overlapping mRNA species transcribed from two promoters, named p5 and pl9. Laughlin et al . , Proc . Nat ' l Acad . Sci . USA 76: 5567 (1979); Carter, in Handbook of Parvoviruses, Vol, I, CRC Press 1990, pp. 227-54) . At least four overlapping Rep proteins expressed from the rep gene have been identified. Mendelson et al . , J . Virol . 60: 823 (1986); Trempe et al . , Virology 161: 18 (1987). Rep78 and Rep68 are gene products transcribed from the p5 promoter and Rep52 and Rep40 are gene products transcribed from the pl9 promoter.

In addition to their activity on the origin of replication and at the site of integration into the human genome, the AAV-2 rep genes affect transcription as well. The AAV gene mediates both complex positive and negative regulator effects. See Carter et al . , (1990), supra , at pp. 227-54. In the presence of helper virus, rep activates expression of AAV genes. Tratschin et al . , Mol . Cell . Biol . 6: 2883 (1986); Labow et al . , J . Virol . 60: 251 (1986); Trempe and Carter, J . Virol . 62: 68 (1988) . Most importantly, AAV-2 rep down-regulates expression from a variety of heterologous promoters, including the human immunodeficiency virus type 1 (HIV-l) long terminal repeat (LTR) . Labow et al . , Mol . Cell. Biol. 7: 1320-1325 (1987); Antoni et al . , J. Virol. 65: 396-404 (1991); Rittner et al . , J. Gen. Virol. 53: 2977- 2981 (1992) . Interestingly, down-regulation of HIV LTR by AAV-rep does not involve known transcription control elements Spl, or NF-&B. Thompson et al . , Virol., 204: 304-11 (1994); Oelze et al . , J. Virol. 68(2): 1229-1233 (1994) . General reviews on control elements of LTR are presented in Cullen et al., FASEB J. 5: 2361-2368 (1991) and Karn et al., Trends Genet. 8: 365-368 (1992). What seems to be involved is an AAV-HIV homology region (AAH) located within the TAR coding sequence which can account for at least part of the rep-mediated inhibition of transcription from the LTR promoter. Oelze, supra.

In addition to viral gene down-regulation, the AAV-2 _Rep68/78 gene has been shown to suppress transformation by bovine papillomavirus and activated EJ-Harvey-ras gene. Hermonat, Virol. 172: 253-61 (1989); Hermonat, Cancer Res. 51: 3373-77 (1991) . Activated H-ras.has been identified in many human malignancies, including carcinomas of the bladder, lung, breast, and urinary tract, as well as melanomas. Der et al., Proc. Natl. Acad. Sci., USA 79: 3637-40 (1982); Goldfarb et al . , Nature 296: 404-09 (1982); Krontiris et al . , Proc. Natl. Acad. Sci., USA 78: 1181-84 (1981) ; Parada et al . , Nature, 297: 474-78 (1982); Santos et al . , Nature 298: 343-47 (1982); Yuasa et al . , Nature 303: 775-79 (1983); Kraus et al . , Proc. Nat'l Acad. Sci., USA 81:5384-88 (1984); Fujita et al . , Nature 309: 464-66 (1984); Albino et al . , Nature 308: 69-72 (1984); Sekiya et al. , Proc. Nat'l Acad. Sci. USA 81: 4771-75 (1984).

Other viral genes that can activate and repress transcription have been described. The herpes simplex virus type l (HSV-1) infected cell protein 4 (ICP4) acts as either a repressor or transactivator of HSV-1 genes. DeLuca et al . , Nucl. Acids Res. 15: 4491-511 (1985a); DeLuca et al ., Mol. Cell. Biol. 5: 1997-2008 (1985b); DeLuca et al . , J. Virol. 62: 732-43 (1988); Hubenthal- Voss et al., J. Virol. 62: 454-62 (1988); Paterson et al., Virologoy 166: 186-96 (1988); Smith et al., J. Virol. 61: 1092-97 (1987). ICP4 protein suppresses the HSV-l α0 and α4 genes, the latter encoding ICP4, by binding to a specific motif, ATCGTCΝΝΝCΝGΝΝ, within their promoters. DeLuca et al . , 1988, supra; Muller, J. Virol. 61: 858-65 (1987); Roberts et al . , J. Virol. 62: 4307-20 (1988); Kristie et al . , Proc. Nat'l Acad. Sci., USA 83: 3218-22 (1986a); Kristie et al . , Proc. Nat'l Acad. Sci., USA 83: 4700-04 (1986b); Muller, (1987), supra. The E2 gene of BPV-1 can also transactivate and repress transcription by binding to the ACCΝ₆GGT motif, the BPV-1 enhancer element. Lambert et al., Cell 50: 69-78 (1987a); Lambert et al . , Cancer Cells 5: 15-22 (1987b); Hubbert et al . , Proc. Nat'l Acad. Sci., USA 85: 5864-68 (1988); Lambert et a . , J. Virol. 63: 3151-54 (1989); McBride et al. , J. Biol. Chem. 266: 18411-14 (1991); Androphy et al., Nature 325: 70-73 (1987); Moskaluk et al., Proc. Nat'l Acad. Sci., USA 84: 1215-18 (1987); McBride et al . , (1991), supra . E2R, an N-terminal truncation of E2, and E8E2, a fusion protein expressed from a spliced message, both contain the DNA binding domain of E2 but lack the transactivating domain. By binding to E2 enhancer elements, E2R or 2E8 block the binding of full length E2 and thus, repress transcription.

Thomson et al . , Nature 351: 78-80 (1991), have reported an HHV-6A protein that has some homology to AAV- 2 i?ep68/78. HHV-6 isolates have been shown to belong to two very closely related groups on the basis of differences in molecular and biological properties. Ablashi et al . , Arch . Virol . 129: 363-366 (1993). Variant A viruses are characterized by HHV-6 strain U1102 and GS, and variant B viruses by strain Z29. Downing et al . , Lancet 2: 390 (1987); Salahuddin et al . , Science 234: 596-601 (1986); Lopez et al . , J . Infect . Dis . 157: 1271-73 (1988) .

A unique component of HHV-6 strain U1102 contains an open reading frame (ORF) encoding a 490 amino acid protein which has 24% identity to the first common 490 amino acids of the nonstructural proteins Rep 78 and 68 of the human parvovirus AAV-2. Thomson et al . , 1991, supra . A similar ORF has been found in the Z29 strain of HHV-6. Rep68 and Rep78 proteins, 536 and 621 amino acids respectively, were shown to have identical N-terminal ends for 528 amino acids, but differ in the C-terminal region because they are translated from differentially spliced messages.

Indeed, the AAV-2 and the HHV-6 derived genes appear to have maintained at least some shared functional domains. HHV-6 ts and AAV-2 Rep68/78 suppress transcription of the same promoters, H-ras and HIV-l, but not MSVLTR. Araujo et al . , J . Virol . 69: 4933-40 (1995); Hermonat, (1991), supra ; Antoni et al . , J . Virol . 65: 396-404 (1991); Rittner et al . , (1992), supra ; Oelze et al . , (1994), supra ; Thomson et al . , (1994), supra . Therefore, it is likely that they both interact with the same target(s) . The cloned HHV-6 gene complemented replication of a rep-deficient AAV-2 genome. Thomson et al . , (1994), supra . AAV-rep was shown to inhibit CAT activity from constructs where the CAT gene was downstream of the HIV LTR promoter in both fibroblast and T-cell lines. Unexpectedly, and in contrast, HHV-6 gene product activated CAT activity expressed from the same constructs, but such activation occurred only in the fibroblast cell line and not in T-cells. HHV-6 rep is therefore believed to be a multifunctional regulatory protein with properties related to, but distinct from, those of AAV-2 rep. Thomson et al . , (1994) supra . Coinfection of CD4+ cells with HHV-6 was reported to both enhance and suppress HIV replication. Lusso et al . , Nature 337: 370-373 (1989) and Carrigan et al . , J. Infect . Dis . 162: 844-851 (1990), respectively. The i?ep68/78 target in the origin of replication for AAV-2 has been identified by binding and DNase footprinting studies as (GCTC)₃. Chiorini et al . , J^". Virol . 68: 797-804 (1994). Both the H-ras promoter sequence and the HIV-l TAR region contain sequences related to (GCTC)3. Oeize et al . , (1994), supra ; Batchu et al . , Cancer Lett . 86: 23-31 (1994). The related sequence within TAR is adjacent to mutations in the TAR "Bulge" and "Loop" that abrogate ts suppression. While Rep78 protein binding to the GCTC cluster in the H-ras promoter has been demonstrated, no mutation studies of this region showing loss of i?ep68/78 (or ts) suppression have been reported. Batchu et al . , (1994), supra . A search for the existence of the GCTC motif (or its compliment, GAGC) within the BPV-1 and HPV-16 sequences cloned into the CAT reporter constructs used in the ts suppression studies shown in Figs. 15A-D revealed four in pl066, 24 in p805-88, and none in pURR16CAT. Moreover, none of the motifs found in the BPV-1 sequences existed in clusters. Thus, the GCTC motif can not be the only critical element required for ts suppression.

Ts protein could act by binding to nucleic acid sequences, other proteins, or both. With regards to the HIV-l promoter, mutation studies indicate that the TAR region, particularly the "Bulge," is critical. Araujo et al . , (1995), supra . Because TAR is downstream of the mRNA start site, ts binding to RNA or DNA could be involved. Additionally, binding to cellular transcription factor(s) probably occurs. Both the HIV-l tat protein and cellular nuclear proteins including a 140 kDa protein TRP185, and 44 kDa TRBP bind to TAR. Rounseville et al . , J . Virol . 66: 1688-94 (1992); Wu et al . , Genes Dev. 5: 2128-40 (1991); Gatignol et al . , Science 251: 1597-1600 (1991) . In the case of the H-ras promoter, the cluster of GCTC motifs that binds Rep68 protein is upstream of any of the approximately 30 mRNA start sites or transcription factor binding sites that have been reported. Batchu et al . , (1994), supra ; Lu et al . , J. Biol . Chem . 269: 5391-403 (1994).

The sites of action of the ts gene product are sequence motifs that are fairly similar to each other in the case of the H-ras and LTR promoters. The fact that two promoters from unrelated organisms are both transcriptionally regulated by the ts gene product by interaction with homologous sequences suggests that the ts-recognized sequences have regulatory functions of their own, and that gene expression regulation employing those functions are likely found elsewhere. This idea is made more likely by the fact that the motifs discussed above are found within TAR, a regulatory region itself. Furthermore, the site of action of the AAV-2 rep gene product and the ts gene product may be similar, both interacting with a region within the TAR sequences of LTR. Ts interacts with the H-ras promoter in a region that appears to contain nucleotide motifs homologous to motifs in the TAR region. Summary of the Invention It is an object of the present invention to provide a vector for use in gene therapy that contains the HHV-6 ts gene. It is another object of the present invention to provide for use of the HHV-6 ts gene , or a fragment thereof, in the treatment of disease states associated with oncogenic transformation or lentivirus infection. It is still another object of the present invention to treat disease states associated with oncogenic transformation or lentivirus infection with at least a portion of the HHV-6 ts gene.

It is yet another object of the present invention to provide methods of characterizing disease states. In accomplishing these and other objects, there is provided, in accordance with one aspect of the present invention, a method of gene therapy, comprising the steps of (A) providing a construct containing a polynucleotide sequence encoding the ts protein and (B) delivering the vector to cells of a subject at risk of a disease state associated with oncogenic transformation or lentivirus infection, such that upon expression of said polynucleotide sequence in said cells the disease state is treated. Preferably, the vector is chosen from the group consisting of a viral vector, a lipidic vector, a plasmid, and an ex vivo transformed cell. The vector can be delivered before, during or after development of the disease state. -lo¬

in accordance with another aspect of the present invention, the disease state is a cancer, including cancer associated with a member of the ras gene family and those associated with viruses, such as the papilloma viruses, HTLV-1, HIV and lentiviruses that contain an LTR-like sequence, including human lentiviruses.

In accordance with another aspect of the present invention, there is provided a vector that comprises a polynucleotide sequence encoding the ts protein and that is suitable for gene therapy such that upon delivery of said construct to cells of a subject at risk of a disease state associated with oncogenic transformation or lentivirus infection expression of the polynucleotide sequence is effected in said cells, treating the disease state. Preferably, the vector is appropriately formulated for delivery to a subject, and thus can serve as a therapeutic agent.

In accordance with still another aspect of the present invention, there is provided a method of characterizing a disease state, comprising the steps of administering a ts protein to a cell affected by the disease state and determining whether the ts protein can treat the disease state. The disease state can be imparted on the cell by transforming the cell with a disease gene. Preferably, the administering step comprises transforming the cell with a ts gene.

In accordance with yet another aspect of the present invention there is provided a method of gene therapy. comprising the step of a administering a therapeutic composition comprising a ts protein or polynucleotide sequence encoding the ts protein to a subject at risk for a disease state associated with oncogenic transformation or lentivirus infection.

The polynucleotide sequence encoding the ts protein may comprise at least a transcription-suppressing portion of the HHV-6 ts gene, and may include the entire HHV-6 ts gene. In accordance with a further aspect of the present invention, there are provided transcription-suppressing polypeptides and polynucleotides encoding them.

These and other aspects of the invention will become apparent to the skilled artisan in view of the teachings contained herein.

Brief Description of the Drawings Figure 1 shows the location of the fragments used to test suppression of H-ras transformation on the HHV-6 genome. Specifically, the locations of HD12, ZVH14, and ED9 fragments within HHV-6 are indicated. ts2.6 is a subfragment of HD12. The ts protein is designated as a filled box with arrow indicating the direction of transcription. Jpnl repeats are shown as a hatched box. Only relevant SacII sites within HD12 are shown. Figures 2A-B depict the HHV-6 ts protein. Figure 2A shows the amino acid sequence of the HHV-6 ts protein compared to the amino acid sequence of AAV-2 Rep 68/78. Figure 2B is a schematic comparison of HHV-6 to AAV-2 Rep 68/78 showing regions of alignment (solid rectangles) as identified by MACAW software.

Figure 3 shows the effect of selected HHV-6 fragments on H-ras transformation. pEJ-H-ras with or without pZVH14 or pHD12 was transfected into NIH 3T3 cells. Fourteen days post-transfection, dishes (four for each experimental condition) were stained and counted for transformed foci (the total number of transformed foci for all four dishes is shown) . A schematic of the H-ras reporter is shown with its four exons (shaded rectangles) and endogenous promoter (solid rectangle) . Data shown are representative of three experiments.

Figures 4A-C shows the effect of the ts gene on H- ras transformation. Figure 4A is a schematic presentation of ts2.6. Horizontal arrows indicate location of ORFs greater than 75 amino acids within ts2.6. The 180 amino acid ORF is the N-terminal end of a 1121 amino acid ORF within HD12. ORFs on different levels are in different reading frames. Vertical arrows represent the positions of translation termination linkers inserted into the suppressor protein within ts2.6. Figure 4B is a Northern blot analysis of total RNA extracted from transiently transfected cells and probed with the StyI fragment within ts (See Figure 1) . Shown at the left are the sizes of molecular weight markers and at the right, the calculated size of the observed transcript. Figure 4C shows only that wild-type ts constructs suppressed H-ras transformation. pEJ-H-ras was transfected alone or with increasing amounts of ts constructs as depicted in Figure 2. The data are representative of two experiments. Figures 5A-B show the effect of the ts gene on transcription of the H-ras gene. Figure 5A depicts the effect of pHD12 or pts2.6 on H-ras transformation when H- ras is expressed from the MSV LTR promoter. pMSVLTR-ras was transfected alone or with increasing amounts of pHD12 or pts2.6. At fourteen days post-transfection, dishes (four per experimental condition) were stained and transformed foci were counted. A schematic of the reporter pMSVLTR-ras with its four exons (shaded rectangles) and heterologous MSV LTR promoter (solid box) also is shown. Data are representative of three indepen¬ dent experiments. Figure 5B the effect of pHD12 on CAT expression from the endogenous H-ras promoter. pHD12 was co-transfected with prasCATl or pMSVLTR-CAT. The amount of each plasmid DNA used is shown below the graph. CAT activity was measured in extracts prepared at 48 hours post-transfection.

Figures 6A-B show the effect of pHD12 on expression from the HIV-l LTR promoter. Figure 6A is a schematic depiction of the HIV-l LTR promoter showing its genetic elements and the LTR wild-type and upstream mutant constructs used. Point mutations are designated by vertical arrows and deletions by dashed lines. Figure 6B shows the effect of ts on HIV-l LTR CAT expression. CD- 12 or upstream LTR mutant construct DNA was electroporated into 12D7 cells alone (shaded bar) or with the indicated concentrations of pRc-ts DNA (filled bar) . CAT activity was assayed from extracts made 24 hours post-electroporation. Data shown are representative of a minimum of two experiments for each HIV-l LTR CAT construct.

Figures 7A-B show the relation between HIV-l TAR and ts suppression. Figure 7A depicts the TAR mRNA structure expressed from wild-type (CD-12) and TAR mutant CAT constructs. Relevant wild-type and mutated TAR sequences are shown below. Deleted bases are indicated by the dashed line. Figure 7B illustrates the effect of TAR mutations on ts suppression of HIV-l LTR expression. CD- 12, TM29-CAT, or TM26-CAT was electroporated into 12D7 cells alone (shaded bar) or with the indicated concentra¬ tions of pRc-ts DNA (filled bar) . CAT activity was assayed from extracts made 24 hours post-electroporation. Data shown are representative of a minimum of two experiments for each HIV-l LTR CAT construct.

Figure 8 schematically depicts the relationship of HHV-6A ts protein (solid rectangle) to AAV-2 Rep proteins (shaded rectangle) . The line indicates the region of the Rep68 splice and the open rectangle indicates the translation from an alternate reading frame caused by the ins42 linker insertion.

Figures 9A-B depict data relating to suppression of transformation. Figure 9A shows that stable ts sense and antisense cell lines were established by transfection of NIH 3T3 cells with pRc-ts and pRc-ts(A), respectively, followed by selection with G418. These cell lines and parental NIH 3T3 were transfected with pEJ-H-ras (containing the H-ras gene expressed by its endogenous promoter) or pMSVLTR-ras (containing the H-ras gene expressed by the MSVLTR promoter) . Two weeks later dishes were stained and scored. Figure 9B depicts the same cell lines that were transfected with prasCATl (containing the chloramphenicol acetylase (CAT) gene expressed by the endogenous H-ras promoter) or pMSVLTR- CAT (containing the chloramphenicol acetylase gene expressed by the MSVLTR promoter) . Forty eight hours later, extracts were made and tested for the level of CAT activity. The percent of acetylated chloramphenicol is indicated above each lane.

Figure IOA and Figure 10B, respectively, depict data from a comparison of stable 12D7 cell lines, which were transfected with HIV-l LTR-CAT (containing the CAT gene expressed by the HIV-l promoter) or TM26-CAT (containing CAT expressed by the TM26 TAR mutant HIV-l promoter) .

Figure 11 depicts data relating to HHV-6 ts suppressed Papillomavirus-Induced Transformation. pBPV with or without pts2.6 was transfected into NIH 3T3 cells. Three weeks post-transfection, dishes (four for each experimental condition) were stained and counted for transformed foci. The total number of transformed foci for all four dishes is shown. Figures 12A-C depict data relating to reproducible suppression of H-ras and Papillomavirus Transformation in Stable ts NIH 3T3 Cell Lines. Additional ts cell lines were established and transfected with pEJ-H-ras (Figure 12A) , pBPV-69T (Figure 12B) , or MSVLTRras (Figure 12C) , as described previously.

Figures 13 A-C illustrate growth characteristics of NIH 3T3 cells expression ts protein. Stable ts expressing 3T3-ts-l, 2, 3, 4, and 5 cell lines were established by transfection of NIH 3T3 cells with pRc-ts, followed by selection with G418 and clonal isolation. Figure 13 A is a Southern blot analysis. DNA was extracted from 3T3-ts cell lines, digested with Hindlll , separated by agarose gel electrophoresis, transferred to GeneScreen, and probed with ³²P-labeled ts Hindlll fragment DNA. The position of the expected 1.8 kbp ts Hindlll fragment is indicated. Figure 13B is a Western blot analysis. Protein extracts of NIH 3T3 and 3T3-ts cell lines were separated by 4-20% SDS-PAGE electrophoresis and Western blotted using anti-ts rabbit polyclonal serum, Ab-679. As a positive control, in vitro transcribed and translated ts protein was produced employing pET14b-ts (ts cloned into the pET14b bacterial expression vector; Novagen) as the template. The ts/antibody complex was detected by the "Western Light" chemiluminescent detection system (TROPIX, Inc.) employing goat anti-rabbit IgG conjugated to alkaline phosphatase. Figure 13C depicts the growth curves. 5 X 10⁴ cells/dish of each cell line were seeded into replicate dishes. On days 2, 3, 4, 5 and 6 the cells were resuspended by trypsinization and counted in a hemocytometer. Figure 14 demonstrates that retroviral ts NIH 3T3 cell lines are resistant to transformation by H-ras. Cell lines were established by infecting NIH 3T3 cells with LNCX or LNCts retrovirus, followed by G418 selection. These cell lines and parental NIH 3T3 were transfected with pEJ-H-ras and analyzed as described for Figure 13.

Figures 15A-D illustrate that bovine and human papillomavirus promoters were suppressed in ts expressing cell lines. pl066 (Fig. 15A) , p805-88 (Fig. 15B) , pCHC6CAT (Fig 15. C) and pURR16CAT (Fig. 15D) were transfected into cell lines NIH 3T3, 3T3-ts-3, 3T3-ts-4, and 3T3-ts-5. After 48 hours, cell extracts were prepared and assayed for CAT activity. The percent o acylated chloramphenicol is shown. Figures 15A-D also comprise a schematic representation of the promoter CAT constructs. The arrow designates the transcription start site.

Detailed Description of the Preferred Embodiments

The present invention relates to methods, vectors and constructs that utilize a portion of the HHV-6 genome to make a polypeptide that can be used for treating disease states associated with oncogenic transformation or lentivirus infection, including human lentiviruses. Such diseases include cancer. This protein, called the "transcription suppression" or ts protein, has an amino acid sequence as set forth in Figure 2A. HHV-6 strain U1102 contains a 1,473 base pair ts gene. Both a 12 kbp HHV-6 Hindlll fragment and a SacIIJPpuMI subfragment containing ts suppress H-ras transformation. Araujo et al . , J. Virol . 69: 4933-40 (1995).

Among the diseases that can be treated with the ts protein are cancers associated with viruses, such as papilloma viruses, HTLV-1, HIV and lentiviruses that contain an LTR-like sequence. The term "treat" in its various grammatical forms herein refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state or progression.

Typically, the ts gene will be administered to subject at risk for the disease state. Such subjects include those already suffering from the disease, as well as subjects who, by virtue of genetic predisposition, viral infection or exposure to environmental factors, such as radiation or chemical mutagens, could develop the disease state. Suitable subjects include humans and other animals that can suffer from disease states caused by oncogenic transformation or lentivirus infection. For example, cattle, which are susceptible to the bovine papilloma virus, can be treated according to the present invention. The ts protein employed in the present invention can have the amino acid sequence set forth in Figure 2A. Changes in the amino acid sequence are contemplated in the present invention, however. For example, the ts protein can be altered by changing the DNA encoding the protein. Preferably, only conservative amino acid alterations are undertaken. Illustrative amino acid substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine.

Additionally, variants and fragments of the ts protein can be used in the present invention. Variants include analogs, derivatives, muteins and mimetics of the natural ts protein that retain the ability to cause the beneficial results described herein. Fragments of the ts protein refer to portions of the amino acid sequence of the ts polypeptide that also retain this ability. The variants and fragments can be generated directly from the ts protein itself by chemical modification by proteolytic enzyme digestion, or by combinations thereof. Additionally, methods of synthesizing polypeptides directly from amino acid residues also exist.

Non-peptide compounds that mimic the binding and function of the ts protein ("mimetics") can be produced by the approach outlined in Saragovi et al . , Science 253: 792-95 (1991) . Mimetics are peptide-containing molecules which mimic elements of protein secondary structure. See, for example, Johnson et al . ,"Peptide Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al . , Eds., (Chapman and Hall, New York, 1993) . The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions For the purposes of the present invention, appropriate mimetics can be considered to be the equivalent of the ts protein itself.

More typically, at least in the case of gene therapy, variants and fragments are created by recombinant techniques employing genomic or cDNA cloning methods. Site-specific and region-directed mutagenesis techniques can be employed. See CURRENT PROTOCOLS IN MOLECULAR BIOLOGY vol. 1, ch. 8 (Ausubel et al . eds., J. Wiley & Sons 1989 & Supp. 1990-93); PROTEIN ENGINEERING (Oxender & Fox eds., A. Liss, Inc. 1987). In addition, linker-scanning and PCR-mediated techniques can be employed for mutagenesis. See PCR TECHNOLOGY (Erlich ed. , Stockton Press 1989) ; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 & 2, supra . Protein sequencing, structure and modeling approaches for use with any of the above techniques are disclosed in PROTEIN ENGINEERING, loc . cit . and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols. 1 & 2, supra . If the compounds described above are employed, the skilled artisan can routinely insure that such compounds are amenable for use with the present invention in view of the screening techniques described herein. For example, testing can be undertaken in cells that possess activated H-ras or appropriately transformed 12D7 cells. The present invention also relates to a characterizing process. In such a process, cells are co¬ transformed with a construct containing ts-encoding sequences and a known disease-causing gene. A reduction in disease phenotype in the cells suggests that the disease-causing gene can be treated with ts therapy. Alternatively, cells that already display the disease phenotype can be transformed with the ts-containing construct to determine whether the ts protein reduce the disease phenotype.

Protection of NIH 3T3 cells by ts from H-ras transformation has been accomplished in three ways: co¬ transfection, stable transfection, and retroviral insertion. Both stable transfection and retroviral insertion were most effective, reducing the transformation frequency by greater that 90%. Retroviral insertion is more advantageous because the efficiency of gene transfer is much higher and gene rearrangements during transfer are less likely to occur. The data presented herein indicate the potential for using ts in the treatment of certain human cancers.

The data set forth herein below demonstrate that an HHV-6 gene is responsible for transformation suppression. Because this gene acts at the level of transcription, as demonstrated with both the H-ras promoter and LTR promoter, the gene is designated "ts" (for transcription suppression. The invention is further explained by the following, non-limiting examples.

EXAMPLE 1: PRODUCTION AND CHARACTERIZATION OF THE

TRANSFORMATION SUPPRESSOR (ts) PROTEIN

A 12 kbp Hindlll C fragment (pHD12) containing the ts gene, as well as a non-overlapping 9 kbp EcoRI D fragment (pED9) , from HHV-6A strain U1102 were obtained.

(Figure 1). See Martin et al . , J . Gen . Virol . 72: 157-68

(1991). A construct was made which contained the 2.6 kb subfragment of HD12, designated pts2.6, and included the ts gene under control of its endogenous promoter. This construct was obtained by first ligating the 4 kb SacII fragment of pHD12 into the unique SacII site of the pBluescript SK+ (pBS) (Stratagene) . A pBS/HD12-Sac I clone, with the SacII fragment in the appropriate orientation was then digested at the Smal site (within the multiple cloning site of pBS) and the PpumI site

(within the HD12 SacII region) and ligated after filling the 5* overhangs with T4 DNA polymerase. The ts gene also was cloned in both the sense and antisense orientations into the mammalian expression vector, pRc- RSV (Invitrogen) using a 1,803 bp PCR amplified sequence; the PCR primers, AAAAAAAGCTTCTTGGGAGGCGCAAACGG and AAAAAAAGCTTCAGCACCCTTGATGATGC, containedflankingHindlll restriction enzyme sites which were used for cloning.

The amino acid sequence of the HHV-6 ts protein is depicted in Figure 2A. Thomson et al . , Nature 351: 78-80 (1991) reported a 490 amino acid ORF within the HD12 fragment of HHV-6A with 24% identity to the amino acid sequence of the i?ep68/78 gene of AAV-2. Re-analysis of these sequences by the Gap program of the Genetics Computer Group revealed 51% similarity (Figure 2A) between HHV-6 ts and AAV-2 Rep68/78 , which refers to amino acid residues that have similar properties. Regions of homology can be found throughout the sequence. Furthermore, analysis with MACAW software (Schuler et al . , Prot . Struct . Funct . Genet . 9: 180-90 (1991)) revealed four regions of significant alignment (P=<1.5 X IO^"6) (Figure 2B) .

EXAMPLE 2: ACTIVITY OF THE TS PROTEIN

To examine whether the HHV-6A ts inhibited H-ras transformation in a manner similar to the AAV-2 Rep68/78 gene (Hermonat et al . , Cancer Res . 51: 3373-77 (1991)), pHD12 was tested to suppress H-ras transformation in the NIH 3T3 cell focus forming assay (Figure 3) . pEJ-H-ras with or without pZVH14 or pHD12 was transfected into NIH 3T3 cells. pZVH14 is a non-overlapping 8.7 kbp fragment from strain HHV-6 strain GS. See Josephs et al . , Science 234: 601-03 (1986). pEJ-Harvey-ras contains the activated H-ras gene isolated from the EJ human bladder carcinoma cell line. See Santos et al . , Nature 298: 343- 47 (1982) .

For the transformation, NIH 3T3 cells were maintained as subconfluent monolayers in Dulbecco's Modified Eagle's Medium (Mediatech) supplemented with 7% calf serum (Hyclone) , penicillin (100 units/ml) and streptomycin (100 μg/ml) . Cells were transfected by the calcium phosphate method as described by Chen et al . , Mol . Cell Biol . 7: 2745-52 (1987), with minor modifications. Briefly, 5 x IO⁴ cells were seeded into 6 well (35mm) plates and transfected with a total of 9 μg/well of plasmid including carrier pBS, all previously purified by double banding in CsCl. Four independent wells were transfected for each experimental condition. Cells were subcultured into 10 cm dishes at 48 hours post- transfection. Cells were stained with 5% Geimsa at 14 days post-transfection and foci were counted. ED9 and ZVH14, non-HD12-overlapping HHV-6 subfragments of approximately the same length as HD12 (see Figure 1) , were used as negative controls in the H-ras transformation suppression studies. Although, ZVH14 has been reported to morphologically transform NIH 3T3 cells after four weeks (Razzaque, Oncogene 5: 1365-70 (1990)), it was still considered useful as a control for these experiments in which H-ras transformation is scored after only 1 to 2 weeks post-transfection.

When pEJ-H-ras was co-transfected with increasing pHD12 concentrations, a dose-dependent suppression (up to 66%) of transformed foci was observed (Figure 3) . Although the specific number of transformed foci induced by H-ras alone varied from experiment to experiment, the dose dependent suppression of transformation by pHD12 was consistently observed and the level of suppression by 6 μg ranged between 60 and 80%.

To show the specificity of pHD12, similar sized fragments from other regions of the HHV-6 genome (Figure 1) were co-transfected with pEJ-H-ras (pZVH14, Figure 3; pED9, Figure 4) . In these cases, no effect on H-ras transformation was observed. Furthermore, no toxicity was observed between cells transfected with pEJ-H-ras alone or those co-transfected with pEJ-H-ras and pHD12, pZVH14, or pED9. To demonstrate that the HHV-6A ts was the element within pHD12 responsible for suppression of H-ras transformation, a 2.6 kb subfragment of HD12 was cloned into pBS. pts2.6 contained the 1473 bp coding region for the ts including 615 bp upstream of the putative translation initiation codon and 507 bp downstream of the stop codon. Three other ORFs greater than 75 amino acids exist within ts2.6. Because of the presence of the other ORFs, three translation termination linker (TTL) mutants within the ts gene, designated pts2.6/TTL-25, pts2.6/TTL- 125, and pts2.6/TTL-245, were constructed.

The three TTL mutants of the pts2.6 plasmid were: (1) pts2.6/TTL-25, constructed by insertion of the translation termination oligodeoxynucleotide 5'- CTAGCTAGCTAG (New England Biolabs) into the filled Ncol site within codon 26 of the ts gene; (2) pts2.6/TTL-125, constructed by insertion of the linker into the filled Pad site within codon 126; and (3) pts2.6/TTL-245, constructed by insertion of the linker into the filled

Mlul site within codon 246. The presence of the linker was confirmed by restriction analysis employing the Nhel site (GCTAGC) within the linker and by DΝA sequencing.

The TTL mutation placed after codon 125 terminates translation only in ts . pts2.6 and its TTL mutant constructs were used to demonstrate that the protein was required for suppression of transformation. Transient transfections of ΝIH 3T3 cells employing pHD12, pts2.6, or the TTL mutant constructs were performed to demonstrate that the ts transcript was expressed. The expected transcript (approximately 1.8 kb) was detected by Northern blot analysis using an internal 1.1 kbp Styl fragment of the ts gene as the probe (see Figure 4B) . Northern blot analysis was performed by extracting total cellular RNA isolated from NIH 3T3 cells 24 hours post- transfection by lysis in guanidine thiocyanate. Chirgwin et al . , Biochem . 18: 5294-99 (1979). RNA was pelleted by centrifugation through a CsCl cushion. Total RNA was separated on 1.0% agarose-for aldehyde gels and transferred to GeneScreen Plus membranes. Blots were probed with the l.l kbp Styl fragment of pts2.6, which was ³²P-labeled using the random primer method (Amersham) . Hybridization at 42°C overnight was followed by two washes with 2X SSC, 0.1% SDS at room temperature and a single wash with 0.1X SSC, 0.1% SDS at 48°C.

The transcript was identified after transfection by ts plasmids including HD12, pts2.6, and all TTL mutants. RNA from NIH 3T3 cells transfected with pBS alone showed no ts transcripts.

Because all constructs expressed ts transcripts, these plasmids could be used to determine if this protein was responsible for suppression of H-ras transformation. See Figure 4C. The dose response for suppression of H- ras transformation by pHD12 and pts2.6 was similar. However, none of the TTL mutants, especially TTL-125 which affected no other ORF than the ts gene, exhibited suppression of transformation. Moreover, another HHV-6A fragment lacking the ts gene, pED9, showed no suppression of H-ras transformation. These confirm that the sequence in HHV-6A is the transformation suppressor (ts) gene. Furthermore, the TTL mutant results suggest the importance of the C-terminal end of the ts protein downstream of amino acid 245. EXAMPLE 3: CHARACTERIZATION OF THE TS PROTEIN

ACTIVITY

The ts gene exerts its suppressor effect either on the endogenous H-ras promoter or directly on the H-ras p21 protein. To distinguish between these possibilities, a construct containing the murine osteosarcoma virus

(MSV) long terminal repeat (LTR) promoter expressing the activated H-ras p21 protein was employed. The MSV LTR promoter was chosen because it contains a TATA box and no Spl binding sites, unlike the H-ras promoter, which contains ten Spl binding sites and no TATA box. In this study, pMSVLTR-ras was employed, which contained the activated H-ras gene under the control of the murine osteosarcoma virus (MSV) long terminal repeat (LTR) . See Hermonat, Cancer Res . 51: 3373-77 (1991) .

When pMSVLTR-ras was co-transfected with either pts2.6 or pHD12 into NIH 3T3 cells, no suppression of transformation was observed (Figure 5A) . Concurrent experiments, utilizing pEJ-H-ras, with its endogenous promoter exhibited the expected suppression of transformation by pHD12 and pts2.6.

To confirm that pHD12 inhibited H-ras transcription, transient transfections were carried out employing constructs containing the CAT gene under the control of the H-ras or MSV LTR promoter (Figure 5B) . pMSVLTR-CAT and prasCATl contained the CAT gene expressed by the MSV¬ LTR or the endogenous ras promoter (Ishii et al., Science 232: 1410-43 (1986)), respectively. NIH 3T3 cells were transfected by the calcium phosphate method (Chen, (1987) supra) with 5 μg of the reporter CAT plasmid prasCATl or pMSVLTR-CAT in the presence or absence of 10 μg pHDl2. Cellular extracts were prepared 48 hours post-transfection and the protein levels determined. Extracts (15 μg) were incubated in 52 μl reactions in 0.25 M Tris (pH 7.75) with 0.2 μCi ^Re¬ labeled chloramphenicol and 0.1 mM Acetyl Coenzyme A, for 3 hours at 37°C. After autoradiography, spots were cut out, counted, and the percentage of acetylated chloramphenicol was calculated.

When prasCATl was co-transfected with pHD12, a 4.5 fold decrease in CAT acetylation was observed. In contrast, pMSVLTR-CAT showed no decrease in CAT activity when co-transfected with pHD12, further supporting the observation that ts suppressed transcription from the endogenous H-ras promoter.

The endogenous H-ras promoter contains six Spl- binding sites, several other transcriptional regulatory elements, but no TATA box element. Honkawa et al . , Mol . Cell . Biol . 7 : 2933-40 (1987); Ishii et al . , Science 232: 1410-43 (1986); Ishii et al . , Science 232: 1378-81 (1985); Lee et al . , J. Mol . Biol . 220: 599-611 (1991); Lu et al . , J. Biol . Chem . 269: 5391-402 (1994); Nagase et al . , Gene 94: 249-53 (1990). Different multiple transcription start point regions have been reported depending on the H-ras promoter construct being studied. While mutational studies have indicated that Spl-binding sites and other transcriptional regulatory elements were important for H-ras transcription, the relative effect of a particular mutation was sensitive to the promoter construct within which it occurred. Therefore, studies for defining the critical elements within the H-ras promoter and/or transcription initiation complex that respond to HHV-6A ts would be very difficult to interpret. On the other hand, the HIV-l LTR promoter contains defined transcriptional regulatory elements, including Spl site(s) that are required for both basal and activated transcription. Furthermore, the AAV-2 Rep68/78 gene has been demonstrated to suppress transcription by the HIV-l promoter. Antoni et al . , J. Virol . 65: 396-404 (1991); Parada et al . , Nature 297: 474-78 (1982); Rittner et al .J . Gen . Virol . 73: 2977-81 (1992); Thomson et al . , Virol . 304-11 (1994). Therefore, the HIV-l promoter was chosen to elucidate the transcriptional regulatory elements required for ts suppression. The effect of ts on the HIV-l promoter was analyzed in a CD4+ human T-cell line, 12D7, the natural host for both HIV-l and HHV-6. Cell line 12D7 is a clonal derivative of A3.01 CEM cells. Kashanchi et al . , J. Virol . 68: 3298-307 (1994). The cells were maintained in RPMI 1640 (Sigma) supplemented with 10% fetal calf serum (Gibco BRL) , penicillin (100 units/ml) and streptomycin (100 μg/ml) . Cells were electroporated by the method of Kashanchi et al . , Nucl . Acids Res . 20: 4673-74 (1992). After electroporation, cells were cultured at 37°C, 8% C0₂ for 20 hours. Harvested cells were extracted for CAT assays.

In these studies, pRc-ts was used with ts under the control of a strong promoter for human cells; pRc-ts(A), the anti-sense construct was used as a negative control. In a series of co-electroporation experiments, ts consistently suppressed CAT activity expressed from the wild-type HIV-l promoter construct, CD-12 (Figure 6A) in a dose dependent manner (Figures 6B and 7B) ; the range of suppression by 20 μg of pRC-ts was between 70 and 97%. Moreover, neither pRc-RSV (the parental vector) nor pRc- ts(A) (the anti-sense construct) suppressed CAT activity from CD-12 when the same range (as pRc-ts) of input DNA was used for co-electroporation) . These data demonstrated that ts suppressed expression of the HIV-l promoter in 12D7 cells. Finally, no ts suppression was observed when pCHC6-CAT (CAT expressed by the CMV immediate early promoter) was electroporated into 12D7 cells as the reporter construct, demonstrating promoter- dependent specificity for ts suppression in human T- cells.

A series of HIV-l LTR mutant constructs was utilized to define the critical elements of the HIV-l promoter required for ts suppression. To examine the role of Spl sites, two mutants were tested: LTR_N1Spldel-CAT contained a deletion of all three Spl sites as well as a second deletion of one of the two NFKB sites; LTR_Spι_m-CAT had double point mutations in each of the Spl sites (Figure 6A) .

Dose dependent suppression of CAT expression by ts was still consistently observed with these mutant CAT constructs to similar extent as with CD-12, which contained the CAT gene expressed by the U3/R (-453 to +80) HIV-l LTR. Therefore, Spl sites were not critical for ts suppression. To test other upstream regions of the HIV-l LTR, three deletion constructs, CD-23, -52, and-54 were used (Figure 6A) . CD-23, -52, and -54 deletion CAT constructs contain -117 to +80, -65 to +80, and -48 to +80 of the HIV-l LTR. See Ensoli et al . , EMBO J. 8: 3019-27 (1989).

Like the Spl mutants, none of the upstream deletions reversed ts suppression (Figure 6B) . Moreover, neither pRc-RSV nor pRc-ts(A) suppressed CAT expression from any of the above upstream HIV-l LTR mutants, including the Spl mutants. Because the CD-54 construct contains only the minimal HIV-l promoter sequences, no other upstream transcriptional regulatory element is required for ts suppression.

The downstream TAR region is also an important transcriptional regulatory element for the HIV-l promoter. Accordingly, two TAR mutations, TM29 and TM26 were examined for their effect on ts suppression. See Rounseville et al . , J. Virol . 66: 1688-94 (1992). TM29- CAT is deleted in the "Bulge" sequence and TM26-CAT has transversion base substitutions in all of the nucleotides of both the "Bulge" and "Loop" sequences (Figure. 7A) . CAT expression from either TM29- or TM26-CAT was never suppressed by pRc-ts in repeated experiments (Figure 7B) . Therefore, TAR is one of the critical element(s) for ts suppression.

EXAMPLE 4: COMPARISON OF THE DATA FROM EXAMPLES 2-3

TO OTHER DATA

The above data demonstrated that the putative ts protein in HHV-6A was functionally a transformation suppressor of H-ras in rodent cells. The data showed that the transformation suppressing activity localized to a 2.6 kbp subfragment, ts2.6, within HHV-6A HD12 and was abolished by the insertion of translation termination linkers into the ts gene at codons 25, 125, or 245 (Figure 4). Furthermore, like Rep68/78 , ts inhibited H- ras at the level of transcription and inhibited HIV-l LTR expression.

The data presented also demonstrated that ts , like its AAV-2 Rep68/78 counterpart, was functionally a suppressor of transcription from the HIV-l promoter in human T-cells (Figure 6B) . Furthermore, none of the upstream transcriptional regulatory elements was required for ts suppression because it was observed with the CD-54 construct which contains only the minimal promoter. In fact, sequences in the NF/cB/Spl region (-117 to -65) may actually interfere with ts suppression because constructs LTR_Nι_Spι_dd-CA , CD-52, and C-54, in which this region had been deleted either internally or terminally exhibited greater ts suppression than CD-23 or CD-12 (Figure 6A) . Finally, TAR mutant constructs, TM29- and TM26-CAT were insensitive to ts suppression, demonstrating that the downstream TAR element was critical. Because the transversion mutations within the TAR "Bulge" and "Loop" of TM26 would not be expected to significantly alter the three dimensional structure of the TAR containing RNA, changes to the primary structure are probably more important. The observation that ts suppress both the H-ras and HIV-l LTR promoters but not MSVLTR may be due to a common binding site for ts protein. An indication of such a site comes from studies which identified the Rep binding site, (GCTC)₃ within the AAV-2 inverted terminal repeat. See Chiorini et al . , J . Virol . 68: 797-804 (1994). Batchu et al . , Cancer Lett . 86: 23-31 (1994), have identified a Rep protein binding sequence within the H-ras promoter with three GCTC motifs in close proximity. In contrast, MSVLTR does not contain a sequence with multiple GCTC motifs. Moreover, the HIV-l TAR region has a sequence with 75% homology to the (GCTC)₃ Rep binding site that is immediately downstream of the "Loop". See Oelze et al . , J. Virol . 68: 1229-133 (1994). This sequence also may be required for ts suppression of HIV-l expression. Recently, Oelze et al . , supra have reported that TAR and negative regulatory element (NRE) were both required for complete inhibition by AAV-2 Rep of basal HIV-l expression. In the present studies, HIV-l LTR mutant constructs (CD-23, CD-52, or CD-54) lacking the NRE exhibited equal or greater ts suppression of basal expression.

Thomson et al . , Virology 204: 304-311 (1994), have reported that, like Rep68/78 , the HHV-6A ts mediates AAV- 2 replication. However, the function of the ts gene in the life cycle of HHV-6 is still not known, even though the HHV-6 origin of replication and origin-binding protein have been identified. See Dewhurst et al . , J. Virol . 67: 7680-783(1993); Dewhurst et al . , J. Virol . 68: 6799-6803 (1994).

The observation that the ts gene downregulates transcription stands in sharp contrast to Thomson et al . , Virology 204: 304-311 (1994), who observed that a homologue of the rep gene transactivated an HIV-l LTR CAT construct in both Vero (African green monkey kidney) and 1BR (human skin fibroblast) cells, but had no effect in the human CD4+ T-cell line (J. Jahn) , whereas the Rep68/78 gene itself suppressed expression in all cell lines. While the transactivation results are interesting, their relevance is questionable because neither Vero nor 1BR cells are the natural hosts for either HHV-6 or HIV-l. In the studies disclosed in the present invention, not only did ts repeatedly suppress wild-type HIV-l, but it also suppressed five different HIV-l LTR mutant constructs in the human CD4+ T-cell line, 12D7. Pellet and colleagues (personal communication) also have detected a Rep68/78 homologue in HHV-6B strain Z-29 with greater than 96% nucleotide and amino acid identities to HHV-6A ts . Almost all of the different amino acid pairs represent either similar or functionally analogous changes. Furthermore, Pellet and colleagues have detected a HHV-6B Rep homologue protein at 36 hours post-infection indicating that this protein is a natural viral product. Taken together the sequence comparisons for U1102 and Z-29 show a very high degree of conservation of the ts gene in HHV-6. Moreover, Blast analysis (Altschul et al . , J . Mol . Biol . 215: 403-10 (1990)) of the SWISS-PROT, PIR, and GenPept databases indicated that a ts gene does not exist in any other known herpesvirus.

The analysis of ts in HHV-6A strain U1102 also may provide new insight as to the domains within AAV-2 -ep68/78 necessary for transformation suppression and gene regulation. Hermonat and colleagues reported that a linker insertion/frameshift mutation (ins42) within the i?ep68 splice (Figure 8) basically eliminated transformation suppression of both H-ras and bovine papillomavirus. Hermonat et al . , J . Virol . 51: 329-339

(1984); Hermonat, Virology 172: 253-261 (1989); Hermonat, Cancer Res . 51: 3373-3377 (1991).

From this data, the author concluded that i?ep78 was the gene responsible for transformation suppression activity. However, Antoni et al . J . Virol . 65: 396-404 (1991) , showed that the Rep78 protein was expressed at much higher levels than Rep68. Therefore, it is difficult to decide from the transformation data with the ins42 mutant whether a low level of suppression by intact Rep68 protein might also have occurred. In any event, the ins42 results imply the importance of the C-terminal end of Rep78.

The data presented herein show that a ts gene with similarity only to the N-terminal portion of Rep68/78 upstream of the ins42 mutation (Figure 8) was capable of ts activity and suppression of HIV-l LTR expression. Therefore, these data suggest that the C-terminal end of the Rep78 protein may not be important. Possibly, the 3 dimensional structure of the Rep78 ins42 mutant protein rather than truncation of C-terminal sequences was responsible for loss of activity.

EXAMPLE 5: EXPRESSION OF TS IN ADDITIONAL NIH 3T3

CELL LINES

NIH 3T3 cells were stably transfected with pRc-ts containing the ts gene under control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) using the following methodology:

Plasmids:

The pRc-ts and pRc-ts(A) constructs contain the ts gene cloned into pRc-RSV (Invitrogen) under the control of the RSV promoter in the sense and antisense orientations, respectively, as described in Araujo et al . , (1995) , supra . pET14b-ts was constructed by cloning a 1509 bp PCR amplified product, synthesized with GGGAGGCGCAAACCATATG and AAGGATCCGTGGTCTTTTAAGATCTG primers, into the Ndel and BamHl sites of the bacterial expression vector, pET14b (Novagen) . pLNCts was constructed by subcloning the Hindlll fragment containing ts from pRc-ts into the Hindlll site of the retroviral vector, PLNCX. Miller et al . , Biotechniques 7 : 980-90 (1989) . pCHC6-CAT, containing CAT expressed by the HCMV immediate early promoter, as provided by Dr. Francis Kern. pBPV-69T contains the 69% transforming region of BPV-l. Lowy et al . , Nature 287: 72-74 (1980). The BPV-1 CAT constructs pl066 and p805-88 contain the E6 and E2 promoters, respectively. Spalholz et al . , J . Virol . 61: 2128-37 (1987); Spalholz et al . , J . Virol . 65: 743-53 (1991) . pURR16CAT contains the CAT gene under the control of the HPV-16 E6 promoter. To construct pURR16CAT, the initiating ATG of the E6 gene was mutated by PCR to AGTG, creating a PstI site. The PstI fragment containing bp 7004-7103 of HPV-16 was cloned into the PstI site of pBLCAT3. Luckow et al . , Nucleic Acids Res . 15: 5490 (1987).

Cells, transfections, and transformation:

NIH 3T3 cells were maintained as a subconfluent monolayer in Dulbecco's modified Eagle's medium

(Mediatech) supplemented with 7% calf serum (Hyclone) , penicillin (100 U/ml) , and streptomycin (100 μg/ml) . Transient transfections were performed by the calcium phosphate method as described in Chen (1987) , supra .

To establish stably transfected cell lines, four μg pRc/RSV, prc-ts, or pRc-ts(A) (ts cloned in the antisense orientation) were linearized within the ampicillin resistance gene with Seal and transfected using Lipofectamine (Gibco BRL) into 2 X IO⁵ NIH 3T3 cells in a 60 mm dish according to the manufacturer's protocol. After 48 hours, transfected cells were subcultured into 10 cm dishes and selected with 500 μg/ml geneticin 24 hours later. Independently selected 3T3-pRc, 3T3-ts and 3T3-ts-(A) colonies were isolated and maintained in culture media supplemented with 200 μg/ml geneticin. pLNCX is a retroviral vector carrying the HCMV "immediate early" promoter for expression in mammalian cells and a neomycin resistance marker. (Miller et al . , (1980) , supra . pLNCX and pLNCts were transfected into GP+E 86 ecotropic packaging cells. Supernatants, containing LNCX, LNCts and LNCtsTTL₁₂₅ retrovirus, were harvested 2 days post-transfection and used to infect PA137 a photropic packaging cells. Clonal producer populations were selected by G418: the titer of each of 50 producer clones for each retrovirus was assayed by determining the frequency of G418 resistance of infected NIH 3T3 cells. The 3 highest titer producer clones (> 10⁶ infectious particles/ml) for each retrovirus were selected for further use. Furthermore, four 3T3-LNCts and one 3T3-LNCX independently derived colonies were isolated to develop into cell lines.

For transformation studies, 5 X IO⁴ cells were seeded into 35 mm dish and transfected with 3 μg of pEJ- H-ras, pMSVLTRras, or 1 μg of pBPV-69T DNA. Schiller et Ai m , Proc . Nat ' l Acad . Sci . , USA 81: 7880-84 (1984). Four independent dishes were transfected overnight for each plasmid. Cells from each dish were subcultured into a 10 cm dish at 48 hr post-transfection. After 12 days, the dishes were stained with 5% Giemsa and the observed foci were counted. For co-transfection studies, cells were transfected with 1 μg of pBPV-69T plus indicated amounts of pts2.6 or pts2.6/TTL constructs. When necessary, the total amount of plasmid DNA was adjusted to 5 μg with pBluescript (Stratagene) DNA.

To determine growth rates, 5 X 10⁴ cells/dish of each line to be tested were seeded into replicate 10 cm dishes. On succeeding days, cells were resuspended by trypsinization and mixed with an equal volume of trypan blue. Cells excluding trypan blue were counted in a hemacytometer.

Western blot analysis:

Polyclonal rabbit antibody, Ab-679, provided by

Chemicon International, was raised against the oligopeptide, DGNAPKIDDWCTYAKTKKN, corresponding to amino acids 135 to 153 of the ts protein. The peptide was produced on an Applied Biosystems peptide synthesizer. Jn vitro transcription and translation reactions were performed according to manufacturer's protocol (Promega) for 2 hr at 30°C using either pETl4b DNA as a negative control or pET14b-ts. Protein extracts from mid-log phase cells were prepared as described (Seed and Sheen, 1988) . Protein was separated by 4-20 % SDS-PAGE, transferred to the PVDF Immobilon-P membrane (Millipore) , Western blotted with Ab-679, anti-ts serum (1:5,000), and detected with the "Western-Light" chemiluminescent detection system (TROPIX) employing goat anti-rabbit IgG conjugated to alkaline phosphatase.

CAT assay:

NIH 3T3 or ts expressing cells were transfected with one of the reporter CAT plasmid prasCATl (20 μg) , pMSVLTR-CAT (20 μg) , pCHC6-CAT (5 μg) , pl066 (20 μg) , p80588 (20 μg) , or pURR16CAT (20 μg) . After 48 hours, extracts were prepared and the protein levels were determined. Extracts, 10 μg for prasCATl, pMSVLTR-CAT and pCHC6-CAT, and 20 μg for pl066, p805-88, and pURR16CAT were incubated in 52 μl reaction mixtures in 0.25 M Tris (pH 7.75) with 0.05 pCi of ¹⁴C-labeled chloramphenicol and 0.02 mM acetyl coenzyme A for 3 hours at 37°C. Acetylated chloramphenicol species were separated by thin layer chromatography. After autoradiography, spots were cut out and counted, and the percent acetylated chloramphenicol was calculated. Using these methods, five independent stable cell lines containing the ts gene were selected by geneticin. Negative control cell lines were established in a similar manner after transfection with either pRc-RSV, the parental vector, or pRc-ts(A), with ts cloned in the antisense orientation. Southern blot analysis was performed to examine the retention and state of ts DNA in the selected cell lines.

After digestion with Hindlll which released the 1.8 kbp ts fragment from PRC-RSV vector sequences, one to two copies per cell of intact ts DNA was observed in each of the 3T3-ts cell lines (Fig. 13A) and in the 3T3-ts-l(A) line (data not shown) .

To determine if the selected cell lines also expressed ts protein, Western blot analysis using Ab-679, a polyclonal antibody raised against a synthetic 19 amino acid peptide from the ts gene (aa 135-153) was performed. The ability of Ab-679 to detect ts protein was confirmed with in vitro transcribed and translated ts protein. Ts protein (56 kDa) was observed after transcription/translation of pET14b-ts (Fig 13b, lane 2) while no similar protein was seen for the pET14b vector plasmid (lane 1) . With extracts of all five 3T3-ts lines, Ab-679 detected a 56 kDa protein (lanes 3-7) not seen in extracts of control parental NIH 3T3 cells (lane 8) or in 3T3-ts-l(A) cells (data not shown) . Thus, the 3T3-ts cell lines all produced ts protein. Growth curves were measured for the 3T3-ts cell lines (Fig. 13C) to determine if expression of ts protein affected cellular growth. Lines 3T3-ts-l, 2, 4, and 5 had growth rates that were approximately the same as that of NIH 3T3 cells; 3T3-ts-3 grew at a slightly lower rate. These results indicate that expression of ts protein did not affect cell growth.

EXAMPLE 6: EFFECT ON TS ON STABLY-TRANSFORMED CELL

LINES Stable NIH 3T3 cell lines were then established by transfection with pRc-ts or pRc-ts(A) , an antisense clone. 3T3-ts-l yielded 10% of EJ-H-ras induced foci compared to NIH 3T3 or 3T3-ts(A)-l (Fig. 9A) . In contrast, no reduction of foci was observed in 3T3-ts-l after transfection with pMSVLTR-ras. Moreover, the expression of the CAT gene expressed from transfected prasCATl was significantly reduced in pRc-ts-1 as compared to NIH 3T3 or 3T3-ts(A)-l (Fig. 9B) . On the other hand, expression of CAT expressed from pMSVLTR-CAT was similar in all three cell lines. These results confirm that ts suppressed H-ras transformation by inhibiting H-ras expression.

Stable 12D7 cell lines were also constructed. Figures IOA and B depict data from a comparison of cell lines expressing vector alone (12D7/RC-1) , wild-type ts

(12D7/ts-2) and mutant ts (12D7/ts-TTL-2) . The cells were transfected by electroporation with LTR-CAT

(containing the CAT gene expressed by the HIV-l promoter) or TM26-CAT (containing CAT expressed by the TM26 TAR mutant HIV-l promoter) . Twenty four hours later, extracts were prepared and tested for CAT activity. The results show that ts suppresses HIV-l transcription by interacting with the HIV-l TAR element, and indicates an ability to protect CD4+ T-cells from infection by HIV. Additionally, the growth of 12D7/ts-2cell line indicates the ability of ts to be expressed in T-cells, without affecting the viability or reproducibility of those T- cells.

EXAMPLE 7: EFFECT OF TS ON OTHER VIRUSES

The above data show that the ts protein can suppress H-ras. H-ras is elevated in a number of human cancers. See Varmus, Rev . Genet . 18: 553-612 (1984). As discussed above, activated H-ras has been identified in many human malignancies, including carcinomas of the (1) bladder (2) lung (3) breast and (4) urinary tract, as well as melanomas. Furthermore, over 95% of human cervical cancer is associated with the retention and expression of the E6 and E7 genes of human papillomavirus (HPV) . Accordingly, therapies that would lead to the attenuation of the H-ras gene or HPV E6 and E7 will control or cure a significant number of human cancers.

The ability of ts to suppress papillomavirus transformation was investigated using bovine papillomavirus (BPV) transfection of NIH 3T3 cells as a model. pts2.6 was aε effective in suppressing BPV transformation (Figure 11) as it was for H-ras, discussed above (see Figure 4C) . Moreover, TTL mutations in pts2.6 eliminated the ability of ts to suppress papillomavirus transformation, confirming the functional role of the ts gene.

In an independent study, where 3T3-ts-l and four additional 3T3-ts lines were tested, H-ras transformation was inhibited by 98% or greater in all cases (Figure 12A) . These 3T3-ts cell lines were refractile to transformation by H-ras, but only when H-ras was expressed from its endogenous promoter. Moreover, BPV transformation also was inhibited by at least 96% (Figure 12B) , while the identical cells failed to inhibit MSVLTRras transformation (Figure 12C) . Thus, the acquisition of ts protected the NIH 3T3 cells from transformation by BPV-1.

To determine if ts suppression occurred at the level of BPV-1 gene expression, two BPV-1 promoters, p89 or p2443, which express mRNAs that encode the E5 gene were tested. 3T3-ts cell lines were transfected with plasmids containing the CAT gene under control of p89 (pl066) or p2443 (p805-88) . For the p89 construct, CAT activity was lower in the three 3T3-ts cell lines relative to NIH 3T3 cells by 56 to 82%. For the p2443 construct, CAT activity was lower by 73 to 89% (Fig. 15A and B) . Thus, the suppression of BPV-1 transformant by ts was due to suppression of transcription. Moreover, the specificity of ts suppression was demonstrated because similar levels of CAT activity were observed in NIH 3T3 and 3T3-ts cell lines after transfection with either pCHC6CAT, where CAT was expressed from the HCMV immediate early promoter (Fig. 15C) or pMSVLTR-CAT (data not shown) . Ts was also tested for its ability to suppress human papillomavirus type 16 (HPV-16) expression. An HPV 16 p97 CAT construct (pURR16CAT) was tested for expression of CAT activity after transfection in the 3T3-ts cell lines. As in the case of the BPV-1 promoter CAT constructs, the three 3T3-ts cell lines exhibited 70 to 88% less CAT activity (Fig. 15D) than did NIH 3T3 cells after transfection with pURRl6CAT.

This data demonstrate the usefulness of ts against HPV expression and its roles in carcinoma, and strongly implicate ts as being useful for gene therapy against H- ras and papillomavirus-involved cancer.

EXAMPLE 8: GENE THERAPY WITH THE TS PROTEIN

The plasmid, pRc-ts, that was shown above both to suppress H-ras and BPV transformation and to establish 3T3-ts cell lines, is constructed utilizing an 1,803 bp PCR amplified ts sequence with terminal Hindlll sites provided by the PCR primers. This Hindlll ts fragment is isolated from pRc-ts, gel purified, and cloned into the Hindlll site of the retroviral vector construct, pLNCX. Miller et al . , Biotechniqueε 7 : 980-990 (1989). This vector system will be employed because (i) it contains the neomycin phosphotransferase gene expressed from the retroviral LTR as a selectable marker, (ii) it contains a Hindlll cloning site for high expression in human cells of an inserted gene from the human cytomegalovirus immediate early promoter, (iii) it yields high-titer virus stocks after introduction into retroviral packaging cells, and (iv) it avoids homologous overlap with viral DNA sequences present in the packaging cells preventing unwanted helper virus production. For negative controls, the antisense-ts construct, pLNCts(A), are constructed. To produce amphotropic stocks, pLNCts and pLNCts(A) are transfected into GP+E 86 ecotropic packaging cells. Markowitz et al . , J. Virol . 62: 1120-1124 (1988). After 2 days, supernatants containing sense and antisense ts retrovirus are used to infect PA317 amphotropic packaging cells. Miller et al . , Mol . Cell . Biol . 6: 2895-2902 (1986) . This "transinfection" method is chosen because it results in high titers due to more efficient expression by an integrated provirus. Hwang et al . , J. Virol . 50: 417-424 (1984). Clonal producer populations will be selected by G418-resistance.

The highest producing clones are be determined by quantitative analysis of G418 resistant NIH 3T3 colonies subsequent to infection by producer stocks. Fifty producer clones are be screened for each construct to assure the establishment of high titer producer clones. The chosen producer clones are tested to make sure that they are not also producing helper virus. EXAMPLE 9: SUPPRESSION OF H-.RAS TRANSFORMATION IN TS

CELL LINES ESTABLISHED BY RETROVIRAL TRANSFER

Ts expressing NIH 3T3 cell lines also were established by retroviral infection employing LNCts followed by G418 selection. As a control, NIH 3T3 cells were infected with the LNCX vector. After transfection by H-ras, 3T3-LNCts-l, ts-2, ts-3, and ts-4 cells exhibited 93 to 98% fewer transformed foci than did 3T3- LNCX-1 cells (Fig. 14) . Thus, ts can be effectively transferred by a high efficiency retroviral infection system.

EXAMPLE 10: VECTORS FOR USE WITH TS GENE THERAPY

The present invention is amenable for use with a variety of vectors for gene therapy. The invention includes construction of a vector containing the gene encoding the ts protein, and administering such a vector to the target site. Such gene therapy will enhance the efficacy of traditional chemotherapy, which is administered according to established protocols.

The construction of a recombinant vector containing the gene encoding ts according to the invention can be achieved by any of the methods well-known in the art for the insertion of exogenous DNA into a vector. See, e . g. , Maniatis et al . , Molecular Cloning (Cold Spring Harbor Press 2d ed. 1989) . In addition, the prior art teaches various methods of introducing exogenous genes into cells in vivo . See Rosenberg et al . , Science 242: 1575-1578 (1988) and Wolff et al . , Proc . Nat ' l Acad . Sci . USA 86: 9011-9014 (1989) . The routes of delivery include systemic administration and administration in situ . Well-known techniques include systemic administration with cationic liposomes, and administration in situ with viral vectors. Any one of the known gene delivery methodologies is suitable for the introduction of a recombinant vector containing the ts gene according to the invention. A listing of present-day vectors suitable for the purpose of this invention is set forth in Hodgson, Bio /Technology 13 : 222 (1995).

For example, liposome-mediated gene transfer is a suitable method for the introduction of a recombinant vector containing the gene encoding ts according to the invention. The use of a cationic liposome, such as DC- Chol/DOPE liposome, has been widely documented as an appropriate vehicle to deliver DNA to a wide range of tissues through intravenous injection of DNA/cationic liposome complexes. See Caplen et al . , Nature Med . 1:39- 46 (1995) and Zhu et al . , Science 261 : 209-211 (1993). Liposomes transfer genes to the target cells by fusing with the plasma membrane. The entry process is relatively efficient, but once inside the cell, the liposome-DNA complex has no inherent mechanism to deliver the DNA to the nucleus. As such, the most of the lipid and DNA gets shunted to cytoplasmic waste systems and destroyed. The obvious advantage of liposomes as a gene therapy vector is that liposomes contain no proteins. which thus minimizes the potential of host immune responses.

Viral vector-mediated gene transfer also is a suitable method for the introduction of a recombinant vector containing the gene encoding ts . Appropriate viral vectors include adenovirus vectors and adeno- associated virus vectors, retrovirus vectors and herpesvirus vectors.

Adenovirus vectors can be used to introduce the gene encoding ts according to the invention. Adenoviruses are linear, double stranded DNA viruses complexed with core proteins and surrounded by capsid proteins. The common serotypes 2 and 5, which are not associated with any human malignancies, are typically the base vectors. By deleting parts of the virus genome and inserting the desired gene under the control of a constitutive viral promoter, the virus becomes a replication deficient vector capable of transferring the exogenous DNA to differentiated, non-proliferating cells. To enter cells, the adenovirus fiber interacts with specific receptors on the cell surface, and the adenovirus surface proteins interact with the cell surface integrins. The virus penton-cell integrin interaction provides the signal that brings the exogenous gene-containing virus into a cytoplasmic endosome. The adenovirus breaks out of the endosome and moves to the nucleus, the viral capsid falls apart, and the exogenous DNA enters the cell nucleus where it functions, in an epichromosomal fashion, to express the exogenous gene. Detailed discussions of the use of adenoviral vectors for gene therapy can be found in Berkner, Biotechniques 6 : 616-29 (1988) and Trapnell, Advanced Drug Delivery Rev . 12 : 185-99 (1993). Adenovirus-derived vectors, particularly non-replicative adenovirus vectors, are characterized by their ability to accommodate exogenous DNA of 7.5 kb, relative stability, wide host range, low pathogenicity in man, and high titers (IO⁴ to IO⁵ plaque forming units per cell) . See Stratford-Perricaudet et al . , Proc . Nat ' l Acad . Sci . USA 89: 2581 (1992).

Adeno-associated virus (AAV) vectors can be used also to introduce the gene encoding ts according to the invention. As described above, AAV is a linear single- stranded DNA parvovirus that is endogenous to many mammalian species. AAV has a broad host range despite the limitation that AAV is a defective parvovirus which is dependent totally on either adenovirus or herpesvirus for its reproduction in vivo . The use of AAV as a vector for the introduction into target cells of exogenous DNA is well-known in the art. See, e . g . , Lebkowski et al . , Mole . & Cell . Biol . 8:3988 (1988) . In these vectors, the capsid gene of AAV is replaced by a desired DNA fragment, and transcomplementation of the deleted capsid function is used to create a recombinant virus stock. Upon infection the recombinant viruε uncoats in the nucleus and integrates into the host genome. Another suitable virus-based gene delivery mechanism is retroviral vector-mediated gene transfer. In general, retroviral vectors are well-known in the art. See Breakfield et al . , Mole . Neuro . Biol . 1:339 (1987) and Shih et al . , in Vaccines 85: 177 (Cold Spring Harbor Press 1985) . A variety of retroviral vectors and retroviral vector-producing cell lines can be used to introduce DNA encoding ts . Appropriate retroviral vectors include Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. These vectors include replication-competent and replication-defective retroviral vectors. In addition, amphotropic and xenotropic retroviral vectors can be used. In carrying out the invention, retroviral vectors can be introduced to a tumor directly or in the form of free retroviral vector producing-cell lines. Suitable producer cells include fibroblasts, neurons, glial cells, keratinocytes, hepatocytes, connective tissue cells, ependymal cells, chromaffin cellε. See Wolff et al . , Proc . Nat ' l Acad . Sci . USA 84: 3344 (1989) .

Retroviral vectors generally are constructed such that the majority of its structural genes are deleted or replaced by exogenous DNA of interest, and such that the likelihood is reduced that viral proteins will be expressed. See Bender et al . , J . Virol . 61:1639 (1987) and Armento et al . , J . Virol . 61 : 1647 (1987). The necessity for host cell replication for retroviral gene expression is not a problem with tumor cells, which are highly replicative, A few normal tissues that a replicative, such as endothelial cells that line the blood vessels that supply blood to the tumor, theoretically are most likely to be transduced by a retroviral vector. In addition, it is also possible that a retroviral vector would integrate into white blood cells both in the tumor or in the blood circulating through the tumor.

The spread of retroviral vector to normal tissues, however, is limited. The local administration to a tumor of a retroviral vector or retroviral vector producing cells will restrict vector propagation to the local region of the tumor, minimizing transduction, integration, expression and subsequent cytotoxic effect on surrounding cells that are mitotically active.

Both replicatively deficient and replicatively competent retroviral vectors can be used in the invention, subject to their respective advantages and disadvantages. For instance, for tumors that have spread regionally, such as lung cancers, the direct injection of cell lines that produce replication-deficient vectors may not deliver the vector to a large enough area to completely eradicate the tumor, since the vector will be released only form the original producer cells and their progeny, and diffusion is limited. Similar constraints apply to the application of replication deficient vectors to tumors that grow slowly, such as human breast cancers which typically have doubling times of 30 days versus the 24 hours common among human gliomas. The much shortened survival-time of the producer cells, probably no more than 7-14 days in the absence of immunosuppression, limits to only a portion of their replicative cycle the exposure of the tumor cells to the retroviral vector.

The use of replication-defective retroviruses for treating tumors requires producer cells and is limited because each replication-defective retrovirus particle can enter only a single cell and cannot productively infect others thereafter. Because these replication- defective retroviruses cannot spread to other tumor cells, they would be unable to completely penetrate a deep, multilayered tumor in vivo . See Markert et al . , Neurosurg. 77: 590 (1992). The injection of replication- competent retroviral vector particles or a cell line that produces a replication-competent retroviral vector virus may prove to be a more effective therapeutic because a replication competent retroviral vector will establish a productive infection that will transduce cells as long as it persists. Moreover, replicatively competent retroviral vectors may follow the tumor as it metastasizes, carried along and propagated by transduced tumor cells.

The risks for complications are greater, with replicatively competent vectors, however. Such vectors may pose a greater risk then replicatively deficient vectors of transducing normal tissues, for instance. The risks of undesired vector propagation for each type of cancer and affected body area can be weighed against the advantages in the situation of replicatively competent verses replicatively deficient retroviral vector to determine an optimum treatment.

Both amphotropic and xenotropic retroviral vectors may be used in the invention. Amphotropic virus have a very broad host range that includes most or all mammalian cells, as is well known to the art. Xenotropic viruses can infect all mammalian cells except mouse cells. Thus, amphotropic and xenotropic retroviruses from many species, including cows, sheep, pigs, dogs, cats, rats, and mice, inter alia can be used to provide retroviral vectors in accordance with the invention, provided the vectors can transfer genes into proliferating human cells in vivo.

Clinical trials employing retroviral vector therapy treatment of cancer have been approved in the United States. See Culver, Clin . Chem . 40 : 510 (1994). Retroviral vector-containing cellε have been implanted into brain tumorε growing in human patientε. See Oldfield et al . , Hum . Gene Ther . 4 : 39 (1993). These retroviral vectors carried the HSV-1 thymidine kinase (HSV-tk) gene into the surrounding brain tumor cells, which conferred sensitivity of the tumor cells to the antiviral drug ganciclovir. Some of the limitations of current retroviral based cancer therapy, as described by Oldfield are: (1) the low titer of virus produced, (2) virus spread is limited to the region surrounding the producer cell implant, (3) possible immune response to the producer cell line, (4) possible insertional mutagenesis and transformation of retroviral infected cells, (5) only a single treatment regimen of pro-drug, ganciclovir, is possible because the "suicide" product kills retrovirally infected cells and producer cells and (6) the bystander effect is limited to cells in direct contact with retrovirally transformed cells. See Bi et al.. Human Gene Therapy 4 : 725 (1993) .

Another suitable virus-based gene delivery mechanism is herpesvirus vector-mediated gene transfer. While much less is known about the use of herpesvirus vectors, replication-competent HSV-1 viral vectors have been described in the context of antitumor therapy. See Martuza et al . , Science 252 : 854 (1991).

It is expected that one skilled in the art having the benefit of the foregoing disclosure and the references cited therein would recognize the relative strengths and weaknesses of each gene delivery system in determining an appropriate method for the introduction of a recombinant vector containing the gene encoding ts according to the invention into appropriate cells. EXAMPLE 11: EVALUATION OF TS GENE THERAPY EFFICACY.

To test the efficacy of an above-described LNCts gene therapy reagent, known anchorage independent and tumorigenic human cancer derived cell lines either (i) containing an activated H-ras gene or (ii) from cervical cancer containing known HPV type E6 and E7 genes are tested to determine if the delivery of the ts gene to these cells will reverse or suppress their anchorage independence and tumorigenic phenotype. Two human cell lines with activated H-ras genes that arise from different tissue types are studied initially to determine if ts gene therapy can be effective against H-ras-involved cancer. For example, Hs 578T cells, derived from a carcinoma of the breast, and T24 cells, derived from the primary tumor of a transitional cell bladder carcinoma, as well as three cervical cancer cell lines, are studied.

HeLa cells are obtained from an adenocarcinoma, and contain HPV 18 sequences. CaSki and SiHa, isolated respectively from an epidermoid and a squamous carcinoma, contain HPV 16 DNA (CaSki has high copy number whereas SiHa has a low copy number) . All cell lines can be obtained from the American Type Culture Collection.

To test the effect of LNCts, each cell line is plated at a density of 10^s cells per 100mm dish, allowed to grow overnight, and infected with a multiplicity of infection of between 3 and 10. After 48 hours, the antibiotic G418 will be added to the cultures to select LNCts infected cells. The appropriate concentration of G418 for optimal selection of each cell line is pre¬ determined for each cell line studied using infection of LNCX virus. Antisense ts cell lines also are established as negative controls. The selected cell lines is examined by Southern and Northern blot analysis and/or RT-PCR to verify the presence and expression of the ts gene.

To determine whether the LNCts infected cells have lost their ability to grow in an anchorage dependent manner, 5 X 10³ to 5 X 10⁵ cells are seeded into agarose top agar. After 3 weeks of culture, the dishes will be assessed for the presence of colonies. For tumorigenicity studies, 10⁷ selected cells will be injected dorsally into nu/nu mice (5 per cell line) . The animals will be examined weekly for the appearance of tumors. When tumors reach 1 cm in diameter, they will be excised; half the tissue will be fixed in formalin for histologic examination and half will be frozen for DNA, RNA, and protein analysis of ts sequences. Cell lines infected with either LNCX or LNCts(A) virus will be used as controls for the anchorage independence and tumorigenicity studies.

It is to be understood that the description, specific examples and data, while indicating preferred embodiments, are given by way of illustration and exemplification and are not intended to limit the present invention. Various changeε and modifications within the present invention will become apparent to the skilled artisan from the discussion and disclosure contained herein.

Claims

What Is Claimed Is:

1. A method of gene therapy, comprising the steps of (A) providing a pharmaceutically acceptable vector comprising a polynucleotide sequence encoding a ts polypeptide and (B) delivering said vector to cells of a subject at risk of or suffering from a disease state associated with oncogenic transformation or lentivirus infection, such that, upon expression of said polynucleotide sequence in said cells, said disease state is treated.

2. A method as claimed in claim 2, wherein said polynucleotide comprises the HHV-6 ts gene, or a portion thereof.

3. A method according to claim 1, wherein said vector is chosen from the group consisting of a viral vector, a lipidic vector, a plasmid, and an ex vivo transformed cell.

4. A method according to claim 1, wherein said disease state is treated by delivery of said vector to said cells prior to disease development in said subject.

5. A method according to claim 1, where said disease state is treated by delivery of said vector to said cells subsequent to disease development in said subject.

6. A method according to claim 1, wherein said disease state is a cancer.

7. A method according to claim 1, wherein said disease state is a cancer associated with a member of the ras gene family.

8. A method according to claim 1, wherein said disease state is a cancer associated with a human papilloma virus.

9. A method according to claim 1, wherein said disease state is a cancer associated with a bovine papilloma virus.

10. A method according to claim 1, wherein said disease state is a cancer associated with a HTLV-1 virus.

11. A method according to claim 1, wherein said lentivirus is HIV.

12. A method according to claim 1, wherein said lentivirus contains an LTR-like sequence.

13. A vector that comprises a polynucleotide sequence encoding a ts polypeptide and that is suitable for gene therapy such that, upon delivery of said vector to cells of a subject at risk of or suffering from a disease state associated with oncogenic transformation or lentivirus infection, expression of said polynucleotide sequence is effected in said cells to treat said disease state.

14. A vector according to claim 1, wherein said polynucleotide comprises the HHV-6 ts gene, or a portion thereof.

15. A vector according to claim 12, wherein said vector is a viral vector, a lipidic vector, a plasmid, or an ex vivo transformed cell.

16. A therapeutic agent comprising a transcription- suppressing polypeptide.

15. A method of gene therapy, comprising the step of administering a therapeutic composition comprising a ts polypeptide or a polynucleotide sequence encoding said ts polypeptide to a subject at risk for or suffering from a disease state associated with oncogenic transformation or lentivirus infection.

16. A method of characterizing a disease state, comprising the steps of: administering a ts polypeptide to a cell affected by said disease state; and determining whether said ts polypeptide can treat said disease state.

17. A method according to claim 16, where said cell is transformed with a gene to cause said disease state.

18. A method according to claim 16, wherein said administering step comprises transforming said cell with a ts gene.