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Title:
EXPRESSION OF CYCLIN G1 IN TUMORS
Document Type and Number:
WIPO Patent Application WO/1997/016209
Kind Code:
A1
Abstract:
A method of treating a tumor (in particular osteosarcoma or Ewing's sarcoma) in a host by administering to a host or to the tumor cells an agent which inhibits cyclin G1 protein in an amount effective to inhibit cyclin G1 protein in tumor cells of the host. The agent may be an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin G1 protein, or an antibody or fragment or derivative thereof which recognizes cyclin G1 protein. Also contemplated within the scope of the present invention are (i) the immortalization of cell lines by transducing cells with a polynucleotide encoding cyclin G1 protein; (ii) increasing the receptiveness of cells to retroviral infection by transducing cells with a polynucleotide encoding cyclin G1 protein; and (iii) the detection of cancer by detecting cyclin G1 protein or a polynucleotide encoding cyclin G1 protein in cells. In addition, the present invention provides expression vehicles, such as, for example, retroviral vectors and adenoviral vectors, which include polynucleotides which encode agents which inhibit cyclin G1 protein, and expression vehicles which include a polynucleotide encoding cyclin G1 protein.

Inventors:
GORDON ERLINDA M (US)
HALL FREDERICK L (US)
ANDERSON W FRENCH (US)
Application Number:
PCT/US1996/017442
Publication Date:
May 09, 1997
Filing Date:
October 31, 1996
Export Citation:
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Assignee:
UNIV SOUTHERN CALIFORNIA (US)
GORDON ERLINDA M (US)
HALL FREDERICK L (US)
ANDERSON W FRENCH (US)
International Classes:
G01N33/574; A61K31/00; A61K39/395; A61K45/00; A61K48/00; A61P9/00; A61P35/00; C07K14/47; C07K16/18; C12N15/09; G01N33/577; A61K38/00; (IPC1-7): A61K39/44; A61K39/395; C07H21/04; C07K16/18; C07K16/30; G01N33/53; G01N33/574
Foreign References:
Other References:
ONCOLOGY REPORTS, 1994, Vol. 1, WU L. et al., "Molecular Cloning of the Human CYCG1 Gene Encoding a G-Type Cyclin: Overexpression in a Human Osteosarcoma Cells", pages 705-711.
CANCER RESEARCH, 01 December 1995, Vol. 55, No. 23, SKOTZKO M. et al., "Retroviral Vector-Mediated Gene Transfer of Antisense Cyclin G1 (CYCG1) Inhibits Proliferation of Human Osteogenic Sarcoma Cells", pages 5493-5498.
JOURNAL OF BIOLOGICAL CHEMISTRY, 15 March 1996, Vol. 271, No. 11, HORNE M.C. et al., "Cyclin G1 and Cyclin G2 Comprise a New Family of Cyclins with Contrasting Tissue-Specific and Cell Cycle-Regulated Expression", pages 6050-6061.
See also references of EP 0858346A4
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Claims:
WHAT IS CLAIMED IS:
1. A method of treating a tumor in a hoεt comprising: administering to a host an agent which inhibits cyclin Gl protein, said agent being administered in amount effective to inhibit cyclin Gl protein in tumor cells of said host.
2. The method of Claim 1 wherein said agent is an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein.
3. The method of Claim l wherein said agent is an antibody or fragment or derivative thereof which recognizes cyclin Gl protein.
4. The method of Claim 3 wherein said antibody is a singlechain antibody.
5. The method of Claim 3 wherein said antibody is a monoclonal antibody.
6. The method of Claim 3 wherein said antibody is a polyclonal antibody.
7. The method of Claim 2 wherein said antisense polynucleotide is administered to said host by transducing tumor cells of said host with an expression vehicle including a polynucleotide encoding an antisenεe polynucleotide which iε complementary to at least a portion of a polynucleotide encoding cyclin Gl protein.
8. The method of Claim 7 wherein said expression vehicle is a retroviral vector.
9. The method of Claim 1 wherein said tumor is a cancerous tumor.
10. The method of Claim 9 wherein said cancerous tumor is osteogenic sarcoma.
11. The method of Claim 9 wherein said cancerous tumor is Ewing's sarcoma.
12. The method of Claim 3 wherein said antibody or fragment of derivative thereof which recognizes cyclin Gl protein is administered to said host by transducing tumor cells of εaid host with an expresεion vehicle including a polynucleotide encoding said antibody or fragment or derivative thereof which recognizes cyclin Gl protein.
13. The method of Claim 12 wherein said expression vehicle is a retroviral vector.
14. A method of immortalizing nontumor cells comprising: transducing said nontumor cells with a polynucleotide encoding cyclin Gl protein or a derivative or ana1ogue thereof.
15. The method of Claim 14 wherein said polynucleotide encoding cyclin Gl or a derivative or analogue thereof is contained within a retroviral vector.
16. A method of enhancing transduction of cells with a retroviral vector that includes a polynucleotide encoding a therapeutic agent, comprising: transducing said cells with a first expresεion vehicle that includeε a polynucleotide encoding cyclin Gl protein, wherein said first expression vehicle is not a retroviral vector; and transducing said cells with a second expression vehicle that includes a polynucleotide encoding a therapeutic agent, said second expression vehicle being a retroviral vector.
17. The method of Claim 16 wherein said first expression vehicle is an adenoviral vector.
18. The method of Claim 16 wherein said cells are transduced with said first expression vehicle and said second expression vehicle in vivo.
19. The method of Claim 16 wherein said cellε are transduced with said first expression vehicle and said second expression vehicle in vi tro.
20. The method of Claim 16 wherein said cells are transduced with said first expression vehicle prior to transduction of said cells with said second expression vehicle.
21. The method of Claim 16 wherein said cells are tranεduced with said first expression vehicle and said second expression vehicle concurrently.
22. An expression vehicle including a polynucleotide encoding an agent selected from the group consisting of (i) an agent which inhibits cyclin Gl protein; and (ii) cyclin Gl protein.
23. The expression vehicle of Claim 22 wherein said expression vehicle is a viral vector .
24. The vector of Claim 23 wherein said vector is a retroviral vector.
25. The vector of Claim 24 wherein εaid agent is an agent which inhibits cyclin Gl protein.
26. The vector of Claim 25 wherein said agent which inhibits cyclin Gl protein is an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein.
27. The vector of Claim 25 wherein said agent which inhibits cyclin Gl protein is an antibody or fragment or derivative thereof which recognizes cyclin Gl protein.
28. The vector of Claim 24 wherein said agent is cyclin Gl protein.
29. The vector of Claim 23 wherein said vector is an adenoviral vector.
30. The vector of Claim 29 wherein said agent is an agent which inhibits cyclin Gl protein.
31. The vector of Claim 30 wherein said agent which inhibits cyclin Gl protein is an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein.
32. The vector of Claim 30 wherein said agent which inhibits cyclin Gl protein is an antibody or fragment or derivative thereof which recognizes cyclin Gl protein.
33. The vector of Claim 29 wherein said agent is cyclin Gl protein.
34. A producer cell which produces the retroviral vector of Claim 24.
35. A method of detecting cancer, comprising: contacting cells with an agent which binds to (i) cyclin Gl protein and/or (ii) a polynucleotide encoding cyclin Gl protein; and determining binding of said agent to εaid cyclin Gl protein and/or said polynucleotide encoding cyclin Gl protein.
36. The method of Claim 35 wherein said agent is a polynucleotide which hybridizes to a polynucleotide encoding cyclin Gl protein.
37. The method of Claim 35 wherein εaid agent is an antibody or a fragment or derivative thereof which recognizeε cyclin Gl protein.
38. The method of Claim 35 wherein said cancer is osteogenic sarcoma.
39. The method of Claim 35 wherein said cancer is Ewing's εarcoma.
40. A method of preventing reεtenoεiε, comprising: administering to a host an agent which inhibits cyclin Gl protein, said agent being administered in an amount effective to prevent reεtenoεiε in a host .
Description:
EXPRESSION OF CYCLIN GI IN TUMORS

This application is a continuation-in-part of application Serial No. 08/551,486, filed November 1, 1995, the contents of which are hereby incorporated by reference.

This invention relates to the expression of cyclin GI in tumors. More particularly, this invention relates to: (i) the treatment of tumors such as osteogenic sarcoma or Ewing's sarcoma, by inhibiting cyclin GI protein in the tumor cells; (ii) the prevention of restenosis by inhibiting cyclin GI protein in cells at the site of an invasive vascular procedure or vascular lesion; (iii) the immortalization of cells by transducing such cells with a polynucleotide encoding cyclin GI protein; (iv) making cells more receptive to infection or transduction by a retroviral vector by transfecting the cells with a polynucleotide encoding cyclin GI protein, prior to or concurrently with said retroviral transduction or infection; and (v) a method of detecting cancer by determining the level of expression of cyclin GI protein in cells. This invention also relates to expression vehicles, preferably retroviral vectors and adenoviral vectors, which include polynucleotides encoding agents which inhibit cyclin GI protein, such as antisense polynucleotides, and antibodies or fragments or derivatives thereof which

recognize cyclin GI protein, and to expression vehicles which include a polynucleotide encoding cyclin GI protein. BACKGROUND OF THE INVENTION

Genes encoding a new class of proteins known as cyclins have been identified as a new class of protooncogenes, and cyclin-dependent kinase (or Cdk) inhibitors have been identified as tumor suppressors, thereby uniting the molecular mechanisms of cellular transformation and tumorigenesis with the enzymology governing cell cycle control. (Hall, et al. , Curr. Qpin. Cell Biol., Vol. 3, pgs.

176-184 (1991 ) ; Hunter, et al . , Cell, Vol. 55, pgs. 3071-1074

(1991) ; Hunter, et al . , Cell. Vol. 79; pgs. 573-582 (1994) ;

Elledge, et al . , Curr. Qpin. Cell Biol. , Vol 6, pgs. 874-878

(1994) ; Peter, et al . , Cell. Vol. 79, pgs. 181-184 (1994)) .

The sequential expression of specific cyclins and the essential functions of specific Cdk complexes have been defined (Wu, et al . , Int. J. Oncol. , Vol. 3, pgs. 859-867

(1993); Pines, et al . , New Biologist, Vol. 2, pgs 389-401

(1990) ; Pines, Cell Growth and Differentiation, Vol. 2, pgs.

305-310 (1991) ; Reed, Ann. Rev. Cell Biol. , Vol. 8, pgs. 529-

561 (1992); Sherr, Cell, Vol. 79, pgs. 551-555 (1994)) , thereby providing direct links to the fundamental mechanisms of DNA replication, transcription, repair, genetic instability, and apoptosis. (D'Urso, et al . , Science, Vol.

250, pgs. 786-791 (1990); Wu, et al. , Oncogene, Vol. 9, pgs

2089-2096 (1994) ; Roy, Cell. Vol. 79, pgs. 1093-1101 (1994) ;

Meikrantz, et al., Proc. Nat. Acad. Sci. , Vol. 91, pgs. 3754-

3758 (1994)) . Both the universal Cdk inhibitor p21/WAFl/CIPl

(Xiong, et al . , Nature, Vol. 366, pgs. 701-704 (1993);

Harper, et al . , Mol. Biol. Cell, Vol. 6, pgs. 387-400

(1995)) , and cyclin GI (Wu, et al . , Oncol. Reports, Vol. 1, pgs, 705-711 (1994)) are induced by the wild-type p53 tumor suppressor protein in the initiation of DNA repair and/or apoptosis. (El-Deiry, et al . , Cell. Vol. 75, pgs 817-825

(1993) ; El-Deiry, et al . , Cancer Res.. Vol. 54, pgs. 1169-

1174 (1994)) . Thus, the molecular components regulating critical cell cycle checkpoints represent strategic targets for potential therapeutic intervention in the treatment of cell proliferation disorders, including pediatric bone cancers, in which the Rb and the p53 tumor suppressor genes often are inactivated. (Hansen, et al . , Proc. Nat . Acad. Sci.. Vol. 82, pgs. 6216-6220 (1985); Toguchida, et al . , Nature. Vol. 338, pgs. 156-158 (1989); Toguchida, et al . , Cancer Res.. Vol. 48, pgs. 3939-3943 (1988) ; Diller, et al . , Mol. Cell. Biol. , Vol. 10, pgs. 5772-5781 (1990)) . Previous studies have characteri ed the progressive profile of cyclin expression and Cdk activation (Wu, 1993; Carbonaro-Hall, et al . , Oncogene. Vol. 8, pgs 1649-1659 (1993) ; Hall, et al . , Oncogene. Vol. 8, pgs. 1377-1384 (1993); Williams, e al., J. Biol. Chem.. Vol. 268, pgs. 8871-8880 (1993) ; Albers, e al., J. Biol. Chem.. Vol. 268, pgs. 22825-22829 (1993)), as well as the p53-independent induction of p21/WAFl/CIPl (Wu, et al . , Oncol. Reports, Vol. 2, pgs 227-231 (1995)), in MG-63 osteosarcoma cells. Also, antisense oligonucleotide strategies directed against cyclin Dl effectively inhibit cell cycle progression in these osteosarcoma cells. (Wu, 1993) .

Metastatic carcinoma is an important target for gene therapy aε it is associated with poor outcome. Colorectal cancer, for example, is the second leading cause of cancer death in the United States after lung cancer, followed by breast and pancreatic cancer (Silberberg et al . , Cancer Clin.. Vol. 40, pgs. 9-26 (1990)) . Of these carcinomas, pancreatic cancer has the worst prognosis. The median survival of patients with metastatic pancreatic cancer is three to six months and virtually all the patients are dead within a year (Merrick et al . , Gastroenterol. Clin. N. Amer., Vol. 19, pgs. 935-962 (1990)) . Approximately 40% of patients will have metastatic disease either to the liver or the peritoneal cavity or both at the time of diagnosis.

Chemotherapy for metastatic disease is ineffective despite multimodal therapy. Hence, alternative approaches to metastatic carcinoma would be desirable.

Wu, et al . (Oncol. Reports, Vol. 1, pgs. 705-711 (1994) ) , hereinabove mentioned, discloses the deduced amino acid sequence and cDNA sequence for human cyclin GI protein. Wu, et al . , also disclose that higher levels of cyclin GI expression were found in osteosarcoma cells and in Ewing's sarcoma cells than in normal diploid human fibroblasts. Although Wu, et al . , state that the overexpression of cyclin GI protein in human osteosarcoma cells provides a potential link to cancer, Wu, et al . , do not disclose the treatment of cancer by interfering with or inhibiting the function of cyclin GI protein in cancer cells.

SUMMARY OF THE INVENTION

Applicants have discovered that by interfering with and/or inhibiting the function or expression of cyclin GI protein in cancer cells, one may inhibit, prevent, or destroy the growth and/or survival of such cancer cellε. Thus, the present invention is directed to the treatment of a tumor (preferably a cancerous tumor) by inhibiting cyclin Gl protein, preferably through the administration of antisense oligonucleotides to a polynucleotide encoding cyclin Gl protein, or antibodies to cyclin Gl protein.

In addition, the present invention is directed to (i) the prevention of restenosis by inhibiting cyclin Gl protein in cells at the site of an invasive vascular procedure or vascular lesion; (ii) the immortalization of cells by transducing cells with a polynucleotide encoding cyclin Gl protein; (iii) the transducing of cells with a polynucleotide encoding cyclin Gl protein in order to make cells more receptive to transduction or infection with a retroviral vector; and (iv) a cancer assay which involves detection of

cyclin Gl protein and/or a polynucleotide encoding such protein.

The present invention also is directed to expression vehicles which include polynucleotides encoding agents which inhibit cyclin Gl protein, and to expression vehicles which include a polynucleotide encoding cyclin Gl protein. Such expression vehicles include, but are not limited to, viral vectors such as retroviral vectors and adenoviral vectors .

BRIEF DESCRIPTION OF THE DRAWINGS

The invention now will be described with respect to the drawings, wherein:

Figure 1 is a nucleotide sequence of human cyclin Gl CDNA;

Figure 2 depicts the staining of MG-63 osteogenic sarcoma cells following transduction of such cells with a retroviral vector including a B-galactosidase, or lacZ gene;

Figure 3 is a graph of the degrees of confluency (%) in mixtures of MG-63 cells which were transduced with a retroviral vector including a Herpes Simplex Virus thymidine kinase (TK) gene, and cells which were not transduced with such vector;

Figure 4 is a schematic of the retroviral vectors GlaDlSvNa, GlaGlSvNa, Glp21SvNa, and GlXSvNa;

Figure 5 is a graph of the cell counts in cultures of MG-63 cells transduced with GlXSvNa, GlaDlSvNa, GlaGlSvNa, or Glp21SvNa;

Figure 6 is a Western Blot of expression of p29 cyclin Gl protein in MG-63 cells transduced with GlXSvNa, GlaGlSvNa, or GlaDlSvNa;

Figure 7 depicts the morphological appearance of MG-63 cells by light microscopy at 72 hours after transduction of such cells with GlXSvNa, GlaGlSvNa, GlaDlSvNa, or Glp21SvNa;

Figure 8 depicts the detection of apoptotic cells in cultures of MG-63 cells transduced with GlXSvNa, GlaGlSvNa, GlaDlSvNa, or Glp21SvNa;

Figure 9A depicts FACS analysis of PI-stained nuclei 48 hours after transduction of VX2 carcinoma cells with a retroviral vector bearing antisense cyclin Gl (GlaGlSvNa) , compared with that of the control (GlXSvNa) vector.

Figure 9B depicts FACS analysis of Pi-stained nuclei 48 hours after transduction of MG-63 osteosarcoma cells with retroviral vectors bearing antisense cyclin Gl (GlaGlSvNa) compared with the control (GlXSvNa) vector.

Figure 10: Cytostatic effects of retroviral vectors bearing antisense cyclin Gl and wild type p53 in transduced VX2 undifferentiated carcinoma cells. Cell densities were measured, by cell counting, in cell cultures of VX2 cells at serial intervals after retroviral vector transduction prior to G418 selection.

Figure 11: Morphological appearance of VX2 cells 10 days after transduction with retroviral vectors bearing antisense cyclin Gl (GlaGlSvNa) , wild type p53 (Glp53SvNa) or the control (GlXSvNa) vector after G418 selection.

Figure 12 : Inhibition of VX2 tumor growth in nude mice following intratumoral injection retroviral vector bearing antisense cyclin Gl. The percentage increase in tumor size, plotted on the vertical axis, is expressed as a function of time (days) , plotted on the horizontal axis.

Figure 13A: Gross appearance of representative VX2 tumor-bearing mice one week after treatment with retroviral vectors bearing antisense cyclin Gl (GlaGlSvNa) or the control vector (GlXSvNa) .

Figure 13B: Hematoxylin-eosin stain of formalin-fixed tumor sections one week following treatment with retroviral vectors bearing antisenεe cyclin Gl (GlaGlSvNa) or the control vector (GlXSvNa) . 40X magnification.

Figure 14 is a graph of tumor sizes in mice injected with MNNG/HOS cells, followed by injection of the retroviral vectors GlXSvNa or GlaGlSvNa. Tumor volumes are measured at 0, 4, 6, 8, 10, and 12 days after injection of the retroviral vectors .

Figure 15: (A) Aortic smooth muscle cells expressing nuclear-targeted β-galactosidase (cells with blue nuclei) following transduction with the GlnBgSvNa vector; (B) Cytostatic and cytocidal effects of antisense cyclin Gl and wild type p53 in transduced aortic SMC. Cell densities were measured by direct cell counting in cultures of aortic SMC harvested at serial intervals after transduction with retroviral vectors bearing antisense Gl (GlaGlSvNa) and wild type p53 (Glp53SvNa) as well as the control vector (GlXSvNa) ; (C) 3 H-thymidine incorporation in cultured aortic SMC after transduction with retroviral vectors (n = 3 each group) . Radioactivity is expressed as dpm per well. Results are expressed as arithmetic mean ± 1 standard deviation;

Figure 16: The morphological appearance of aortic SMC, observed by light microscopy at 24 hrs after tranεduction with control and antisense cyclin Gl retroviral vectors (A = GlXSvNa control vector; B-D = GlaGlSvNa) . Detection of apoptosis in vascular SMC after antisense cyclin Gl retroviral vector transduction; (E) GlXSvNa control vector- transduced cells, (F) GlaGlSvNa antisense cyclin Gl vector- transduced cells. The dark-staining apoptotic bodies are noted both within and out of the syncytial cells;

Figure 17: Cytocidal "bystander" effect in antisense cyclin Gl vector-transduced aortic SMC. Incorporation of non- transduced, fluoreεcently labeled aortic SMC into multicellular syncytia when overlaid onto an SMC culture previously transduced with an antisense cyclin Gl vector. A and B, low magnification; C and D, high magnification; A and C, phase contrast; B and D, UV light. A representative multinuclear syncytium incorporating cells containing the

fluorescent label is identified by the arrow. (E) Quantification of syncytia formation over time in vascular SMC transduced with retroviral vectors: GlXSvNa, control vector; GlaGlSvNa, vector bearing antisense cyclin Gl gene; Glp53SvNa, vector bearing wild type p53;

Figure 18: (A) High density cultures of aortic SMC scraped with a 200 μl pipet tip to create a 1 mm track devoid of cells, (B) The appearance of the "wound" margin immediately upon scraping and washing to remove detached cells, (C) Aortic SMC expressing nuclear targeted β- galactosidase along the margins of the track, (D) Proliferation and migration of GlXSvNa control vector- transduced aortic SMC into the track at 24 hrs after injury, (E) Apoptotic and degenerative changes in GlaGlSvNa vector- transduced aortic SMC with marked syncytia formation; and

Figure 19: Test of efficacy of an antisense cyclin Gl vector in the rat carotid artery injury model of restenosis. The elastin layer of the tunica media is identified (in A-D) by Verhoeff's stain. The neointima, comprised of proliferating smooth muscle cells (reddish yellow staining cells) , is identified by Siris red stain. A and C = non- treated arterial segments; B and D = antisense cyclin Gl vector-treated arterial segments. E and F = higher magnification of non-treated and aGl-treated arterial segments, respectively; G = Analysis of neointima to media ratios of non-treated (NT) , control (GIX) and antisense cyclin Gl (aGl) -treated arterial segments are represented as vertical bars. DETAILED DESCRIPTION OF THE INVENTION

In accordance with an aspect of the present invention, there is provided a method of treating a tumor in a host . The method comprises administering to a host or to the tumor an agent which inhibits cyclin Gl protein. The agent is administered in an amount effective to inhibit cyclin Gl protein in the tumor cells.

The term "treating a tumor" as used herein means that one provides for the inhibition, prevention, or destruction of the growth of the tumor cells.

The term "inhibit cyclin Gl protein" as used herein, means that the agent inhibits or prevents the expreεεion of a polynucleotide encoding cyclin Gl protein, or inhibits or prevents the function of cyclin Gl protein.

Agents which inhibit cyclin Gl protein which may be employed include, but are not limited to, polynucleotides (including antisense oligonucleotides or polynucleotide fragments or sequences which are complementary to at least a portion of a polynucleotide encoding cyclin Gl protein) which bind to a polynucleotide encoding cyclin Gl protein to prevent expression of a polynucleotide encoding cyclin Gl protein, and antagonists to cyclin Gl protein, such as, for example, antibodies or fragments or derivatives thereof which recognize cyclin Gl protein, and cyclin-dependent kinase inhibitors.

In one embodiment, the agent which inhibits cyclin Gl protein is a polynucleotide which binds to a polynucleotide encoding cyclin Gl protein, and in particular is an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein. A nucleotide cDNA (Figure 1) and deduced amino acid sequence of human cyclin Gl protein is described in Wu, et al . , Oncology Reports. Vol. 1, pgε. 705-711 (1994) , which is incorporated herein by reference.

The term "polynucleotide" aβ used herein means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single- and double-stranded DNA, as well as single- and double-stranded RNA. The term also includes modified polynucleotides such as methylated or capped polynucleotides.

In general, the antisense polynucleotide which is complementary to at least a portion of a polynucleotide

encoding cyclin Gl protein includes at least 15 nucleotides, preferably at least 18 nucleotides, and more preferably from 18 to 20 nucleotides. In one embodiment, the antisense polynucleotide is complementary to the entire length of the polynucleotide encoding cyclin Gl protein.

In one embodiment, the antisense polynucleotide is complementary to, and thus capable of binding or hybridizing to, at least a portion of mRNA encoding cyclin Gl protein, thereby inhibiting translation of such mRNA. In another embodiment, the antisense polynucleotide is complementary to, and thus capable of binding or hybridizing to, single- stranded or double-stranded DNA encoding cyclin Gl protein, thereby preventing the transcription of such DNA to mRNA, or inhibiting the replication of such DNA. The antisense polynucleotide may bind to any portion of the DNA or mRNA encoding cyclin Gl protein, but preferably such antisense polynucleotide binds at the 5' end of the DNA or mRNA.

In another embodiment, the antisense polynucleotide may be a ribozyme that promotes the cleavage of mRNA encoding cyclin Gl. As used herein, the term "ribozyme" means any single strand of polynucleotide that forms a secondary structure which promotes the catalytic cleavage of a target mRNA molecule once specific sequence-based recognition of the target mRNA is achieved.

The antisense oligonucleotide may be synthesized according to techniques known to those skilled in the art, such as, for example, by an automatic oligonucleotide synthesizer. The antisense oligonucleotide then is administered to a host in an amount effective to inhibit the expression of a polynucleotide encoding cyclin Gl protein in tumor cells of a host. The antisense oligonucleotide may be administered in an amount of from about O.lμM to about lOμM, preferably from about lμM to about 5μM. The host may be an animal host, and in particular a mammalian host, including human and non-human primate hosts. The antisense

oligonucleotide in general is administered to the host systemically in conjunction with an acceptable pharmaceutical carrier, such as physiological saline. Alternatively, the antisense oligonucleotides may be contained within liposomes, which are administered to the host systemically in conjunction with an acceptable pharmaceutical carrier. Such systemic administration may be, for example, by intravenous, intraarterial, or intraperitoneal administration. Alternatively, the antisense oligonucleotide may be administered directly to the tumor.

The antisense oligonucleotides may be modified in order to stabilize the oligonucleotide against degradation by nucleases and/or to enhance the ability of the antisense oligonucleotide to penetrate the tumor cells. Such modification may be accomplished by substituting at least one of the phosphodiester bonds of the antisense oligonucleotide with a structure which provideε for increased εtabilization of the antisense oligonucleotide against degradation by nucleases and/or enhances the ability of the antisense oligonucleotide to penetrate the tumor cells. Such substitutions may include phosphorothioate and phosphorodithioate bonds, phosphotriesters, alkyl or aryl phosphonate bonds, such as methylphosphonate bonds, short chain alkyl or cycloalkyl structures or short chain heteroatomic or heterocyclic structures, such as, for example, CH 2 -NH-0-CH 2 , CH 2 -N(CH 3 ) -0-CH 2 , CH 2 -0-N(CH 3 ) -CH 2 , CH 2 - N(CH 3 ) -N(CH 3 ) -CH j , and 0-N(CH 3 ) -CH 2 -CH 2 , as well as morpholino structures. Examples of such modifications are described in PCT Application No. W093/05182, published March 18, 1993, and in U.S. Patent No. 5,034,506, iεεued July 23, 1991. Examples of alkyl or aryl phosphonate bonds also are described in U.S. Patent Nos. 4,469,863 and 4,511,713. Alternatively, at least one nucleotide of the antisense oligonucleotide may be conjugated to a moiety which may be an amino acid; a dipeptide mimic, a sugar; a sugar phosphate; a

neurotransmitter; a hydrophilic polymer such as poly- hydroxypropylmethacrylamide, dextrans, polymaleic anhydride, a cyclodextrin, a starch, or polyethyleneimine. Examples of such moieties are described in PCT Application No. WO94/01448, published January 20, 1994. Further examples of moieties which may be employed in modifying the antisense oligonucleotide include, but are not limited to, alkyl- or arylphosphorates, carbamates, sulfamates, and (thio) formacetal.

The above modifications may be made to the antisense oligonucleotide during synthesis of the antisense oligonucleotide by means known to those skilled in the art. In a preferred embodiment, when the antisense oligonucleotide is administered directly or in a liposome, the antisense oligonucleotide includes at least one phosphorothioate or phosphorodithioate linker moiety, which may be attached to the backbone of the antisense oligonucleotide during synthesis by techniques known to those skilled in the art.

In another embodiment, the antisense oligonucleotide is administered to the host by transducing tumor cells of the host with a polynucleotide encoding an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein.

The polynucleotide encoding an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein may be contained within an appropriate expression vehicle which is transduced into the tumor cell. Such expression vehicles include, but are not limited to, plasmids, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors) , and viral vectors.

In one embodiment, the vector is a viral vector. Viral vectors which may be employed include RNA virus vectors (such as retroviral vectors) , and DNA virus vectors (such as adenoviral vectors, adeno-associated virus vectors, Herpes

Virus vectors, and vaccinia virus vectors) . When an RNA virus vector is employed, in constructing the vector, the polynucleotide encoding the antisense polynucleotide is in the form of RNA. When a DNA virus vector is employed, in constructing the vector, the polynucleotide encoding the antisense polynucleotide is in the form of DNA.

In one embodiment, the viral vector is a retroviral vector. Examples of retroviral vectors which may be employed include, but are not limited to, 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. The vector is generally a replication incompetent retrovirus particle.

Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.

These new genes have been incorporated into the proviral backbone in several general ways. The most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR) . Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors

one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. Alternatively, two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site.

Efforts have been directed at minimizing the viral component of the viral backbone, largely in an effort to reduce the chance for recombination between the vector and the packaging-defective helper virus within packaging cells. A packaging-de ective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itεelf.

Examples of retroviral vectors which may be employed include retroviral vectors generated from retroviral plasmid vectors derived from retroviruses including, but not limited to, Moloney Murine Leukemia Virus vectors such as those described in Miller, et al . , Biotechniques. Vol. 7, pgs. 980- 990 (1989) , and in Miller, et al . , Human Gene Therapy, Vol. 1, pgs. 5-14 (1990) .

In a preferred embodiment, the retroviral plasmid vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol. In a preferred embodiment, the retroviral plasmid vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 08/340,805, filed November 17, 1994, and in PCT Application No. W091/10728, published July 25, 1991, and incorporated herein by reference in their entireties.

When a retroviral plasmid vector including such cloning sites is employed, there may alεo be provided a εhuttle cloning vector which includeε at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector. The shuttle cloning vector also includes at least one desired gene which is capable of being tranεferred from the shuttle cloning vector to the retroviral vector.

The shuttle cloning vector may be conεtructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having endε correεponding to the reεtriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.

The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmidε εuch as pBR322; pUC 18; etc.

The retroviral plasmid vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechnicrueε, Vol. 7, No. 9, 980-990 (1989), or any other promoter ( e. g. , cellular promoterε εuch as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters) . Other viral promoters which may be employed include, but are not limited to, adenovirus promoters., TK promoters, and B19 parvovirus promoters. The selection of a

suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The retroviral plasmid vector then is employed to transduce a packaging cell line to form a producer cell line. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, xp-2 , ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ CRE, ^ CRIP, GP+E-86, GP+envAml2, and DAN cell lines, as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990) . The retroviral plasmid vector containing the polynucleotide encoding the antisense polynucleotide, which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein, transduces the packaging cells through any means known in the art. Such means include, but are not limited to, eiectroporation, the use of liposomes, and CaP0 4 precipitation.

The packaging cells thus become producer cells which generate retroviral vectors which include a polynucleotide encoding an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein. Such retroviral vectors then are transduced into the tumor cells, whereby the transduced tumor cells will produce the antisense polynucleotide, which is complementary to at least a portion of the polynucleotide encoding cyclin Gl protein.

The retroviral vectors are administered to a host in an amount which is effective to inhibit, prevent, or destroy the growth of the tumor cells through inhibition of the expression of the polynucleotide encoding cyclin Gl protein in the tumor cells. Such administration may be by systemic administration as hereinabove described, or by direct injection of the retroviral vectors in the tumor. In general, the retroviral vectors are administered in an amount of at least 1x10 s cfu/ml, and in general, such an amount does not exceed lxlO 9 cfu/ml. Preferably, the retroviral vectors are administered in an amount of from about lxlO 6 cfu/ml to

about lxlO 8 cfu/ml. The exact dosage to be administered is dependent upon a variety of factors including the age, weight, and sex of the patient, and the size and severity of the tumor being treated.

The retroviral vectors also may be administered in conjunction with an acceptable pharmaceutical carrier, such as, for example, saline solution, protamine sulfate (Elkins- Sinn, Inc., Cherry Hill, N.J.) , water, aqueouε buffers, such as phosphate buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis, MO) . The selection of a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein.

In another alternative, retroviral producer cells, such as those derived from the packaging cell lines hereinabove described, which include a polynucleotide encoding an antisenεe polynucleotide, which iε complementary to at least a portion of a polynucleotide encoding cyclin Gl protein, may be administered to a host. Such producer cells may, in one embodiment, be administered systemically (e.g., intravenously or intraarterially) at a point in close proximity to the tumor, or the producer cells may be administered directly to the tumor. The producer cell line then produces retroviral vectors including a polynucleotide encoding an antisenεe polynucleotide which is complementary to a polynucleotide encoding cyclin Gl protein, in vivo, whereby εuch retroviral vectorε then transduce the tumor cells.

In another embodiment, the agent which inhibits cyclin Gl protein is an antagonist to cyclin Gl protein which binds to and inhibitε cyclin Gl protein. Exampleε of antagonists to cyclin Gl protein include, but are not limited to, antibodies or fragments or derivatives thereof which recognize cyclin Gl protein, and small moleculeε, εuch as, for example, cyclin-dependent kinase inhibitors which bind to and inhibit the function of cyclin Gl protein.

In one embodiment the antagonist is an antibody or fragment or derivative thereof which recognizes cyclin Gl protein. The term "fragment or derivative thereof," means an antibody having deletions and/or subεtitutions of amino acid residues with respect to the unmodified antibody, yet such fragment or derivative recognizes cyclin Gl protein. Such antibody may be a monoclonal or polyclonal antibody. In one embodiment, the antibody is a single chain antibody.

Preferably, the antibody is administered to the host such that the antibody or fragment or derivative thereof enters the tumor cells. In a preferred embodiment, the antibody or fragment or derivative thereof which recognizes cyclin Gl protein is administered to the host by transducing tumor cells of the host with a polynucleotide encoding the antibody or fragment or derivative thereof which recognizes cyclin Gl protein. The polynucleotide may be contained in an appropriate expression vehicle such as those hereinabove deεcribed. In one embodiment, the polynucleotide is contained in a retroviral vector, which may be a retroviral vector as hereinabove described.

The vector, which includes the polynucleotide encoding an antibody or fragment or derivative thereof which recognizes cyclin Gl protein is administered to the hoεt in an amount effective to inhibit the function of the cyclin Gl protein in the tumor cellε in the hoεt. When a retroviral vector iε employed, such retroviral vector is administered in an amount of from about lxlO 6 cfu/ml to about 1x10 s cfu/ml. Such vector may be administered systemically (such as, for example, by intravenous, intraarterial, or intraperitoneal administration) or, alternatively, the vector may be administered directly to the tumor. The vectors then transduce the tumor cells, whereby the antibody or fragment or derivative thereof which recognizes cyclin Gl protein is expressed in the tumor cells. Such antibody or fragment or derivative thereof will bind to the cyclin Gl protein in the

tumor cells, thereby inhibiting the function of the cyclin Gl protein in the tumor cells.

Tumors which may be treated in accordance with the present invention, through the inhibition of cyclin Gl protein, include non-malignant, as well as malignant, or cancerous tumors . Cancerous tumors which may be treated include, but are not limited to, osteogenic εarcoma and Ewing'ε εarcoma and other neoplaεtic disorders in which cyclin Gl is expressed, such as, glioblastoma, neuroblaεtoma, breast cancer, prostate cancer, leukemias, lymphomaε (including Hodgkin's and non-Hodgkin' ε lymphoma) , fibroεarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, liver cancers such as hepatocellular carcinoma, and adenocarcinomas .

The above treatments, in which cyclin Gl is inhibited, also may be employed in combination with other treatments of tumors, such as, for example, (i) radiation; (ii) chemotherapy; or (iii) the transduction of the tumor cells with a polynucleotide encoding a negative selective marker, such as, for example, a viral thymidine kinase gene, followed by the administration of an interaction agent, such as, for example, ganciclovir, which kills the cells transduced with the polynucleotide encoding the negative εelective marker.

In one embodiment, an agent which inhibits cyclin Gl protein is administered to a host in accordance with one of the methods hereinabove described. The growth of any tumor cells which contain the agent will be inhibited, prevented or destroyed. In addition, the tumor cells are tranεduced with a polynucleotide encoding a negative εelective marker or "εuicide" gene. The polynucleotide encoding the negative selective marker may be contained in an expreεsion vehicle such as those hereinabove described. Once the tumor cells are transduced with the polynucleotide encoding the negative selective marker, an interaction agent is administered to the host, whereby the interaction agent interacts with the

negative εelective marker in order to prevent, inhibit, or destroy the growth of the tumor cells.

Negative selective markers which may be employed include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase; and cytosine deaminase.

In one embodiment, the negative selective marker is a viral thymidine kinase selected from the group consisting of Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase. When such viral thymidine kinases are employed, the interaction or chemotherapeutic agent preferably is a nucleoside analogue, for example, one selected from the group consisting of ganciclovir, acyclovir, and 1-2-deoxy-2-fluoro- jS-D-arabinofuranosil-5-iodouracil (FIAU) . Such interaction agents are utilized efficiently by the viral thymidine kinases as substrates, and such interaction agentε thuε are incorporated lethally into the DNA of the tumor cells expressing the viral thymidine kinases, thereby resulting in the death of the tumor cells.

In another embodiment, the negative εelective marker iε cytosine deaminase. When cytosine deaminase is the negative selective marker, a preferred interaction agent iε 5- fluorocytoεine. Cytoεine deaminaεe convertε 5-fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, the tumor cells which express the cytosine deaminase gene convert the 5-fluorocytosine to 5-fluorouracil and are killed.

The interaction agent is administered in an amount effective to inhibit, prevent, or destroy the growth of the transduced tumor cells. For example, the interaction agent may be administered in an amount from 5 mg to 10 mg/kg of body weight, depending on overall toxicity to a patient.. The interaction agent preferably is administered systemically, such as, for example, by intravenous administration, by

parenteral administration, by intraperitoneal administration, or by intramuscular adminiεtration.

When an expreεsion vehicle, such as those hereinabove described, including a negative selective marker is administered to tumor cells, a "bystander effect" may result, i.e., tumor cells which were not originally tranεduced with the nucleic acid εequence encoding the negative selective marker may be killed upon administration of the interaction agent. Although Applicants do not intend to be limited to any theoretical reasoning, the transformed tumor cells may be prodticing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent, or is taken up by adjacent, non-transformed tumor cells, which then become susceptible to the action of the interaction agent. It also is possible that one or both of the negative selective marker and the interaction agent are communicated between tumor cells.

Agents which inhibit cyclin Gl protein alεo may prevent vaεcular restenosis after invasive vascular procedures such as angioplaεty, vascular graftε, εuch as arterial grafts, or coronary bypasε εurgery. Thuε, in accordance with another aspect of the present invention, there is provided a method of preventing restenoεiε which compriεeε adminiεtering to a hoεt, or to the site of an invaεive vaεcular procedure or vaεcular leεion, an agent which inhibits cyclin Gl protein. The agent is administered in an amount effective to prevent restenosis in a host. The agent may be administered during or after the invasive vascular procedure. The term "invasive vascular procedure" as uεed herein meanε any procedure which involves repair, removal, replacement and/or redirection (e.g., bypass or shunt) of a portion of the vascular system including but not limited to arteries and veins. Such procedures include, but not limited to, angioplasty, vascular grafts such as arterial grafts, removals of blood clots,

removals of portions of arteries or veins, and coronary bypass surgery.

Agents which inhibit cyclin Gl protein which may be employed include, but are not limited to, those hereinabove described. Preferably, the agent which inhibits cyclin Gl protein is an antisense polynucleotide which is complementary to, and thus is capable of binding or hybridizing to, at least a portion of a polynucleotide encoding cyclin Gl protein as hereinabove described. Such antisense oligonucleotide may have a length as hereinabove deεcribed and be administered in an amount effective to prevent restenosis. Such amount may be as hereinabove described. The antisense oligonucleotide may be administered intravascularly and may be administered directly to the site of the invasive vascular procedure or the vascular lesion.

In a preferred embodiment, the antisenεe oligonucleotide iε adminiεtered to the host by transducing vascular cells at the site of an invasive vascular procedure or a vascular lesion with a polynucleotide encoding an antisense polynucleotide which is complementary to at least a portion of a polynucleotide encoding cyclin Gl protein. Such polynucleotide encoding the antisense polynucleotide may be contained in an appropriate expression vehicle as hereinabove described, which is transduced into the cells of the εite of an invaεive vascular procedure or vaεcular leεion. In one embodiment, the expression vehicle is a viral vector εuch as thoεe hereinabove described. In one embodiment, the viral vector is a retroviral vector, which may be as hereinabove described.

When a retroviral vector is employed, such retroviral vector is administered in an amount hereinabove described, and is administered intravascularly. In one embodiment, the retroviral vector is administered to the site of the invasive vascular procedure or vascular lesion. The vectors transduce the vascular cells at the site of the invasive vascular

procedure or vascular lesion, whereby the antisense oligonucleotide is produced in such cells, thereby inhibiting the expression of a polynucleotide encoding cyclin Gl in such cells and thus preventing restenosiε by preventing the proliferation of εuch cellε.

In another embodiment, the agent which inhibits cyclin Gl protein is an antagonist to cyclin Gl protein which binds to and inhibits cyclin Gl protein as hereinabove described, and in one embodiment may be an antibody or fragment or derivative thereof which recognizes cyclin Gl protein.

The antibody is administered to the host such that the antibody or fragment or derivative thereof enters the cells of the site of the invasive vascular procedure or vascular lesion. Preferably, the antibody or fragment or derivative thereof which recognizes cyclin Gl protein is administered by transducing cellε at the site of the invasive vascular procedure or of a vascular leεion with a polynucleotide encoding the antibody or fragment or derivative thereof which recognizeε cyclin Gl protein. The polynucleotide may be contained in an appropriate expression vehicle such as those hereinabove described. In one embodiment, the expression vehicle is a retroviral vector as hereinabove deεcribed, which may be adminiεtered in an amount as hereinabove described. Such vector is administered intravascularly as hereinabove described, and may be administered directly to the site of an invasive vascular procedure or vascular lesion.

This method is applicable to the prevention and treatment of restenoεiε and the prevention or treatment of vaεcular leεionε following a variety of invasive vascular procedures, including but not limited to, cardiovascular angioplasty, arterial grafts, and coronary bypasε εurgery. This method also applies to the prevention and treatment of vascular lesions including, but not limited to, lesions of

the femoral, carotid, or renal arteries, particularly renal arteries associated with renal dialysiε fiεtulaε.

In accordance with another aεpect of the present invention, there iε provided a method of immortalizing non- tumor cells which compriεes transducing the non-tumor cells with a polynucleotide encoding cyclin Gl protein or a derivative or analogue thereof. The term "derivative or analogue thereof" as used herein means that the protein may be a protein which has deletions and/or substitutions of amino acid residues with respect to the native cyclin Gl protein sequence, yet retains the same biological properties as native, or unmodified cyclin Gl protein. Although the scope of this aspect of the present invention is not intended to be limited to any theoretical reasoning, Applicants have discovered that overexpression of cyclin Gl protein in non- tumor cells, would contribute to cell immortalization and permanent cell lines that would retain the ability to respond to subsequent cell cycle events, and avoiding the use of viral oncogenes which cause cell transformation.

The polynucleotide encoding cyclin Gl protein or a fragment or derivative thereof may be contained in an appropriate expression vehicle, which may be as hereinabove described. In one embodiment, the expresεion vehicle iε a retroviral vector, which may be aε hereinabove deεcribed.

Non- umor cells which may be transduced in accordance with this aspect of the present invention include, but are not limited to, fibroblasts, hepatocyteε, muscle cells, endothelial cells, and epithelial cells.

In accordance with yet another aspect of the present invention, there is provided a method of enhancing transduction of cells with a retroviral vector. The method comprises transducing the cells with a first expreεsion vehicle including a polynucleotide encoding cyclin Gl protein. The first expression vehicle is not a retroviral vector. The cells also are transduced with a second

expression vehicle which preferably includes a polynucleotide encoding a therapeutic agent. The second expression vehicle is a retroviral vector. This method can be uεed to tranεduce cellε in vivo or ex vivo or in vi tro.

The firεt expreεεion vehicle may be any expresεion vehicle which is not a retroviral vector. Such expreεεion vehicleε include, but are not limited to, plasmid vectors, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors) , and viral vectors other than retroviral vectors, including, but not limited to, adenoviral vectors, adeno-asβoσiated virus vectors, Herpes virus vectors, and vaccinia virus vectors.

In a preferred embodiment, the first expression vehicle is an adenoviral vector. Although this embodiment is not to be limited to any theoretical reasoning, cyclin Gl protein is induced in very early Gl phase, when cell activation occurs. The transduction of the cells with an adenoviral vector including a polynucleotide encoding cyclin Gl protein provides transient overexpression of cyclin Gl protein in the cells, thereby activating the cells, and enabling increased integration of the retroviral vector including the polynucleotide encoding the therapeutic agent into the cells. Such method is applicable particularly to the introduction of retroviral vectors into cells with low replication indices and low transduction efficiency.

The adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome (Shenk et al. , Curr. TOP. Microbiol. Immunol., 111(3) : 1-39 (1984) . Alternatively, the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome haε been deleted.

In the preferred embodiment, the adenoviral vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding

cyclin Gl protein; and a promoter controlling the DNA sequence encoding cyclin Gl protein. The vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins promoted by the adenoviral major late promoter.

In one embodiment, the vector also is free of at least a portion of at least one DNA sequence selected from the group consisting of the E2 and E4 DNA sequences.

In another embodiment, the vector is free of at leaεt the majority of the adenoviral El and E3 DNA sequences, and iε free of a portion of the other of the E2 and E4 DNA sequences.

In still another embodiment, the gene in the E2a region that encodes the 72 kilodalton binding protein iε mutated to produce a temperature sensitive protein that is active at 32°C, the temperature at which the viral particles are produced. This temperature sensitive mutant is described in Ensinger et al . , J. Virology. 10:328-339 (1972) , Van der Vliet et al . , J. Virology, 15:348-354 (1975), and Friefeld et al . , Virology. 124:380-389 (1983) .

Such a vector, in a preferred embodiment, is constructed first by constructing, according to standard techniques, a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elementε, " which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter) ; a multiple cloning site (which may be aε herein described) ; a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome. The vector also may contain a tripartite leader sequence. The DNA segment corresponding to the adenoviral genome serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such sequence may encompass, for example, a segment of the adenovirus 5 genome no longer than

from base 3329 to base 6246 of the genome. The plasmid may also include a selectable marker and an origin of replication. The origin of replication may be a bacterial origin of replication. Representative examples of such shuttle plasmidε include pAvS6, which iε deεcribed in publiεhed PCT Application Noε . W094/23582, publiεhed October 27, 1994, and W095/09654, publiεhed April 13, 1995. The DNA sequence encoding cyclin Gl protein may then be inserted into the multiple cloning site to produce a plasmid vector.

This conεtruct iε then used to produce an adenoviral vector. Homologous recombination is effected with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequenceε have been deleted. Such homologous recombination may be effected through co¬ transfection of the plasmid vector and the modified adenovirus into a helper cell line, εuch aε 293 cellε, by CaP0 4 precipitation. Upon such homologous recombination, a recombinant adenoviral vector iε formed that includes DNA sequenceε derived from the εhuttle plaεmid between the Not I εite and the homologous recombination fragment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.

In one embodiment, the homologouε recombination fragment overlaps with nucleotides 3329 to 6246 of the adenovirus 5 (ATCC VR-5) genome.

Through εuch homologous recombination, a vector is formed which includeε an adenoviral 5' ITR, an adenoviral encapεidation εignal; an Ela enhancer aequence; a promoter; a DNA sequence encoding cyclin Gl protein protein; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences; and an adenoviral 3' ITR. The vector also may include a tripartite leader sequence. The vector may then be transfected into a helper cell line, such aε the 293 helper cell line (ATCC No. CRL1573) , which will include the Ela and Elb DNA εequenceε, which are

necessary for viral replication, and to generate adenoviral particles. Transfection may take place by eiectroporation, calcium phosphate precipitation, microinjection, or through proteoliposomes .

The vector hereinabove described may include a multiple cloning site to facilitate the insertion of the DNA sequence encoding the cyclin Gl protein into the cloning vector. In general, the multiple cloning site includes "rare" restriction enzyme sites; i.e., sites which are found in eukaryotic genes at a frequency of from about one in every 10,000 to about one in every 100,000 base pairs. An appropriate vector is thus formed by cutting the cloning vector by standard techniques at appropriate restriction εiteε in the multiple cloning εite, and then ligating the DNA sequence encoding cyclin Gl protein into the cloning vector.

The DNA sequence encoding cyclin Gl protein is under the control of a suitable promoter, which may be selected from those herein described, or such DNA may be under the control of its own native promoter.

In one embodiment, the adenovirus may be conεtructed by uεing a yeast artificial chromosome (or YAC) containing an adenoviral genome according to the method described in Ketner, et al . , PNAS. Vol. 91, pgs. 6186-6190 (1994) , in conjunction with the teachings contained herein. In this embodiment, the adenovirus yeast artificial chromosome is produced by homologous recombination in vivo between adenoviral DNA and yeast artificial chromosome plaεmid vectorε carrying segmentε of the adenoviral left and right genomic termini. A DNA sequence encoding cyclin Gl protein then may be cloned into the adenoviral DNA. The modified adenoviral genome then is excised from the adenovirus yeast artificial chromosome in order to be used to generate adenoviral vector particles as hereinabove described.

The retroviral vector, which is the second expresεion vehicle, may be as hereinabove described. Such retroviral

vector includes a polynucleotide encoding a therapeutic agent. The term "therapeutic" is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents.

Polynucleotides encoding therapeutic agents which may be contained in the retroviral plasmid vector include, but are not limited to, polynucleotides encoding tumor necrosis factor (TNF) genes, such as TNF-α; genes encoding interferons such as Interferon-α, Interferon-?, and Interferon-γ; genes encoding interleukins such aε IL-1, IL-13, and Interleukinε 2 through 14; genes encoding GM-CSF; genes encoding adenosine deaminase, or ADA; genes which encode cellular growth factors, εuch aε lymphokineε, which are growth factors for lymphocyteε; geneε encoding epidermal growth factor (EGF) , and keratinocyte growth factor (KGF) ; geneε encoding εoluble CD4; Factor VIII; Factor IX; cytochrome b; glucocerebrosidase; T-cell receptors; the LDL receptor, ApoE, ApoC, ApoAI and other genes involved in cholesterol transport and metabolism; the alpha-1 antitrypsin (αlAT) gene; the insulin gene; the hypoxanthine phosphoribosyl transferase gene; the CFTR gene; negative selective markers or "suicide" genes, such as viral thymidine kinase genes, such as the Herpes Simplex Virus thymidine kinase gene, the cytomegalovirus virus thymidine kinase gene, and the varicella-zoster virus thymidine kinase gene; Fc receptors for antigen-binding domainε of antibodieε, antisense sequenceε which inhibit viral replication, such as antiεenεe εequences which inhibit replication of hepatitis B or hepatitis non-A non-B virus; antisense c-myb oligonucleotides; and antioxidants such as, but not limited to, manganese superoxide dismutase (Mn-SOD) , catalase, copper-zinc-superoxide dismutase (CuZn-SOD) , extracellular superoxide dismutase (EC-SOD) , and giutathione reductase; tissue plasminogen activator (tPA) ; urinary plasminogen activator (urokinase) ; hirudin; the phenylalanine hydroxylase

gene; nitric oxide synthetase; vasoactive peptides; angiogenic peptides; the dopamine gene; the dystrophin gene; the 3-globin gene; the α-globin gene; the HbA gene; protooncogeneε such as the ras, src, and bcl genes; tumor- suppressor genes εuch aε p53 and Rb; the LDL receptor; the heregulin-α protein gene, for treating breaεt, ovarian, gastric and endometrial cancers; monoclonal antibodies specific to epitopes contained within the 3-chain of a T-cell antigen receptor; the multi-drug resiεtance (MDR) gene; polynucleotides encoding ribozymes; antisense polynucleotides; genes encoding εecretory peptidec which act as competitive inhibitors of angiotensin converting enzyme, of vascular smooth muscle calcium channels, or of adrenergic receptors, and polynucleotides encoding enzymes which break down amyloid plaques within the central nervous system. It is to be understood, however, that the scope of the present invention is not to be limited to any particular therapeutic agen .

The polynucleotide encoding the therapeutic agent is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; the histone promoter; the polIII promoter, the /3-actin promoter; inducible promoters, such as the MMTV promoter, the metallothionein promoter; heat shock promoters; adenovirus promoters; the albumin promoter; the ApoAI promoter; B19 parvovirus promoters; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; human growth hormone promoters, and the MxIFN inducible promoter. The promoter also may be the native promoter which controls the polynucleotide encoding the therapeutic agent. It is to be understood, however, that the scope of the present invention is not to be limited to specific foreign genes or promoters.

The first expression vehicle, which preferably is an adenoviral vector, which includes a DNA sequence encoding cyclin Gl protein or an analogue derivative thereof, and the retroviral vector, which includes a polynucleotide encoding therapeutic agent, may transduce cells in vivo or in vitro.

In one embodiment the cells are transduced with the first expression vehicle, which preferably is an adenoviral vector, prior to transduction of the cells with the second expresεion vehicle (i.e., the retroviral vector) . In another embodiment, the cells are transduced with the first expression vehicle and the second expression vehicle concurrently.

When administered in vivo, the adenoviral vector is administered in an amount effective to transduce the desired cells with the polynucleotide encoding cyclin Gl protein. The adenoviral vector may be administered syεtemically, εuch aε, for example, by intravenous, intraarterial, or intraperitoneal administration. Altematively, the adenoviral vector may be administered by direct, nonsystemic injection to a deεired tissue, organ, or mass of cells, such as, for example, a tumor. In general, the adenoviral vector is administered at a multiplicity of infection of from about 1 to about 10.

The retroviral vector is administered to the animal host in vivo in an amount effective to produce a therapeutic effect in the animal.

The animal may be a mammal, including human and non¬ human primates. The retroviral vectors may be adminiεtered εyεtemically, for example, intravenously or intraarterially or intraperitoneally, or by direct nonsystemic injection into a desired tissue, organ or mass of cellε, εuch aε, for example, a tumor.

The retroviral vectorε are administered to an animal in an amount effective to produce a therapeutic effect in the animal. In general, the retroviral vectors are administered

in an amount of at least 1x10 s cfu/ml, and in general such amount does not exceed lxlO 9 cfu/ml. Preferably, the retroviral vectors are administered in an amount of from about 1x10 s cfu/ml to about lxlO 8 cfu/ml. The exact dosage to be administered is dependent upon various factors, including the age, height, weight, and sex of the patient, the diεorder being treated, and the severity thereof.

The retroviral vectors and the adenoviral vectors each are administered to the patient in a pharmaceutically acceptable carrier, such as, for example, a physiological saline solution. Other pharmaceutical carriers include, but are not limited to, mineral oil, alum, and lipid vesicles such as liposomes. The selection of a suitable pharmaceutical carrier is deemed to be within the scope of those skilled in the art from the teachings contained herein.

In one embodiment, the eukaryotic cells which are transduced in vivo with the retroviral and adenoviral vectors are primary human cells. The gene encoding a therapeutic agent can be any gene having clinical usefulnesε, for example, therapeutic or marker geneε. Preferably, the primary human cells are blood cells. The term "blood cells" as used herein is meant to include all forms of nucleated blood cells as well as progenitors and precursors thereof.

The gene carried by the blood cells can be any gene which directly enhances the therapeutic effects of the blood cells. The gene carried by the blood cells can be any gene which allows the blood cells to exert a therapeutic effect that it would not ordinarily have, such as a gene encoding a clotting factor (e.g., Factor VIII or Factor IX) useful in the treatment of hemophilia. The gene can encode one or more products having therapeutic effects. Examples of suitable genes include those that encode cytokines such as TNF, interleukins (interleukins 1-12) , interferons (α, β, γ- interferons) , T-cell receptor proteins and Fc receptors for binding to antibodies.

The retroviral vectors are useful in the treatment of a variety of diseaseε including but not limited to adenoεine deaminase deficiency, sickle cell anemia, thalassemia, hemophilia, diabetes, α-antitrypsin deficiency, brain disorders such as Alzheimer'ε diεeaεe, and other illnesses such as growth diεorderε and heart diεeaεeε, for example, thoεe cauεed by alterations in the way cholesterol is metabolized and defectε of the immune εystem.

In one embodiment, the retroviral vectors may include a negative selectable marker, such as, for example, a viral thymidine kinase gene, and more particular y,, the Herpes Simplex Virus thymidine kinaεe (TK) gene. Such retroviral vectorε may be adminiεtered in conjunction with the adenoviral vectors hereinabove described to tumor cells (in particular to cancer cells) in a human patient in vivo . The adenoviral vectors and the retroviral vectors then transduce the tumor cells. After the retroviral vectors have transduced the tumor cells, the patient is given an interaction agent, such as gancyclovir or acyclovir, which interacts with the protein expressed by the negative selectable marker in order to kill all replicating cells (i.e., the tumor cells) which were transduced with the retroviral vector including the negative selectable marker.

The adenoviral vectors and the retroviral vectors mentioned hereinabove also may be administered in an animal model for determining the effectiveneεε of a gene therapy treatment. For example, an adenoviral vector including a polynucleotide encoding cyclin Gl protein and a retroviral vector including a polynucleotide encoding a therapeutic agent, may be administered to animals of the εame species. The retroviral vector is administered to the animals in varying amounts. From determining the effectiveneεε of the gene therapy treatment in the animal, one may determine an effective amount of the retroviral vector to be administered to a human patient.

In another embodiment, the adenoviral vectors, which include a DNA sequence encoding cyclin Gl protein, are administered in vivo to a patient in conjunction with retroviral producer cells which generate retroviral vectors including a polynucleotide encoding a therapeutic agent.

Such an embodiment is applicable particularly to the treatment of tumors (including malignant and non-malignant tumors) such as, for example, liver tumors, bone tumors, and lung tumors. For example, the producer cells may include a retroviral plasmid vector including a negative selectable marker. The adenoviral vectors and the retroviral producer cells then are administered to the tumor, whereby the producer cells generate retroviral vector particles including the polynucleotide encoding the negative selectable marker. The adenoviral vectors and the retroviral vector particles generated by the retroviral producer cells transduce the tumor cells, whereby the tumor cells produce the negative selectable marker. Upon administration of an interaction agent to the patient, the transduced tumor cells are killed.

Altematively, the adenoviral vectors and the retroviral vector may transduce eukaryotic cells, in vi tro, whereby the eukaryotic cellε are cultured in vi tro for the in vi tro production of the therapeutic agent, or, altematively, the transduced eukaryotic cells may be adminiεtered to a host as part of a gene therapy procedure, whereby the transduced eukaryotic cells express the therapeutic agent in vivo in a host.

As stated hereinabove, the above methods of the present invention may be accomplished through the use of appropriate expression vehicles containing either a polynucleotide encoding an agent which inhibits cyclin Gl protein (when one desireε to treat a tumor by inhibiting cyclin Gl protein) , or a polynucleotide encoding cyclin Gl protein (when one deεires to immortalize a cell line or enhance retroviral transduction of cells) . Thus, in accordance with another aspect of the

present invention, there is provided an expression vehicle which includes a polynucleotide encoding an agent which, in one embodiment, is an agent which inhibits cyclin Gl protein. Such agentε include thoεe hereinabove deεcribed, such as, for example, antisense polynucleotides or antibodies or fragments or derivatives thereof which recognize cyclin Gl protein, or a cyclin-dependent kinase inhibitor. In another embodiment, the polynucleotide encodes cyclin Gl protein.

The expresεion vehicle may be εelected from those hereinabove described, and preferably may be a viral vector, including PNA virus vectors and DNA virus vectors as hereinabove described.

In one embodiment, the viral vector is an RNA virus vector, and preferably is a retroviral vector, such as those hereinabove deεcribed. In another embodiment, the viral vector is a DNA virus vector, and preferably is an adenoviral vector, such as those hereinabove deεcribed.

In accordance with a further aεpect of the present invention, there is provided a method of detecting cancer by detection of increased expression of cyclin Gl protein, with such increased expression being detected by detecting increased amounts of polynucleotides encoding cyclin Gl protein, or by detecting increased amounts of cyclin Gl protein, as compared with normal, non-cancerous cells. The method comprises contacting cells with an agent which binds to (i) cyclin Gl protein and/or (ii) a polynucleotide encoding cyclin Gl protein. Binding of the agent to cyclin Gl protein and/or a polynucleotide encoding cyclin Gl protein then is determined.

The cyclin Gl protein is expresεed intracellularly, and to assay for the increased expression of cyclin Gl protein, appropriate procedures are employed prior to contacting the cells with agent which binds to cyclin Gl protein and/or a polynucleotide encoding cyclin Gl protein, to enable binding of the agent in the assay. Such procedures include, but are

not limited to, the fixation of a hiεtological εample of cells prior to the assay.

Agents which may be employed in this aspect include, but are not limited to, polynucleotides (e.g., DNA or RNA probes) which hybridize to a polynucleotide encoding cyclin Gl protein, and antibodies or fragments or derivatives thereof which recognize cyclin Gl protein.

In one embodiment, the agent is a polynucleotide which hybridizes to a polynucleotide encoding cyclin Gl protein.

In another embodiment, the agent is an antibody or fragment or derivative thereof which recognizes cyclin G protein. Such antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and single chain antibodies.

Certain propertieε of cancer may be determined through the analyεiε of the amount of binding to cyclin Gl protein expressed in the cells, or the amount of binding to a polynucleotide encoding cyclin Gl protein present in the cells. A determination an elevated amount of binding of the agent to cyclin Gl protein as compared to that observed in normal cells, or to a polynucleotide encoding cyclin Gl protein may be indicative of the presence of cancer cells. Cancers which may be determined in accordance with this method include osteogenic εarcoma and Ewing's sarcoma, and other neoplastic disorders in which cyclin Gl is expressed, such as those hereinabove described.

The determination of binding of the agent to cyclin Gl protein or to a polynucleotide encoding cyclin Gl protein may be determined by a variety of assay methods known to those skilled in the art. Such assays include, but are not limited to, direct and indirect sandwich assays, colorimetric assays and ELISA assays.

In the above assays, the agent which binds to the cyclin Gl protein or to the polynucleotide which encodes cyclin Gl protein, or a binder which binds to the agent when an

indirect sandwich assay is employed, is coupled to a detectable label or marker. Such labels or markers include, but are not limited to, radioactive isotopes of, for example, iodine, cobalt or tritium; an enzyme; a fluorescent dye; an absorbing dye; a chemiluminiscent substance; a spin label; biotin; hematoxylin; a colored particle or any other labeling subεtance known to one εkilled in the art.

In one embodiment, fixed cellε, εuεpected of being cancer cellε, are contacted with an antibody which recognizeε cyclin Gl protein. Detection of bound antibody may be determined by an indirect sandwich assay employing a biotin- avidin complex, such as a biotin-streptavidin complex which is bound to the antibody. The avidin is bound to an enzyme, such as, for example, alkaline phosphatase. The sample is contacted with a substrate for the enzyme, which produces a colored reaction product. By measuring the development of the colored reaction product, the amount of cyclin Gl protein in the sample of cells may be determined, thereby determining the presence of cancer and/or the extent and severity thereof.

EXAMPLES

The invention now will be described with respect to the following exampleε, however, the scope of the present invention is not intended to be limited thereby.

Example 1 MATERIALS AND METHODS Cloning of antisense cyclin Gl, antisense cyclin Dl, and p21/WAFl/CIPl Expression Constructs.

The full coding regions of human cyclin Gl (Figure 1)

(Wu, et al . , Oncol. Reports. Vol. 1, pgs. 705-711 (1994)) , cyclin Dl (Xiong, et al . , Cell. Vol. 65, pgs. 691-699

(1991)) , and p21/WAFl/CIPl (Harper, et al., Mol. Biol. Cell..

Vol. 6, pgs. 387-400 (1995); El-Deiry, e al., Cell. Vol. 75, pgs. 817-825 (1993)), including the stop codonε, were

prepared by primer-directed RT-PCR amplification. To create the antisense cyclin Gl (aGl) expression construct, the 586 bp N-terminal fragment, including -65 bp of the untranslated region, was released by double digestion with Xbal/Hpal from the CYCG1 gene originally isolated from a human WI-38 fibroblast (ATCC, Rockville, Maryland) cDNA library, and then cloned by blunt ligation into the pcDNA3 vector (Invitrogen, San Diego, California) at the EcoRV site. The 605 N-terminal region of cyclin Dl (antisense orientation, aDl) and the 495 bp full coding region of WAF1/CIP1 (p21) were released by digestion with Ndel/Ncol and Ndel/EcoRI, respectively, followed by blunt end cloning into the pcDNA3 vector at the EcoRV site. The structure of each construct was confirmed by manual DNA sequence analysiε, uεing a modified dideoxy chain termination method (United Stateε Biochemicalε) .

Construction of Retroviral Vectors Bearing Cell Cycle Control Genes (GlaGlSvNa, GlaDlSvNa, and Glp21SvNa: Retroviral Vector Source, pGlXSvNa; Insert Source, pcDNA3aGl, pcDNA3aDl, pcDNA3p21) .

To create each retroviral vector, pGlXSvNa (Genetic Therapy, Inc., Gaithersburg, Maryland) was digested with NotI, the 5' phosphates were removed by treatment with calf intestinal alkaline phosphatase, and the resulting fragment was then gel-purified (1% agarose) , excised, and electroeluted. pGlXSvNa is a retroviral plasmid vector derived from pGl (described in PCT Application No. WO91/10728 published July 25, 1991), and which includes a retroviral 5' LTR, a retroviral 3' LTR, a multiple cloning region and a neomycin resistance gene under control of the SV40 promoter. pGlXSvNa is described further in PCT Application No. WO95/09654, published April 13, 1995. This procedure generated a 5856 bp long fragment of DNA which cannot relegate or re-circularize. To isolate the aGl, aDl and p21 insert fragments, the respective plasmid DNAs were double

digested with Hindi11/NotI for aGl and EcoRI/NotI for aDl and p21, respectively. These digestε were reεolved on 1% agarose gels yielding the 597 bp Hindlll/NotI fragment of aGl, the 632 bp EcoRI/NotI fragment of aDl, and the 522 bp EcoRI/NotI fragment of p21. These bands were then excised from the agarose gels and electroeluted. The NotI end of each insert was ligated to the NotI end of the digested pGlXSvNa vector, and isolated on 1% agarose gels yielding 6453, 6488 and 6378 bp long fragments for aGl, aDl and p21 respectively. Each fragment was then electroeluted and treated with the Klenow fragment to generate blunt ends, and then ligated to generata closed plasmid DNA including the respective genes of interest. Succeεεful cloning and inεert orientation were determined by restriction analysis. The expected DNA fragments generated by digestion with BεtEII and NotI were 920, 955 and 845 bp for aGl, aDl and p21 inεertε reεpectively, and ± 5500 bp for the vector DNA.

Retroviral Vector Supernatants and Producer Cell Lines.

The β galactoεidase and HStk expresεion vectorε were kindly provided as high titer PA317 packaging cell clones (titers: 1.3 x 10 s and 4.9 X 10 s G418 r colony-forming units, cfu/ml for β galactosidase and HStk vectors respectively) by Genetic Therapy, Inc. (Gaithersburg, MD) . The 3 experimental retroviral plasmid vectors bearing cell cycle control enzyme cDNAs were packaged in PA317 cellε (Miller, et al . , Mol. Cell Biol. , Vol. 6, pgε. 2895-2902 (1986)) and teεted as pooled vector supernatants (vector titer: 1 X 10 s cfu/ml each) . The vectors are referred to as GIBgSvNa, GlTKlSvNa.7, Glp21SvNa, GlaDlSvNa and GlaGlSvNa to indicate the order of promoters and coding regions contained in each vector (Gl vector, Moloney Murine Leukemia Virus long terminal repeat (LTR) sequences; Bg, β galactosidase or lacZ gene; HStk, Herpes Simplex thymidine kinase gene; aGl, antisense human cyclin Gl; aDl, antisense cyclin Dl; p21, Cdk inhibitor

p21/Wafl/Cipl gene; Sv, SV40 early region enhancer/promoter; and Na.7, neo τ gene, clone 7) . Retroviral vector GITKlSvNa 7 is described further in PCT Application No. WO95/09654, published April 13, 1995. Retroviral vector GIBgSvNa was generated from the plasmid pGlBgSvNa. pGlBgSvNa was constructed by digesting pSvNa (PCT Application No. WO95/09654) and pGlBg (PCT Application No. WO91/10728) with Sail and Hindlll. The Sall-Hindlll fragment of pSvNa containing the SV40 promoter and a neomycin resistance gene was ligated to the Sall/Hindlll digested pGlBg to form pGlBgSvNa.

The vector source, GlXSvNa, containing only the SV40 promoter-driven neo τ gene was used as a control for the effects of gene transduction and G418 selection.

Cells, Cell Culture Conditions and Transduction of Cells with la. Z , Cell Cycle Control Genes, and HStk Vectors.

Human osteogenic sarcoma (MG-63, ATCC No. CRL 1427) cells and primary normal diploid human fibroblasts (of hepatic origin) were cultured at a plating density of 2.5 x 10* cells in each of six-well plates, in DMEM supplemented with 10% FBS (D10) . After overnight attachment, the cells were exposed to 1 ml of the respective retroviral vector in the presence of Polybrene (8 μg/ml) for 2 hours, after which 1 ml of fresh D10 was added to each well. Forty-eight hours after transduction with the lacZ vector, gene transfer efficiency was measured by determining the percentage of lacZ positive cells, upon X-gal εtaining and light microεcopy.

Ganciclovir (GCV) Cytotoxicity/Bystander Effects in HStk Vector Transduced MG-63 Cells.

Initial dose-response studies determined the senεitivity of MG-63 cellε and the optimal concentrationε of G418 uεed to εelect transduced cells. Upon G418 selection, varying proportions of HStk-transduced and non-transduced MG-63 cells

(plating density 2.5 X 10* cells) were exposed to 20 μg GCV/ml D10 in each of εix-well plateε, for 10 dayε. Hence, the bystander effects of GCV in HStk-transduced MG-63 were meaεured by determining the degree of confluency of cellε in each well in 10 day cultures. Bystander effects of GCV treatment were compared to those in HStk-transduced NIH 3T3 cells (ATCC No. CRL 1658) .

Evaluation of Cell Growth, Protein Expression, and Apoptosis in MG-63 Cells Bearing Chimeric Retroviral Vectors.

To assess the cytostatic effects of retroviral vectors bearing cell cycle modulators, the cells that were transduced with control vectors or vectors expreεεing cell cycle modulatorε were evaluated for their proliferative potential by counting the number of viable cells in each culture at εerial intervalε (0, 24, 48, 72, 144 and 192 hrε) after transduction. Western analysis of protein expression was performed as described previously (Williams, et al . , J. Biol. Chem.. Vol. 268, pgs. 8871-8880 (1993); Wu, et al . , Oncol. Reports, Vol. 2, pgs. 227-231 (1995)) , using a polyclonal anti-peptide antibody recognizing the C-terminal 18 amino acids of human cyclin Gl (Wu, et al . , 1994) . To analyze the comparative efficacy of antisense Gl, antisense Dl, and p21 expression in the induction of apoptosis in MG-63 cells, the cells initially were examined by light microscopy for morphologic changes associated with apoptosis (cell shrinkage, cytolysis, nuclear fragmentation, and condensation of chromatin) . The relative number of apoptotic cells were further confirmed and quantified using the Apoptag Plus in situ apoptosis detection kit (Oncor, Gaithersburg, MD) , which specifically detects the nascent 3 ' -OH DNA ends generated by endonuclease-mediated DNA fragmentation. The significance of differences among retroviral vectors bearing aGl, aDl, and p2l inserts, and control vectors waε determined by analyεiε of variance.

RESULTS Human Osteogenic Sarcoma aβ a Target for Gene Therapy Using Retroviral Vectors.

Initial studieε were aimed at characterizing the transduction efficiency of human osteoεarcoma cellε, using the GIBgSvNa retroviral vector conεtruct. The apparent transduction efficiency of the retroviral vector was relatively high, approaching 80-90% for the transformed MG-63 cells, as compared to normal diploid fibroblasts in which transduction efficiencies of 20-30% were observed. Figure 2 shows the β -galactoieidase staining MG-63 cells following transduction with the lacZ vector. Next, potential "bystander" cytocidal effects by mixing cells transduced with the Herpes Simplex thymidine kinase (HStk) gene with non- transduced cells followed by exposure to 20 μg/ml ganciclovir (GCV) was examined. Figure 3 is a graph which shows the degree of confluency (%) in mixtures of HStk+ and HStk- MG-63 cells cultured for 10 days in the presence of GCV (20 μg/ml) . The non-transduced cultures containing 100% HStk- cells showed 75% confluency. In contraεt, the cultures containing 10% HStk+/90% HStk- and 30% HStk+/70% HStk- cells showed only 15% confluency, while cultureε containing 50% HStk+/50% HStk- cells achieved 10% confluency, and cultures with greater than 50% HStk-i- cell cultureε achieved <10% confluency. The non- linearity of the survival curve demonstrateε a εignificant byεtander effect of GCV in mixed cultures of MG-63 cells. Both the high transduction efficiency of retroviral vectors and the occurrence of pronounced bystander effects to HStk+/GCV treatment attest to the feasibility of gene therapy for human osteogenic sarcoma using retroviral vectors.

Cytostatic and Cytocidal Effects of the Antisense Cyclin Gl

Retroviral Vector in Cultured Human Osteogenic Sarcoma Cells.

The structure of the experimental retroviral vector constructε are preεented diagrammatically in Figure 4,

including the location of the neomycin phosphotransferase (neo 1 ) gene positioned downstream of the respective genes for 3 cell cycle control proteins, two of which are truncated fragments engineered in antisense orientation. The expected εizeε of the transcripts for antisenεe cyclin Gl, antiεenεe cyclin Dl, and p21 expreεsion vectors are 3421, 3456, and 3346 base pairs, respectively. Transduction of MG-63 cells with each of the test vectors (Figure 5) revealed a marked reduction in the number of viable cells observed at 24 to 168 hours post-transduction, when compared to transduced cultures containing the control vector expresεing only the neo r gene. Cell denεitieε were meaεured, by cell counting, in cultures of MG-63 cells at εerial intervals after transduction with the retroviral vectors bearing antisense cyclin Gl (GlaGl) , antisense cyclin Dl (GlaDl) , and p21(Glp21), as well as the control vector GlXSvNa (G1X) .

As shown in Figure 6, the comparative expression of the p29 cyclin Gl protein was analyzed by Weεtern blotting, and found to be εignificantly reduced in MG-63 cellε bearing the antisense cyclin Gl vector.

Antisense Knock-out Cyclin Gl Induces Apoptosis in Human Osteogenic Sarcoma Cells.

The morphological appearance of MG-63 cells was obεerved by light microscopy at 72 hours after transduction of retroviral vectors bearing antisenεe cyclin Gl, antisense cyclin Dl, p21 inhibitor, and control vector constructε (Figure 7) . In addition to significant decreases in cell densities observed in cultures transduced with vectors containing antisense cyclin Gl, as well as the antisense cyclin Dl and p21 constructs (see Figure 5) , morphological evidence of apoptotic changes were noted, including cell εhrinkage, nuclear segmentation, chromatin condensation, and nuclear fragmentation (Arends, et al . , Int . Rev. Ex p . Pathol .. Vol. 32, pgs. 223-254 (1991) ; Wyllie, et al . ,

International Review of Cytology. Vol. 68, pgs. 251-306 (1980) ) , in cellε transduced with each of these cell cycle control elements. To investigate further the mechanism of cell death, a molecular/immunocytochemical approach (Arends, et al . , Amer. J. Path.. Vol. 136, pgs. 593-608 (1990); Gavrieli, et al . , J. Cell Biol. , Vol. 119, pgs. 493-501 (1992) ) was employed to detect the endonuclease-mediated DNA cleavage fragments that are characteristic of apoptoεis (Bursch, et al . , Biochem. Cell Biol., Vol. 68, pgs. 1071-1074 (1990) ; Compton, Cane. Metast.. Vol. 11, pgs. 105-119 (1992) ) . Figure 8 shows the detection cf apoptotic cells by immunocytochemical analysis of DNA fragmentation in cultures bearing the chimeric vectors containing antiεenεe cyclin Gl, antisense cyclin Dl, and p21 constructs. The induction of apoptosis in each of the cultures transduced with the cell cycle control vectors was determined to be highly significant (antisenεe cyclin Gl, mean incidence = 38.8 ± 5.0 %, n = 6, p < 0.00l; antisense cyclin Dl, mean incidence = 37.4 ± 24.4%, n = 6, p<0.01; and p21, mean incidence = 37.5 ± 8.2%, n = 6; p<0.001) when compared to cultureε tranεduced with the control vector (mean incidence = 3.6 ± 4,1%, n = 6) . Theεe reεultε confirm that the obεerved cytocidal effects of these retroviral-mediated cell cycle blockadeε reεult from apoptosis .

Metastatic osteogenic sarcoma is a target for experimental gene therapies as it is invariably asεociated with a fatal outcome. This type of sarcoma tends to recur locally, spread to other bones or to lungs, which are surgically accessible sites. In fact, recent studies have reported increased survival time in patients who have undergone aggressive metastasectomy (Damron, et al . , Oncology. Vol. 9, pgs. 327-340 (1995)) . The safety and efficacy of therapeutic vectors bearing specific cell cycle control enzymes or HStk could be evaluated by intratumoral injection of producer cells or vector supernatant into

metastatic foci, followed by metaεtaεectomy and histologic examination for evidence of apoptosis, cytolysis or overt cytodifferentiation. The present study reveals a relatively high transduction efficiency of MG-63 osteosarcoma cells for the above-mentioned retroviral vectors in comparison with normal diploid fibroblastε. Interestingly, the apparent transduction efficiency of these cells (80-90%) is far greater than the percentage of cells in S phase in asynchronous cultures (Carbonaro-Hall, e al., Oncogene, Vol. 8, pgs. 1649-1659 (1993)) . Non-transduced MG-63 cells exhibited significant "bystander" cytocidal effects of ganciclovir, when mixed with HStk+ transduced cells, which, together with retroviral transduction suεceptibility, affirm the feasibility of developing gene therapy approaches in the clinical management of metastatic diεease.

Previous studies characterized the precise sequence of cyclin expression in MG-63 osteosarcoma cells (Wu, et al . , Int. J. Oncol.. Vol. 3, pgs. 859-867 (1993) ; Carbanaro-Hall, 1993; Hall, et al. , Oncogene, Vol. 8, pgs. 1377-1384 (1993) ; Williams, et al. , J. Biol. Chem., Vol. 268, pgε. 8871-8880 (1993)) , enabling the temporal localization of a novel Cdk- aεεociated cell cycle block point revealed by the antiproliferative agent rapamycin (Alberε, et al . , J. Biol. Chem.. Vol. 268, pgε. 22825-22829 (1993)) . The reεultε of the preεent study with retroviral vectors confirms the resultε of previous εtudieε using penetrant antisense oligonucleotides (Wu, 1993) : that antisense strategies directed against the cyclin Dl locus effectively inhibit osteosarcoma cell proliferation. The mechanism of cell death observed in cells transduced with each of the experimental constructs (i.e., aGl, aDl, and p21) was determined to be apoptosiε, which is of considerable importance in terms of therapeutic efficacy in vivo.

The physiological function of cyclin Gl and its therapeutic potential is of particular interest, in that this

candidate protooncogene ( CYCG1 ) was first linked to cancer pathogenesis in human osteosarcomas (Wu, 1994) . Moreover, a recent εtudy suggests that cyclin Gl, like p21, is a transcriptional target of the p53 tumor suppreεεor protein (Okamoto, et al . , EMBO J. , Vol. 13, pgε. 4816-4822 (1994)) . However, the initial hypothesis that cyclin Gl might counterintuitively function as an inhibitory εubunit of cyclin-dependent kinases in a p53-mediated pathway to prevent tumorigenesis was discounted by experiments in which enforced overexpression of cyclin Gl failed to cause cell cyclin arrest in either normal or neoplastic cell lineε (Okαmoto, 1994) . In contrast, the present study repreεentε the firεt demonεtration that cyclin Gl is essential for the survival and/or growth of osteoεarcoma cellε. Theεe new data εupport the concept that cyclin Gl is involved in cell activation and or "competence" (Wu, 1994) , and that blockade of cyclin Gl expression by antisense constructs exert profound cytocidal as well as cytostatic effects.

Example 2 Materials and Methodε

Retroviral Vector Supernatantε and Producer Cell Lines.

The β-galactosidase and p53 expression vectors were kindly provided as high titer PA317 packaging cell clones (titers: 1.3 x 10 s and 2 X IO 6 colony-forming units, cfu/ml for β-galactosidase and p53 vectors, respectively) by Genetic Therapy, Inc. (Gaithersburg, MD) . The experimental vector bearing antisense cyclin Gl cDNA was packaged in PA317 cells and grown to high titer clones (vector titer: 1 X IO 6 cfu/ml each) . The vectors are referred to as GIBgSvNa, Glp53SvNa.7, and GlaGlSvNa to indicate the order of promoters and coding regions contained in each vector (Gl vector, Moloney murine leukemia virus long terminal repeat (LTR) sequenceε; Bg, β- galactosidase or lacZ gene; p53, p53 tumor suppressor gene;

aGl, antisense human cyclin Gl; Sv, SV40 early region enhancer/promoter; and Na, neo r gene) . The vector source, GlXSvNa, containing only the SV40 promoter-driven neo r gene was used as a control for the effects of gene transduction and G418 selection.

The vector Glp53SvNa.7 was constructed from pGlXSvNa and the plasmid pp53. Plasmid pp53 was constructed from pBSK- SN3, obtained from PharmaGenics (Allendale, New Jerεey) , which contains a 1.8 kb Xbal fragment that includes the wild type p53 open reading frame as well as 5 ' and 3 ' untranslated regions cloned into the Xbal εite of pEluescriptSK (Stratagene, LaJolla, California) . pBSK-SN3 was digested with Smal and partially digested with Ncol to generate a 1,322 bp fragment containing the p53 open reading frame. The fragment was gel purified and ligated into plasmid pBg (described in published PCT Application No. WO91/10728, published July 25, 1991) , in place of the /3-galactosidase gene between the Ncol and the Xhol sites to yield plasmid pp53.

Plasmid pGlXSvNa was digested with SnaBI and NotI. The SnaBI and NotI sites are located in the polylinker region of the plasmid. The digest generated a fragment having a length of 5,848 base pairs. The ends of the fragment were treated with calf intestinal alkaline phosphatase.

Plasmid pp53 was digeεted with NotI and Smal. the digest generated a 2,081 base pair fragment and a 1,400 base pair fragment. The 1,400 base pair fragment contained the p53 gene. This fragment was isolated and gel purified.

The 5,848 base pair fragment obtained from pGlXSvNa, and the 1,400 base pair fragment obtained from pp53, with each fragment having sticky/blunt ends, were ligated to form pGlp53SvNa. The resulting plasmid was identified and confirmed by several diagnostic restriction analyses. The plasmid pGlp53SvNa then was packaged in PA317 cells to generate the retroviral vector Glp53SvNa.7.

Cells, Cell Culture Conditions and Transduction of Cells with lacZ, antisense cyclin Gl and p53 Vectors.

Rabbit undifferentiated carcinoma (VX2) cells and other primary and established cell lines were cultured at a plating density of 2.5 x 10* cells in each of six-well plates, in DMEM supplemented with 10% FBS (D10) . After overnight attachment, the cells were exposed to 1 ml of the respective retroviral vector in the presence of Polybrene (8 μg/ml) for 2 hours, after which 1 ml of fresh D10 was added to each well. Forty- eight hours after transduction with the lacZ vector, gene transfer efficiency was measured by determining the percentage of lacZ positive cells, upon X-gal staining and light microscopy.

Evaluation of Cell Proliferation and Cell Cycle Kinetics in VX2 Transduced with Retroviral Vectors Bearing Cell Cycle Control Genes.

To assess the cytostatic effects of retroviral vectors bearing cell cycle modulators, the cells that were transduced with control vectors, or vectors expressing antisense cyclin Gl or p53 genes, were evaluated for their proliferative potential by counting the number of viable cells in each culture at serial intervals after transduction. The effect of cell cycle modulators on the cell cycle kinetics of VX2 (carcinoma) as well as MG-63 (sarcoma) cells was tested by FACS analyεis. The survival of transduced VX2 cells in the preεence of G418 also waε evaluated to determine to what extent the antisense cyclin Gl waε cytocidal to the transduced cells.

Development of a Tumor Model In Athymic Nude Mice for In Vivo Gene Therapy Using Retroviral Vectors Bearing Cell Cycle Modulators.

Undifferentiated carcinoma (VX2) tumors have been grown successfully in nude mice by subcutaneous implantation of VX2

cells. Theεe tumors grow rapidly within three weeks, and are surgically accessible for evaluation of changes in tumor volume and morphology. Briefly, VX2 tumors were grown over 5 weeks in athymic nude mice by εubcutaneous injection of 1 x IO 7 VX2 cells. When the tumors reached 100 mm 3 in size, 100 μl of concentrated retroviral vector supernatant (GlaGlSvNa, bearing the antisense cyclin Gl gene or the GlXSvNa control vector, bearing only the neo r gene: vector titer, 1 x IO 8 cfu/ml) was injected intratumorally, under Metofane anesthesia, every day for 2 weeks. Tumor volume was measured every week using a Vernier caliper, and the percentage change in tumor volume was estimated. The significance of differences between the antisense cyclin Gl vector- and control vector-treated tumors was teεted uεing the Student's t test. Additionally, the formalin-fixed tumors were stained with hematoxylin-eosin (H & E) for histologic examination.

Results A wide variety of cell lines were tested for sensitivity to retroviral vectors bearing cell cycle modulators. The results of such testing are given in Table I below.

TABLE I

In Vi tro Transduction Efficiencies and Cytostatic Effects of an Antisenεe Cyclin Gl Retroviral Vector in Cancer and Non- cancer Cellε

Cell Line Cell Type Transduction Cytostatic

Efficiency Effect

(GIBgSvNa) (GlaGlSvNa)

Human Cancer

MG-63 osteosarcoma 80% +

HT29 colon carcinoma 13% +

Bxpc-3 pancreatic carcinomai 9% +

Miapaca pancreatic carcinoma L 19% +

Mnng/Hos osteosarcoma 15% +

EW-1 Ewing's Sarcoma 5% +

MDA-MB 231 breast cancer <1% -

Non-human Cancer

XC-6 (rat) osteosarcoma 22% + Kml2C (rat) colon carcinoma 20% + Kml2C4A (rat) colon carcinoma 15% + Kml2SM (rat) colon carcinoma 15% + C6 (rat) glioma 5% VX-2 (rabbit) undifferentiated CA 6%

Human Non-cancer primary bone marrow stroma 22% primary activated keratocyte 20% primary hepatic fibroblast 23% primary keloid fibroblast 31% primary dermal fibroblast 24% ECU endothelial 5%

Non-human Non-cancer

AIO (rat) aortic smooth muscle 45% NIH3T3 (mouse) fibroblast 30%

Of the cells tested, proliferation of 4 colon cancer cells (HT-29, KM12C4A, KM12C and KM12SM) , Ewings sarcoma (EW- 1), C6 glioma, 2 pancreatic cancer (BxPc3, Miapaca) and 2 osteosarcoma (MG-63, MnngHOS) was inhibited by the antisense cyclin Gl retroviral vector. The HT29, BxPc3, and KM12SM cells were also sensitive to wild type p53. Among the non- cancer cell lines, cytostasis was induced by antisense cyclin Gl and p53 in embryonic rat aortic smooth muscle cells and human skin and keloid fibroblastε, but not in normal human stromal, human liver-derived fibroblastε or human endothelial cells.

FACS analysis was used to investigate the effect of the antisense cyclin Gl retroviral vector on the cell cycle kinetics of sarcomatous and carcinomatous tumor cells. VX2 undifferentiated carcinoma cellε tranεduced with retroviral vectors bearing antisense cyclin Gl showed profound alterations of cell cycle kinetics upon FACS analysis, exhibiting a broadening of peaks that is indicative of

nuclear fragmentation and a reduction of cells in S phase (Figure 9A) . In comparison, FACS analyεiε of MG-63 cells transduced with the antisense cyclin Gl vector showed accumulation of cells in Gl phase, and a significant decrease in the number of cells in S phaεe, εuggesting that the mechanism of cytostasiε in theεe tranεduced cells accompanies a Gl phase cell cycle block (Figure 9B) .

Simultaneous with the altered cell cycle kinetics, the antisense cyclin Gl as well as the p53 vectors inhibited proliferation of VX2 carcinoma cells over 144 hours compared with control vector-treated cells (Figuie 10) . Upon selection of transduced cells with G418, only 5% of the VX2 cells were eliminated (Figure 11) , indicating that the vast majority of cells bearing antisense cyclin Gl and wild type p53 had undergone cell death, presumably via apoptoεis. These data represent the first in vi tro demonstration that antisense cyclin Gl may exhibit antitumor activity in cancers of epithelial origin.

Figure 12 shows inhibition of VX2 tumor growth in nude mice by intratumoral injection of a retroviral vector bearing antisenεe cyclin Gl (GlaGl) when compared to growth of VX2 tumors in mice receiving the control vector (G1X; p <.05 at 7 days; p<.001 at 11 days; and p< .05 at 21 days; n = 3 mice each group) . In 1 of 5 mice treated with antisense cyclin Gl vector, a 12% decrease in tumor εize waε noted one week following treatment. In contrast, tumor growth waε not arrested in the mice treated with the control vector.

The mice treated with the antisense cyclin Gl vector showed grossly smaller tumors than control mice. Figure 13A showε repreεentative antisense cyclin Gl versus control vector- treated mice while Figure 13B showε the histopathologic characteristics of formalin-fixed and H&E stained VX2 tumor sections, harvested at 21 days (one week after completion of treatment) . The sections of tumors that were treated with the control vector showed areas of

increased cell density with anaplastic spindle-shaped cells and numerous mitotic figures. In contrast, the sections of tumors that were treated with the antisense cyclin Gl vector showed areas of decreased cell density with less mitotic figures and notable mononuclear cell infiltration. However, residual tumor cells were noted in sections of tumor that received the antisense cyclin Gl vector, indicating that a population of tumor cells were not effectively transduced. Taken together, the retroviral vector expressing antisenεe cyclin Gl appears to exhibit antitumor effects in vivo in this tumor model of undifferentiated carcinoma.

DISCUSSION

Cancer is a leading target for gene therapy because patients with cancer, particularly those with metastatic disease, often have few or no treatment options, and would be eligible for experimental therapies. The retroviral vector delivery system has been used in 76 of the 106 human trials approved. This vector system utilizes a replication incompetent mouse retrovirus, and thus far, its uεe, both ex vivo and in vivo, has not caused any major side effects.

Other gene therapy εtrategieε include 1) enhancement of the immune response by injection of tumor vaccines containing transduced irradiated tumor cells expresεing cytokineε, MHC Claεs 1 or B7 genes, 2) enforced expression of tumor suppressor genes, 3) knock-out of protooncogene overexpression by antisense vectors, and 4) enforced expression of growth factor receptor genes. In recent years, overexpression or amplification of various cell cycle control genes have been reported in various malignant disorders, indicating that antisense knock-out of these overexpressed genes could be used to re-establish control of cell proliferation, induce cytostasis, inhibit tumor growth and decrease tumor burden.

These conceptε ariεe from initial εtudies of the budding yeast S. cerevisiae wherein extracellular signals that modulate the growth and differentiation act via regulation of a Gl control point termed START (Hartwell, Science, Vol. 183, pgs. 46-51 (1974); Crosε et al . , Ann. Rev. Cell Biol., Vol. 5, pgε. 341-395 (1989)), which iε loosely analogous to the Gl restriction point (R point) observed in animal cells in culture (Pardee, Science. Vol. 246, pgs. 603-608 (1989)) . Therefore, the finding that Gl cyclins (Clns) in S. cerevisiae, in association with a Cdk subunit (Cdc28) , were required for cells to pasε START led to the hypothesis that Gl specific cyclins may indeed function as upstream components of the mammalian S phase Promoting Factor (Draetta, Trends Biochem. Sci.. Vol. 15, pgs. 378-383 (1990) ; Reed, Trends in Genetics, Vol. 7, pgs. 95-99 (1991)) . Screening of human cDNA libraries for genes that could serve to rescue Cln-deficient yeast cells led to the identification and molecular cloning of three novel families of human Gl cyclins (cyclinε C, D, and E: Lew et al . , Cell. Vol. 66, pgs. 1197-1206 (1991) ; Koff eϋ al . , Cell. Vol. 66, pgs. 1217-1228 (1991) ; Xiong et al . , Cell. Vol. 65, pgs. 691-699 (1991) ; Sherr, Cell. Vol. 73, pgs.1059-1065 (1993)) . Subsequent studies have mapped the PRADl/Cyclin Dl gene to chromosome llql3, implicating cyclin Dl as the BCL-1 oncogene that is translocated and overexpressed in B cell neoplasms (Rosenberg et al . , Proc. Nat. Acad. Sci.. Vol. 88, pg. 9638 (1991) ; Witherε et al . , Mol. Cell . Biol .. Vol. 11, pg. 4846 (1991)) and aε the llql3 oncogene that iε amplified and overexpreεssed in squamouε cell, breaεt, esophageal, and bladder cancers (Lammie et al . , Oncogene, Vol. 6, pg. 439 (1991) ; Jiang et al . , Cancer Res .. Vol. 52, pg. 2980 (1992) ; Motokura et al . , Curr. Qpin. Genet . Dev.. Vol. 3, pg. 5 (1993)) . Genetic amplification, increased expresεion, and altered metabolism of cyclin E has also been observed in human cancer cells (Buckley et al . , Oncogene, Vol. 8, pg.

2127 (1993) ; Keyomarsi et al. , Cancer. Res.. Vol. 54, pg. 380 ( 1994)) . More recently, a human G-type cyclin, a Gl cyclin that was markedly overexpresεed in a subset of osteosarcoma cells was isolated (Wu et al . , 1994) . Taken together, these findings affirm that constitutive, ectopic, or deregulated expression of Gl cyclins, which normally link signal transduction pathways to the enzymatic machinery of the cell cycle (Hunter and Pines, Cell, Vol. 66, pgs. 1071-1074 (1991) ; Sherr, (1993)) , may play an important role in neoplastic transformation and tumorigenesis (Hunter and Pines, Cell, Vol. 79, pgs. 573-382 (1994)) , and could b« used as strategic checkpoints for development of novel gene therapy approaches to cancer and hyperproliferative disorderε.

In thiε study, the safety and efficacy of an antisense cyclin Gl retroviral vector supernatant as a potential gene therapy approach to cancer was tested. A wide variety of cancer cells showed sensitivity to antisense knockout cyclin Gl in comparison to wild-type p53. The proliferation of some non-cancerous cells also was inhibited by the antisense cyclin Gl vector, suggesting its potential utiliy in the management of non-malignant fibroproliterative disorders as well. Hence, various cell types εhowed differential sensitivity to cell cycle modulators. The antisense cyclin Gl vector had profound effects on the cell cycle kinetics of both carcinomatous and εarcomatouε tumor cellε, with a net effect of decreased DNA synthesiε, aε evidenced by a reduction of cells in S phase. These data suggest that the mechanism of cytostasis in these transduced cells accompanies a Gl phase cell cycle block. Upon selection of transduced cells with G418, only 5% of the VX2 cells were eliminated, indicating that the vast majority of cells bearing antisense cyclin Gl and wild type p53 had undergone cell death, presumably via apoptosis.

Finally, in vivo tumor growth was inhibited dramatically by succesεive intratumoral injection of a concentrated antisense cyclin Gl retroviral vector supematant. In contrast, tumor growth was not arrested in the mice treated with the control vector. Histologic examination of the tumors one week after cessation of treatment showed areas of increased cell density with anaplastic spindle-shaped cells and numerous mitotic figures in control vector-treated tumors. In contraεt, the εectionε of tumors that were treated with the antisense cyclin Gl vector showed areas of decreased cell density with less mitotic figures and notable mononuclear cell infiltration. Taken together, these findings represent the first demonstration of in vivo antitumor activity of a retroviral vector expresεing antiεense cyclin Gl in a model of undifferentiated carcinoma.

Example 3

Inhibition of In Vivo Tumor Growth by a

Retroviral Vector Bearing Antisense Cyclin Gl in Athymic Nude Mice

Osteosarcoma tumors were grown over two weeks in athymic nude mice by subcutaneous injection of 1X10 7 MNNG/HOS cells.

When the tumors reached 100mm 3 in size., lOOμl of concentrated retroviral vector supernatant (GlXSvNa control vector, bearing only the neo R gene, or GlaGlSvNa, bearing the antiεense cyclin Gl gene: vector titers: each 1X10 8 cfu/ml) were injected intratumorally every day for 10 days.

The tumor volume waε meaεured at intervalε of 0, 4, 6, 8, 10, and 12 days after vector injection. Figure 14 εhowε the tumor volume at each of the above-mentioned intervalε. As εhown in Figure 14, the antiεenεe cyclin Gl vector-treated mouse has a smaller tumor than the control vector-treated mouse.

Hematoxylin and eosin staining of formalin-fixed MNNG/HOS tumor sections for two days following the treatment

with the retroviral vectors bearing the antisense cyclin Gl gene (GlaGlSvNa) or the control vector (GlXSvNa) shows decreased mitotic index (1% for antisenεe cyclin Gl-treated tumors versus 3.5% for control vector-treated tumors) , and increased εtroma formation.

FACS analysis of PI-stained nuclei obtained from MNNG/HOS tumors showed a dramatic decrease in the number of aneuploid cells in the antisense cyclin Gl vector-treated tumors (2%) compared with that in control vector treated tumors (45%) . Further, the diploid population of cells from the antisenεe cyclin Gl vector-treated tumors showed a 77% accumulation of cells in Gl phase versus 49% in GlXSvNa control vector-treated tumors, and a significant decrease in the number of cells in S phase (15% versus 25%) , which suggests that the mechanism of cytostasiε in the tranεduced tumors was accompanied by a Gl phaεe cell cycle block.

Example 4 MATERIALS AND METHODS Retroviral Vectors, Vector Supernatants and Producer Cell Lines.

The cDNA sequence encoding human cyclin Gl (Accession #X77794) is as originally described by Wu et al . , 1994. The experimental vector bearing the antisense cyclin Gl cDNA (Wu, et al . , 1994) was packaged in PA317 cells and grown to high titer clones (vector titer: 1 X 10 s cfu/ml each) . The β- galactosidase and p53 expression vectors were provided kindly as high titer PA317 packaging cell clones (titers: 5 x 10 s and 2 X IO 6 colony-forming unitε, cfu/ml for β-galactosidase and p53 vectors respectively) by Genetic Therapy, Inc. (Gaithersburg, MD) . The vectors are referred to as GlnBgSvNa (described in PCT Application Nos. W095/19427, published July 20, 1995 and W096/22212, published July 25, 1996), Glp53SvNa.7, and GlaGlSvNa to indicate the order of promoters and coding regions contained in each vector (Gl, Moloney

murine leukemia virus long terminal repeat (LTR) sequences; Bg, β -galactosidase gene; p53, p53 tumor suppreεεor gene; aGl, antisense human cyclin Gl; Sv, SV40 early region enhancer/promoter; and Na, neo τ gene) . The retroviral vector supernatants were concentrated further to a titer of 1 X IO 8 cfu/ml by low speed centrifugation. The vector backbone, GlXSvNa, containing only the SV40 promoter-driven neo τ gene was used as a control for the effects of tranεduction and G418 selection.

Cells, Cell Culture Conditions, and Transduction with Retroviral Vectors.

Rat aortic smooth muscle (AIO) cells were obtained from ATCC (Cat. #CRL1476) and maintained as monolayers at a plating density of 2.5 x 10* cells per well, in DMEM supplemented with 10% fetal bovine serum (FBS;D10) . After overnight attachment, the cells were exposed to 1 ml of the respective retroviral vector in the presence of Polybrene (8 μg/ml) for 2 hours, with periodic rocking, after which 1 ml of fresh D10 was added to each well. Forty-eight hours after transduction with the β-galactosidase vector, gene transfer efficiency was measured by determining the percentage of β - galactosidase positive cells, upon expoεure to X-gal ( β - galactosidase) staining as described in Lai, et al., J___ Histochem. Cvtochem. , Vol. 42, pgε. 953-956 (1994) , and visualization by light microscopy.

Analysis of Cell Proliferation, DNA Synthesis, Cyclin Gl Protein Expression and Apoptosis

To assess the cytostatic effects of retroviral vectors bearing cell cycle modulators, the SMC that were transduced with control vectors or vectors expressing antisense cyclin Gl (or p53) gene(s) were evaluated for their proliferative potential by counting the number of viable cells in each culture at serial intervals after tranεduction. Values shown

represent the mean of triplicate ± standard deviation (S.D.) . The effect of cell cycle modulators on DNA synthesis was monitored by the incorporation of 3 H-thymidine into DNA as described in Gordon, et al . , Proc. Nat. Acad. Sci.. Vol. 93, pgs. 2174-2179 (1996) . Briefly, 24 hrs. after transduction with the antisense cyclin Gl or control retroviral vector, the cell cultures were exposed to 3 H-thymidine (1 μCi per well; specific activity, 6.7 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) for 2 hrs. The cells were then placed on ice, rinsed twice with cold phosphate-buffered saline (PBS) , and then rinsed three times with ice-cold 5% trichloroacetic acid (TCA) . The final TCA rinse was removed and the TCA- precipitated material was solubilized with 0.2 ml of IM sodium hydroxide followed by neutralization with an equal volume of IM acetic acid. 3 H-thymidine incorporation into cellular macromolecules was measured by liquid scintillation counting and expressed as radioactivity units in dpm/well. The significance of differences between untreated and vector- treated groups was determined by analyεiε of variance (ANOVA) .

Western Blot analysis of cyclin expression was performed as described in Wu, et al . , Int. J. Oncol.. Vol. 3, pgs. 859- 867 (1993) and Colton, Statisticε in Medicine, pg. 99, Little, Brown & Co., (1974), uεing a polyclonal antipeptide antibody recognizing the C-terminal 18 amino acids of human cyclin Gl (Wu, et al . , 1994) . The occurrence of apoptosis in transduced cell cultures was evaluated with the Apoptag Plus in si tu detection kit (Oncor) , which detects nascent 3 ' -OH DNA ends generated by endonuclease-mediated DNA fragmentation utilizing enzymatic (terminal deoxynucleotidyl transferase;TdT) addition of digoxigenin-labeled nucleotides followed by immunocytochemical detection of the modified DNA fragments (Skotzko, et al., Cancer Res.. Vol. 55, pgs. 5493- 5498 (1995) ) .

Retrovirus-mediated Transfer of the Antisense Cyclin Gl Gene in a Rat Carotid Injury Model of Vascular Restenosis.

Under general anesthesia (ketamine, 10 mg/kg; rompun, 5 mg/kg) , in accordance with a protocol approved by the USC Institution Animal Care and Use Committee (IACUC) , a 2F Intimax arterial embolectomy catheter (Applied Medical Resources Corp., Laguna Hills CA) was used to denude the carotid artery endothelium of Wistar ratε (each weighing 400- 500 gm) . The catheter was inserted into the extemal carotid artery which was ligated distally, and passed into the common carotid artery. The balloon was inflated to a volume of 10 μl and passed 3 times along the length of the common carotid artery. After balloon injury, the embolectomy catheter was removed and the internal carotid artery was ligated tranεiently juεt diεtal to the bifurcation. The diεtal half of the injured segment was likewise transiently ligated, and then exposed to the retroviral vectors for 15 minutes . Each group of animals received an infusion of 100 μl of concentrated high titer antisenεe cyclin Gl vector (n = 7) or a control vector bearing only the neo r gene (n = 4) , after which the ratε were allowed to recover, and fed a regular mouse/rat diet and water ad libitum. For purposeε of analgeεia, the animals were given buprenex, 0.2 mg/kg s.c. every 12 hours for 72 hours post-operatively. The rats were sacrificed 2 weeks after induction of vascular injury by an overdose of sodium pentobarbital (120 mg/kg IM) , and formalin-fixed sections of both injured and non-injured contralateral carotid arteries were stained with hematoxylin- eoεin, Siris red-Verhoeff's elastin stain. Hiεtologic sections were examined by light microscopy, and morphometric evaluation of the neointima versus media surface areas was conducted uεing a digitizing system; the extent of intimal hyperplasia following vascular injury is expresεed as neointima to media ratios. The significance of differences between the neointima to media ratios of non-treated and

vector-treated vessels was determined by paired t-test (Colton, 1974) .

Results Transduction of Aortic SMC with Retroviral Vectors Bearing Cell Cycle Control Genes.

Using a nuclear-targeted β-galactosidase vector (GlnBgSvNa) , the apparent transduction efficiency of rat (AIO) aortic SMC was about 45% (Figure 15A) , which was similar to murine NIH3T3 cells, and somewhat greater than normal human fibroblasts or scar-derived (keloid.) fibroblasts in which transduction efficiencies of 20% and 30%, respectively, were observed. Transduction of aortic SMC with vectors bearing antisense cyclin Gl (aGl) showed a marked decrease in the number of viable cells observed at 24 to 144 hours post-transduction, when compared to transduced cultures containing the empty (control) vector (Figure 15B) . Western Blot analysis confirmed down-regulation of cyclin Gl protein expression in aortic SMC transduced with antisenεe cyclin Gl when compared to the control vector (not εhown) . Proliferation of AIO cellε was alεo inhibited by retroviral mediated overexpression of the p53 tumor suppresεor gene in sense orientation. Both antisense cyclin Gl and p53 vectors inhibited cell cycle progression, as determined by the incorporation of 3 H-thymidine (p<.001 for both aGl and p53 ; Figure 15C) .

Antiεense Cyclin Gl Induceε Degeneration, Multicellular Syncytia Formation, and Apoptosiε in Aortic SMC.

The photomicrographs shown in Figure 16 display the morphological appearance of aortic SMC observed by light microscopy at 24 hours after transduction with control and antisense cyclin Gl retroviral vectors. As εhown in F.igure 16A, the cells transduced with the control vector showed no significant morphologic changes. In contrast, a significant

decrease in cell density was observed in cultureε tranεduced with vectors bearing antisense cyclin Gl, associated with overt degenerative changes, increased multinuclear syncytium formation, and cytolysiε (Figures 16B, 16C, 16D) . Remarkably, the proportion of cells involved in the syncytia far exceeded the transduction efficiency as determined by the transduction and expression of β-galactosidase. Syncytium formation occurred in AIO cultures transduced with the antisense cyclin Gl vector supernatants derived from three different high titer clones, as well as the p53 vector to some extent, but not in the control (GlXSvNa) or β- galactosidaεe vectors. To further investigate the mechanisms of cell death, a molecular and immunocytochemical approach was employed to detect the endonuclease-mediated DNA cleavage fragments that are characteristic of apoptosis. As shown in Figures 16E and 16F, no evidence of apoptosis was observed in cells transduced with the control vector (Figure 16E) ; however, a number of apoptotic cells were observed in the antisense cyclin Gl vector-transduced cultures (Figure 16F) . These results indicate that the cytocidal effects of the antisense cyclin Gl vector in AIO aortic SMC result in part from apoptosis, cell degeneration, and aberrant syncytium formation.

Evidence for a Cytocidal "Bystander" Effect in Aortic SMC Cultures Transduced with Antisenεe Cyclin Gl Retroviral Vectorε.

To confirm that non-tranεduced cellε were incorporated into the multicellular syncytia found in antisenεe cyclin Gl- transduced cultures, we loaded non-transduced AIO cells with a fluorescent marker and overlaid the marked cells on previously transduced cultures two hours after washout of the vector supernatant. The incorporation of non-tranεduced, f1oureεcently-labeled AIO εmooth muεcle cellε into multinuclear syncytia clearly was evident when these marked

cellε were overlaid onto previously transduced AIO cultures (Figures 17A and 17B, low magnification; 17C and 17D, high magnification; 17A and 17C, phase contrast; 17B and 17D, UV light) . A representative multinuclear syncytium incorporating cells containing the flourescent label is identified by the arrow. Twenty-four hours after co-culture with non-transduced, fluorescently-labelled aortic SMC, a considerable number of the multinucleated syncytia were also labelled with the fluoreεcent dye, indicating that cell fusion between the transduced and non-tranεduced cells had occurred. This finding provides additional evidence of a novel cytocidal "bystander effect" distinguishable from the classic "bystander effect" induced by the Herpes Simplex Virus thymidine kinase/ganciclovir syεtem and mediated by gap junctionε preεent in susceptible cells.

The phenomenology of cell fusion was followed over time (Figure 17E, revealing a significant increase in the number of syncytia that increased over 4-8 hours in aortic SMC that were transduced with the antisense cyclin Gl vector (GlaGlSvNa) , when compared to the cells transduced with the control vector (GlXSvNa; p<.001) . An appreciable degree of syncytium formation also was noted in cells that were transduced with the wild-type p53 vector (Glp53SvNa) which also produced marked cytostasiε in AIO cells. However, the number of syncitia observed in p53 transduced cells waε εignificantly leεs than than that observed in aGl transduced cells at 8, 12 and 24 hours (p<.001) .

The Antisense Cyclin Gl Vector Inhibits Proliferation and Migration of Aortic Smooth Muscle Cells in an In Vitro "Tissue" Injury Model.

High density (confluent) monolayer cultures of AIO SMC exhibiting contact inhibition of cell growth can be stimulated to proliferate along a track of cell/tisεue diεturbance exhibiting a characteristic "wound healing"

responεe over a period of 7 dayε. Figure 18A εhows high density cultures of aortic SMC εcraped with a 200 μl pipet tip to create a 1 mm track devoid of cellε. Figure 18B shows the appearance of the "wound" margin immediately upon scraping and washing to remove the detached cells. As shown in Figure 18C, subsequent transduction of the cell cultures (at t = 24 hours) with a nuclear-targeted β-galactosidase vector was greatest at the margins of the "wound", an area of activated SMC proliferation. Figure 18D showε proliferation and migration of aortic SMC into the wound track at t = 24 hourε after injury. In contraεt, apoptotic and other degenerative changeε were obεerved in the SMC that were transduced with the antisense cyclin Gl vector (Figure 18E) . Notably, these degenerative changes were marked by multicellular syncytia formation that was not observed in either the control or β-galactosidase vector. Further, cell proliferation and overt cell migration into the wound track was reduced markedly in the antisense cyclin Gl-transduced cell cultures, evidenced by delayed closure of the wound track (about 7days) compared to the control vector-treated cultureε (about 3 dayε) .

Inhibition of Neointima Formation In Vivo by Infusion of High Titer Antisense Cyclin Gl Vector Supernatant.

Previous studies demonstrated direct transfer of recombinant marker genes into the arterial wall by retroviral vectors with viral titerε of 10*-10 e particleε/ml (Nabel, et al . , Science. Vol. 249, pgs. 1285-1288 (1990)) , and a number of studieε have demonεtrated the efficacy of cytoεtatic gene therapies delivered by other methods in animal models of vascular restenoεiε. In thiε εtudy, high titer retroviral vector εupernatantε (viral titer: 1 X 10 8 cfu/ml) were generated to teεt the efficacy of antiεenεe cyclin Gl delivered by highly concentrated retroviral vectorε in the rat carotid injury model of reεtenosis. Histologic

examination of stained sections obtained from balloon-injured untreated arteries showed substantial neointima formation at 2 weeks, as evidenced by invasion of the tunica intima by proliferating vascular SMC (Figures 19A and 19C) . In contrast, injured arterial segments that were treated with high titer antisense cyclin Gl vector supernatants showed a significant reduction in neointima formation (Figures 19B and 19D) . Morphometric analysis confirmed significant inhibition in neointima formation in injured carotid arteries that were treated with the antisense cyclin Gl retroviral vector (I:M ratio 0.4 + S.D. 0.4) compared to the untreated arterial segments (I:M ratio 1.1 ± 0.4; pc.OOl; Figure 19G) . In control studies, there was no difference between the extent of neointima formation in non-treated arterial segments (I:M ratio 1.3 ± SD 0.5) when compared with high titer vectors containing only the neo r gene (I:M ratio 1.5 ± 0.2) .

DISCUSSION Clinical trials based on the molecular blockade of identified growth factors and/or growth factor receptors implicated in the pathogenesis of intimal hyperplasia have not proven to be effective vehicles for cytostatic vaεcular therapy (Faxon, et al. , J. Amer. Coll. Cardiol.. Vol. 25, pgε. 362-369 (1995)) . Thus, it has been suggested that approaches which target intracellular signalling cascades that are shared by many growth regulatory molecules may be more strategic (Gibbonε, et al . , Science. Vol. 272, pgs. 689- 693 (1996) ) . Accordingly, novel gene therapy approaches to inhibit SMC proliferation and neointima formation have focused recently on cell cycle control mechanisms. Indeed, antisense approaches against cell cycle regulatory genes has been shown to be remarkably effective in limiting neointimal hyperplasia in animal models of lesion formation following both bypasε εurgery (Mann, et al. , Proc. Nat. Acad. Sci.. Vol. 92, pgε. 4502-4506 (1995)) and balloon angioplasty. A single intraluminal delivery of antisense Cdc2 kinase or Cdk2 kinase produced significant inhibition of neointimal hyperplasia Morishita, et al . , Proc. Nat. Acad. Sci.. Vol. 90, pgs. 8474-8478 (1993); Morishita, et al. , J. Clin. Invest.. Vol. 93, pgs. 1458-1464 (1994) ; Abe, Biochem. Biophvε. Reε. Comm.. Vol. 198, pgs. 16-24 (1994)) . An adenoviral vector bearing a nonphoεphorylatable, constitutively active form of Rb also was reported to inhibit SMC proliferation and neointima formation following balloon angioplasty (Chang, et al . , Science. Vol. 267, pgs. 518-522 (1995) ) . Molecular strategies directed against E2F also have been developed, as the concerted induction of numerous cell cycle regulatory genes is regulated by this transcription factor. Oligonucleotideε containing the E2F ciε element εequence function aε "decoys" that bind E2F within the. cell and inhibit neointimal lesion formation in vivo (Morishita, et al . , Proc. Nat. Acad. Sci.. Vol. 92, pgs. 5855-5859

(1995) ) . Further support for the concept of cytostatic gene therapy based on the inhibition of cell cycle control enzymes is provided by recent findings that rapamycin, which inhibits the activation of cell division/cycle enzymes (Albers, et al., Ann. New York Acad. Sci.. Vol. 696, pgs. 54-62 (1993) ; Albers, et al. , J. Biol. Chem.. Vol. 268, pgs. 22825-22829 (1993); Jayaraman, et al., J. Biol. Chem.. Vol. 34, pgs. 25385-25388 (1993), also inhibits vascular lesion formation in both rat and porcine models (Gregory, et al . , Transplantation. Vol. 59, pgs. 655-661 (1995); Marx, et al . , Circ. Res. , Vol. 76 , pgs. 412-417 (1995)) .

Cyclin Gl is a member of the so-called Gl family of cyclins which act in concert with cyclin-dependent protein kinases during the Gl phase of the cell cycle (Wu, et al . , Int. J. Oncol.. Vol. 3, pgs. 859-867 (1993) ; Sherr, Cell. Vol. 79, pgs. 551-555 (1994)) . Induced in early Gl and suspected to participate in the molecular mechanisms of cell activation (Wu et al . , Oncol. Reports, Vol. 1, pgs. 705-711 (1994) ) , cyclin Gl appears to be a transcriptional target of the p53 tumor suppressor gene (Okamoto, et al . , EMBO J. , Vol. 13, pgs. 4816-4822 (1994)) . Cyclin Gl overexpression was first linked to cancer (Wu, et al . , 1994) and, more recently, down-regulation of cyclin Gl expression by retroviral vectors bearing antisense CYCGl was reported to inhibit the growth and survival of human osteosarcoma (MG-63) cells (Skotzko, et al . , 1995) .

In this example, the effects of retroviral vectors bearing an antisense cyclin Gl construct on the proliferation of AIO rat aortic smooth muscle cells were examined. Retroviral vectors bearing the antisense cyclin Gl gene, as well as the p53 gene, in sense orientation, inhibited the survival and proliferation of transduced AIO cells in 2-6 day cultures. Cytostasis was associated with decreased DNA synthesis and dow -regulation of cyclin Gl in vascular SMC transduced with the antisense cyclin Gl vector as compared to

those transduced with the control vector. Morphological examination of the transduced SMC revealed cytolyεis, giant syncytia formation, and overt apoptotic changes evidenced by cell shrinkage, nuclear fragmentation, and chromatin condensation observed in both antisenεe cyclin Gl vector- and p53 vector-transduced AIO cells. However, the number of multinuclear syncytia were found to be significantly higher in the cell cultures treated with the antisense cyclin Gl vector. Pronounced "bystander" effects were noted in AIO cells transduced with the antisense cyclin Gl vector as determined by quantitative cell fusion assays and th<=» fluorescent labeling of non-transduced cells. These findings indicate that the antisense cyclin Gl vector induces a "fusion-promoting factor", possibly a proteaεe or glycoεylaεe, that facilitates cell fusion and syncytia formation, perhaps by augmenting mechanisms related to the fusogenic propertieε of the MoMuLV envelope protein (Jones, et al . , J. Virol., Vol. 67, pgs. 67-74 (1993)).

Cytostatic gene therapies for restenosiε εhow promiεe of additional therapeutic consequences in that the inhibition of cell cycle regulatory genes iε reported to trigger vascular cell apoptosis (Gibbons, et al . , 1996; Laird, et al . , Circulation. Vol. 93, pgs. 529-536 (1996)). In mitotically activated SMC, as in osteosarcoma cells (Skotzko, et al . , 1995) , the cytotoxicity of the cyclin Gl blockade is attributable, at least in part, to the activation of an apoptotic pathway (Figure 16F) . Furthermore, the induction of cell cycle arrest in some circumstanceε alεo appearε to inhibit SMC migration and extracellular matrix production (Biro, et al . , Proc. Nat. Acad. Sci., Vol. 90, pgε. 654-658 (1993)) . In the in vitro "tissue injury" model, both the proliferation and migration of AIO cells that were transduced with the antisense cyclin Gl vector were inhibited in the area of cell injury (Figure 18E) . Taken together with the observations of marked cytotoxicity, cell cycle blockade, and

multicellular syncytia formation, these findings lend additional support for the concept that cyclin Gl may represent a strategic locus for therapeutic intervention in the management of proliferative disorderε.

Once a potential therapeutic gene has been identified, the challenge remainε to deliver the gene transfer vector efficiently to the appropriate physiologic site. In the case of balloon angioplasty, both the denudation of the endothelial lining and the mitogenic activation of neighboring SMC provide favorable conditions for the delivery of retroviral vectors, as the therapeutic genes delivered by retroviral vectors are expressed preferentially in mitotically active cells. In the present εtudy, very high titer supernatants (10 β cfu/ml) were generated to enhance the transduction efficiency of vascular SMC, and hence, the efficacy of retroviral vectors in this experimental model of restenosiε. Indeed, the in vi tro εtudieε of retroviral vector-mediated gene delivery in embryonic AIO SMC, may be particularly relevant to the phyεiology of reεtenoεis, for numerous reports have indicated that embryonic and neointimal SMC exhibit similar responses to mitogenic signals (Schwartz, et al . , The Vascular Smooth Muscle Cell. Schwartz, et al . , eds. pg. 81-139, Academic Press, Inc., New York (1995)) . This study in the rat carotid artery injury model of restenosis demonstrates the efficacy of this approach: Sections of balloon-injured carotid arteries that were treated with an infusion of highly concentrated (10 8 cfu/ml) antisense cyclin Gl retroviral vector supematant showed a signifcant reduction in neointima formation. Taken together, these data support the utility of retroviral vectors bearing cyclin Gl, alone or in combination with p53 or the now- classic Herpes Simplex Virus thymidine kinase/GCV approach, in the development of novel gene therapy strategies to combat vascular restenosis.

The disclosures of all patents, publications, (including published patent applications) , database accesεion numbers, and depository accession numbers referenced in this specification are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication, database accession number, and depository accession number were specifically and individually indicated to be incorporated by reference.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention mav be practiced other than aε particularly deεcribed and εtill be within the scope of the accompanying claims.