Hyaluronic acid mediated adenoviral transduction Number:7,144,870 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides methods of treatment of adenoviral mediated disease, improved methods for transducing cells with adenoviral and related vectors, and improved methods of gene therapy utilizing such methods.<H transduction, adenoviral, Hyaluronic, acid, 7144870.html, Patent, pto, 7144870

Title: Hyaluronic acid mediated adenoviral transduction

Abstract: The present invention provides methods of treatment of adenoviral mediated disease, improved methods for transducing cells with adenoviral and related vectors, and improved methods of gene therapy utilizing such methods.

Patent Number: 7,144,870 Issued on 12/05/2006 to Chaudhuri, et al.

Inventors: Chaudhuri; Saumya-Ray (West Bengal, IN), Hurwitz; Mary Y. (Houston, TX), Holcombe; Vien (Houston, TX), Marcus; Karen T. (Sugarland, TX), Hurwitz; Richard L. (Houston, TX)
Assignee: Research Development Foundation (Carson City, NV)

Appl. No.: 10/367,581

Filed: February 14, 2003

Current U.S. Class: 514/44 ; 435/320.1; 435/325; 536/23.1

Current International Class: C07H 21/04 (20060101); C12N 15/63 (20060101); C12N 5/00 (20060101)

Field of Search: 514/44

References Cited [Referenced By]

U.S. Patent Documents


4141973	February 1979	Balazs
4801619	January 1989	Lindblad
4808526	February 1989	Lawford
4840941	June 1989	Ueno et al.
5670488	September 1997	Gregory et al.
5824544	October 1998	Armentano et al.
5932210	August 1999	Gregory et al.
6194392	February 2001	Falk et al.
6218373	April 2001	Falk et al.
6258791	July 2001	Braun
6271216	August 2001	Mello et al.
6312681	November 2001	Engler et al.
2001/0046965	November 2001	Ayares et al.
2002/0004040	January 2002	Kovesdi et al.

Foreign Patent Documents


2131130	Mar., 1996	CA
WO 97/15330	May., 1997	WO

Other References

Gunther, M et al. Curr Med Chem Anti-Cancer Agents 5(2):157-71, 2005. cite- d by examiner .
Chevez-Barrios et al., "Gene therapy for retinoblastoma: comparison of gene repalcement with RB gene suicide gene," Laboratory Investigation, 80(3):198A, #1167, 2000. cited by other .
Abe et al., "Transduction of a drug-sensitive toxic gene into human leukemia cell lines with a novel retroviral vector (43611)," Proc. Soc. Exp. Biol. Med., 203:354-359, 1993. cited by other .
Banerjee et al. "Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13.sup.1," Cancer Res., 52:6297-6304, 1992. cited by other .
Behbakht et al., "Adenovirus-mediated gene therapy of ovarian cancer in a mouse model," Am. J. Obstet. Gynecol., 175(5):1260-1265, 1996. cited by other .
Bett et al., Packaging capacity and stability of human adenvirus type 5 vectors, J. Virol., 67(10):5911-5921, 1993. cited by other .
Blackwell et al., "Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma," Arch. Otolaryngol. Head. Neck Surg., 125(8):856-863, 1999. cited by other .
Chen et al., "Combination gene therapy for liver metastasis of colon carcinoma in vivo," Proc. Natl. Acad. Sci. USA, 92(7):2577-2581, 1995. cited by other .
Chen et al., "Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo," Proc. Natl. Acad. Sci. USA, 91(8):3054-3057, 1994. cited by other .
Chillon et al., "Group D adenovirous infect primary central nervous system cells more efficiently than those from group C," J. Virol., 73(3):2537-2540, 1999. cited by other .
Chroboczek et al., "The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2," Virology, 186:280-285, 1992. cited by other .
da Cruz et al., "Dynamics of gene transfer to retinal pigment epithelium," Invest. Opthalmol. Vis. Sci., 37(12):2447-2454, 1996. cited by other .
Dorai et al., "A recombinant defective adenoviral agent expressing anti-Bcl-2 ribozyme promotes apoptosis of Bcl-2-expressing human prostate cancer cells," Int. J. Cancer, 82(6):846-852, 1999. cited by other .
Eastham et al., "Prostate cancer gene therapy: herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models," Hum. Gene Ther., 7(4):515-523, 1996. cited by other .
Esandi et al., "Gene therapy of experimental malignant mesothelioma using adenovirous vectors encoding the HSVtk gene," Gene Ther., 4(4):280-287, 1997. cited by other .
Evans, "Latent adenovirus infections of the human respiratory tract," Am. J. Hyg. 67:256-263, 1958. cited by other .
Feldman et al., "Perspectives of arterial gene therapy for the prevention of restenosis," Cardiovasc. Res., 32(2):194-207, 1996. cited by other .
Flomenberg et al., "Increasing incidence of adenovirus disease in bone marrow transplant recipients," J. Infect. Dis., 169:775-781, 1994. cited by other .
Goebel et al., "Adenovirus-mediated gene therapy for head and neck squamous cell carcinomas," Ann. Otol. Rhinol. Laryngol., 105(7):562-567, 1996. cited by other .
Goldstein et al, "Defective lipoprotein receptors and atherosclerosis-lessons from an animal counterpart of familial hypercholesterolemia," New Engl. J. Med., 309(11983):288-296, 1983. cited by other .
Graham and Prevec, "Methods for construction of adenovirus vectors," Mol Biotechnol., 3(3):207-220, 1995. cited by other .
Han et al., "Receptor-mediated gene transfer to cells of hepatic origin by galactosylated albumin-polylysine complexes," Biol. Pharm. Bull., 22(8):836-840, 1999. cited by other .
Hermens and Verhaagen, "Viral vectors, tools for gene transfer in the nervous system," Prog. Neurobiol., 55(4):399-432, 1998. cited by other .
Hoekstra, "Hyaluronan-modified surfaces for medical devices," Medical Device & Diagnostic Industry Magazine, Feb. 1999. cited by other .
Horwitz, In: Virology, 2d edit., Fields (Ed.), Raven Press, Ltd. New York, 1990. cited by other .
Hurwitz et al., "Suicide gene therapy for treatment of retinoblastoma in a murine model," Hum. Gene Ther., 10:441-448, 1999. cited by other .
Irie et al., "Therapeutic efficacy of an adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer," Antisense Nucleic Acid Drug Dev., 9(4):341-349, 1999. cited by other .
Ishibashi et al, "Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery," J. Clin. Invest., 92:883-893, 1993. cited by other .
Ishibashi et al, "Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein receptor-negative mice," J. Clin. Invest., 93:1885-1893, 1994. cited by other .
Kreil, "Hyalurnidases--a group of neglected enzymes," Protein Sci., 4(9):1666-1669, 1995. cited by other .
Laurent et al., (eds.) The Chemistry, Biology and Medical Applications of Hyaluronan and its Derivatives, Wenner-Gren Internatinal Series, vol. 72, c1998. cited by other .
Lee and Spicer, "Hyaluronan: a multifunctional, megaDalton, stealth molecule," Curr. Opin. Cell Biol., 12:581-586, 2000. cited by other .
Lesch, "Gene transfer to the brain: emerging therapeutic strategy in psychiatry?" Biol Psychiatry, 45(3):247-253, 1999. cited by other .
Marienfeld et al., "Autoreplication of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes," Gene Ther., 6(6):1101-1113, 1999. cited by other .
Mincheff et al., "Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial," Eur. Urol., 38(2):208-217, 2000. cited by other .
Morrison et al., "Complete DNA sequence of canine adenovirous type 1," J. Gen. Virol., 78(Pt 4):873-878, 1997. cited by other .
Neumann et al., "Detection of adenovirus nucleic acid sequence in human tonsils in the absence of infectious virus," Virus Res., 7:93-97, 1987. cited by other .
O'Malley et al., "Adenovirus-mediated gene therapy for human head and neck squamous cell cancer in a nude mouse model," Cancer Res., 55(5):1080-1085, 1995. cited by other .
Parks et al., "A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging," J. Virol., 71(4):3293-3298, 1997. cited by other .
Petrof, "Respiratory muscles as a target for adenovirus-mediated gene therapy," Eur. Respir. J., 11(2):492-497, 1998. cited by other .
Reddy et al., "Nucleotide sequence and transcription map of procine adenovirus type 3," Virology, 251(2):414-426, 1998. cited by other .
Robbins et al., "Viral vectors for gene therapy," Trends Biotechnol., 16(1):35-40, 1998. cited by other .
Rooney et al., "Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation," Lancet., 345:9-13, 1995. cited by other .
Rosenfeld et al., "Adenoviral-mediated delivery of herpes simplex virus thymideine kinase results in tumor reduction and prolonged survival in a SCID mouse model of human ovarian carcinoma," J. Mol. Med., 74(8):455-462, 1996. cited by other .
Schmitz et al., "Worldwide epidemiology of human adenovirus infections," Am. J. Epidemiol., 117-455-466, 1983. cited by other .
Shiver et al., "Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity," Nature, 415:331-335, 2002. cited by other .
SIGMA.TM. 2002-2003 Catalog of Biochemical and Reagents for Life Science research. Sigma-Aldrich Co., Inc. P.O. Box 14508, St. Louis, MO 63178, USA, 2002. cited by other .
Spandidos et al., "Expression of the normal H-ras1 gene can suppress the transformed and tumorigenic phenotypes induced by mutant ras genes," Anticancer Res., 10:1543-1554, 1990. cited by other .
Spicer and McDonald, "Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family," J. Biol. Chem., 273:1923-1932, 1998. cited by other .
Spicer, "In vitro assays for hyaluronan synthase," Methods Mol. Biol., 171:373-382, 2001. cited by other .
Stewart et al., "Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial," Gene Ther., 6(3):350-363, 1999. cited by other .
Tanzawa et al., "WHHL-rabbit: a low density lipoprotein receptor-deficient animal model for familial hypercholesterolemia," FEBS Letters, 118(1):81-84, 1980. cited by other .
Vanderkwaak and Alvarez, "Immune directed therapy for ovarian carcinoma," Curr. Opin. Obstet. Gynecol., 11(1):29-34, 1999. cited by other .
Wadell et al., "Genetic variability of adenoviruses," Ann. NY Acad. Sci., 354:16-42, 1980. cited by other .
Watanabe, "Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit)," Atherosclerosis, 36:261-268, 1980. cited by other .
Wilson, "Vehicles for gene therapy," Nature, 365:691-692, 1993. cited by other .
Wilson, "When bad gene transfer if good," J. Clin. Invest., 98(11):2435, 1996. cited by other .
Yotnda et al., "Efficient infection of primitive hematopoietic stern cells by modified adenovirus," Gene Ther., 8:930-937, 2001. cited by other .
Chevez-Barrios et al., "Response to retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir," J. of Clinical Oncology, 23:7927-7935, 2005. cited by other .
Mallam et al., "Efficient gene transfer into retinal cells using adenoviral vectors: dependence on receptor expression," Investigative Ophthalmology & Visual Science, 45:1680-1687, 2004. cited by other .
Shayakhmetov et al., "Efficient gene transfer into human cd34+ cells by a retargeted adenovirus vector," J of Virology. 74:2567-2583, 2000. cited by other .
Bourguignon et al., "Interaction between the adhesion receptor, CD44, and the oncogene product, p 185HER2, promotes human ovarian tumor cell activation," Bio. Chem., 272:27913-27918, 1997. cited by other.

Primary Examiner: Woitach; Joseph
Assistant Examiner: Noble; Marcia S.
Attorney, Agent or Firm: Fulbright and Jaworski L.L.P.

Parent Case Text

BACKGROUND OF THE INVENTION

Claims

We claim:

1. A method of gene delivery to cells of the eye in a subject for treating a cancer of the eye in a subject comprising administering to the eye of said subject an AdV5/F35 vector comprising a transgene that encodes a therapeutic protein, wherein expression of said therapeutic protein results in treatment of cancer of the eye.

2. The method of claim 1, wherein said subject is human.

3. The method of claim 2, wherein said vector is administered into the eye.

4. The method of claim 3, wherein said vector is administered via intravitreous injection.

5. The method of claim 1, wherein said cancer is retinoblastoma.

6. The method of claim 1, wherein said method of gene delivery is combined with administration of a second therapeutic agent.

7. The method of claim 6, wherein said transgene is thymidine kinase.

8. The method of claim 7, wherein said thymidine kinase is herpes simplex virus thymidine kinase.

9. The method of claim 7, wherein said second therapeutic agent is ganciclovir.

10. The method of claim 9, wherein ganciclovir is administered intravenously.

11. The method of claim 10, wherein the number of tumors in said subject is reduced.

12. The method of claim 1, wherein said transgene is p53 or a retinoblastoma gene.

Description

The present application claims the benefit of U.S. Provisional Application Ser. No. 60/357,485 filed Feb. 15, 2002, the entire text of which is herein incorporated by reference.

1. Field of the Invention

The present invention is directed to the fields of molecular biology, gene therapy, and treatment of viral disease. More specifically, the present invention relates to methods of treatment of adenoviral infection and disease, to improved methods for expressing transgenes introduced into cells with adenoviral and related vectors, and improved methods of gene therapy utilizing such methods.

2. Description of Related Art

Wild-type adenoviruses are associated with a variety of human diseases including respiratory, ocular, and gastrointestinal infections. These infections are a major cause of school absenteeism for children and of loss of work productivity for adults. In immuno-compromised individuals, infection with adenovirus currently has no effective antiviral treatment and is frequently fatal. Adenovirus infections may thus be lethal to immunocompromised patients who have received chemotherapy, bone marrow transplants, other organ transplants, or suffer from AIDS. Pediatric bone marrow transplant patients are particularly susceptible, with 10 30% developing adenovirus infection.

There are no anti-viral compounds that are effective against adenovirus infections. Thus, there is a need in the art to develop an effective treatment for adenoviral infection, especially for immunocompromised individuals.

In contrast, non-pathogenic replication-defective adenoviral vectors are useful for many preclinical and clinical gene therapy applications. Human gene therapy is an approach to treating human disease that is based on the modification of gene expression in cells of the patient. It has become apparent over the last decade that the single most outstanding barrier to the success of gene therapy as a strategy for treating inherited diseases, cancer, and other genetic dysfunctions is the development of useful gene transfer and expression vehicles. Eukaryotic viruses have been employed as vehicles for somatic gene therapy. Among the viral vectors that have been cited frequently in gene therapy research are adenoviruses.

Modified adenoviruses that are replication incompetent and therefore non-pathogenic are being used as vehicles to deliver therapeutic genes for a number of metabolic and oncologic disorders. These adenoviral vectors may be particularly suitable for disorders such as cancer that would best be treated by transient therapeutic gene expression since the DNA is not integrated into the host genome and the transgene expression is limited. Adenoviral vector may also be of significant benefit in gene replacement therapies, wherein a genetic or metabolic defect or deficiency is remedied by providing for expression of a replacement gene encoding a product that remedies the defect or deficiency.

Adenoviruses can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. Recombinant adenoviruses types 2 and 5 (Ad2 and AdV5, respectively), which cause respiratory disease in humans, are among those currently being developed for gene therapy. Both Ad2 and AdV5 belong to a subclass of adenovirus that are not associated with human malignancies. Recently, the hybrid adenoviral vector AdV5/F35 has been developed and proven of great interest in gene therapies and related studies (Yotnda et al., 2001).

Recombinant adenoviruses are capable of providing extremely high levels of transgene delivery. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders (Watanabe, 1986; Tanzawa et al., 1980; Golasten et al., 1983; Ishibashi et al., 1993; and S. Ishibashi et al., 1994). Indeed, a recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved for use in at least two human CF clinical trials (Wilson, 1993). Hurwitz, et al., (1999) have shown the therapeutic effectiveness of adenoviral mediated gene therapy in a murine model of cancer (retinoblastoma).

Unfortunately, adenoviral vectors, although effective at transducing target cells, do not necessarily result in the desired level of expression of the transgene in the target cells and tissues. An exception has been noted in the ocular environment where relatively high levels of transgene expression have been observed.

There is therefore a need for effective treatment of wild type adenoviral infection, especially in immunocompromised individuals. There is also a need for methods and compositions that are effective in enhancing the expression of transgenes introduced into a wide variety of cell types and tissue.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods useful in the treatment of adenoviral mediated disease and adenoviral infection and improved expression of transgenes delivered to cells using adenoviral and related vectors.

Therefore, a preferred embodiment of the present invention is a method of treating adenoviral disease comprising identifying a subject in need of treatment for adenoviral disease and administering to the subject a composition comprising an adenoviral inhibitor in an amount sufficient to inhibit or prevent adenoviral disease. Another preferred embodiment is a method for inhibiting adenovirus infection comprising administering to a cell a composition comprising an adenovirus inhibitor in an amount sufficient to inhibit the progress of adenovirus infection.

A related embodiment is a method for treating adenoviral disease comprising identifying a subject in need of treatment for adenoviral infection and inhibiting adenoviral infection by administering to a cell of the subject a composition comprising an adenovirus inhibitor in an amount sufficient to inhibit the progress of adenovirus infection. An additional preferred embodiment is a method for treating adenoviral disease comprising identifying a subject in need of treatment and administering to adenoviral or adenoviral vector transduces cells of the subject an inhibitor of adenoviral-mediated transgene expression.

A further preferred embodiment is a method of inhibiting transgene expression comprising obtaining a cell transduced with an adenovirus or adenoviral vector and contacting the cell with an inhibitor of adenoviral-mediated transgene expression. In further preferred aspects, the cell is part of a tissue. Additional preferred embodiments include those wherein the cell is a vertebrate cell, a mammalian cell, a primate cell, or a human cell. Of course, the cell may be non-human. In particular embodiments, the subject of treatment is human. In others, the subject of treatment is non-human.

In certain preferred embodiments, the adenoviral inhibitor comprises low molecular weight hyaluron. The molecular weight of low molecular weight hyaluron is contemplated to be such that the hyaluron has an average molecular weight that may range from less than 750,000 Da to the lowest molecular weights of hyaluronic acid, i.e. a single repeating unit. Therefore, it is contemplated that the average molecular weight of low molecular weight hyaluron may be less than 750,000; 650,000; 600,000; 550,000; 500,000; 450,000; 400,000; 350,000; 300,000; 250,000; 200,000; 150,000; 100,000; 50,000 Da or less or any range derivable therein.

An alternative means of denoting low molecular weight hyaluron within embodiments of the present invention is where the low molecular weight hyaluron displays a substantially lower molecular weight than high molecular weight hyaluron. In particular embodiments the low molecular weight hyaluron displays a substantially lower molecular weight than high molecular weight hyaluron when compared by agarose gel electrophoresis.

In additional embodiments of the invention, the adenoviral inhibitor comprises degradation products of high molecular weight hyaluron. Such degradation products may be obtained by a number of means. In one embodiment, the degradation products are comprised of out of date hyaluron. Out of date hyaluron is defined as hyaluron that is past the date beyond which the hyaluron would not be acceptable as a clinical composition or as useful as high molecular weight hyaluron. Such dates are typically provided on commercial samples of hyaluron by the manufacturer but may also be determined by routine experimentation. Similarly, in another embodiment, the inhibitor comprises degradation products of vitreous. In one such embodiment, such degradation products are out of date vitreous.

In further embodiments the adenoviral inhibitor comprises the products of treatment of high molecular weight hyaluron with lyase or hyaluronidase. Similarly, in one embodiment the inhibitor comprises vitreous treated with lyase or hyaluronidase.

In further preferred embodiments cells to be treated are incubated in a solution comprising the inhibitor. In specific embodiments thereof, the inhibitor is low molecular weight hyaluron. The concentration of the low molecular weight hyaluron may range from about 30 micrograms per 100 microliters of solution to more than 240 micrograms per 100 microliters. Thus, the concentration of low molecular weight hyaluron may be 30 micrograms per 100 microliters, 60 micrograms per 100 microliters, 120 micrograms per 100 microliters, or 240 micrograms per 100 microliters or any concentration derivable therein. Further, in particular embodiments, the concentration of low molecular weight hyaluron may approach saturation.

Because the scope of the invention includes methods and compositions for the enhancement of adenoviral-mediated transgene expression, a preferred embodiment is a method for enhancing transgene expression in a cell comprising obtaining a cell transduced by an adenoviral vector and contacting the cell with an enhancer of adenoviral-mediated transgene expression.

In certain embodiments the cell may be contacted with the enhancer over a time period of from 2 to 20 hours after transduction, or any range derivable therein. Preferred embodiments include those wherein the cell is contacted with the enhancer over a time of from 2 to 4, 6, or 8 hours after transduction, or any shorter time period within those. In certain embodiments the cell is contacted with the enhancer 2 hours after transduction. Of course, in certain other preferred embodiments, the cell is contacted with the enhancer substantially simultaneously with transduction. In yet further preferred embodiments, the cell is contacted with the enhancer continuously from transduction onwards.

In preferred embodiments of the invention relating to enhancement of adenoviral-mediated transgene expression the enhancer comprises vitreous, high molecular weight hyaluron, or mixtures thereof. In particular embodiments the enhancer comprises low molecular weight hyaluron in combination with vitreous. In further particular embodiments the enhancer is vitreous. In other certain preferred embodiments the enhancer is high molecular weight hyaluron.

Preferred related embodiments comprise the step of incubating a cell transduced by an adenovirus or adenoviral vector in a composition comprising the enhancer. When the enhancer is vitreous, particular embodiments include those wherein the concentration of vitreous in the composition is in the range of 0.5% to 5% (v/v). Specific embodiments include those wherein the concentration of vitreous in the composition is about 0.5%, 2.5%, or 5%.

In yet further preferred embodiments the enhancer comprises high molecular weight hyaluron. The molecular weight of high molecular weight hyaluron is contemplated to be such that the hyaluron has an average molecular weight that may range from more than 750,000 Da to the highest molecular weights of hyaluron, i.e. over several million Da or more. Therefore, it is contemplated that the average molecular weight of high molecular weight hyaluron may be greater than 650,000; 750,000; 1,000,000; or more, or any range derivable therein.

An alternative means of denoting high molecular weight hyaluron within embodiments of the present invention is where the high molecular weight hyaluron displays a substantially higher molecular weight than low molecular weight hyaluron. In particular embodiments the high molecular weight hyaluron displays a substantially higher molecular weight than low molecular weight hyaluron when compared by agarose gel electrophoresis.

When the enhancer is high molecular weight hyaluron, certain embodiments comprise concentrations of high molecular weight hyaluron ranging from 10 micrograms per 100 microliters to more than 100 micrograms per 100 microliters in a composition in which a cell is incubated.

Embodiments of the invention are not limited by the specific adenovirus or adenoviral vector employed. The inventors have discovered a means of enhancing or inhibiting adenoviral infection and transgene expression that is general to adenovirus and vectors derived therefrom. Of course, in specific embodiments the enhancer is contacted to a cell that is infected by adenovirus. In other particular embodiments, it is contemplated that the adenoviral vector be derived from adenovirus 5 or adenovirus 2 and their relatives. Of course, when formulated into a vector, adenoviral constructs may comprise transgenes for expression within cells.

Particular transgenes whose expression are contemplated for enhancement or inhibition by the methods and compositions of the present invention include, but are not limited to transgenes useful in treating cancer. In preferred embodiments the transgene is a gene useful in the treatment of retinoblastoma. In further particular embodiments the transgene is a retinoblastoma (RB) gene or a thymidine kinase (TK) gene. In other embodiments the transgene is a tumor suppressor gene. In particular embodiments, the tumor suppressor gene encodes p53. In other preferred embodiments, the transgene encodes a reporter gene. Reporter genes are well known to those of skill in the art and the choice of reporter gene is not limiting to the invention. Embodiments include those wherein the transgene is a luciferase, a green fluorescent protein, or a Beta-galactosidase gene.

The invention provides for enhancement of adenoviral-mediated transgene expression in cells. In particular embodiments the cell is part of a tissue. In further embodiments the cell is a vertebrate cell, a mammalian cell, a primate cell, or a human cell.

The inventors have additionally discovered a role for the CD44 protein in modulating transgene expression in the presence of hyaluron or vitreous. Therefore, one embodiment is a method of modulating transgene expression comprising contacting a cell transfected with an adenoviral vector and with at least one antibody specific to CD44. Related embodiments include the step of contacting the cell with high molecular weight hyaluron or vitreous either before, during, or after contacting the cell with at least one antibody specific to CD44. In certain embodiments the antibodies include at least one monoclonal antibody. In additional embodiments the antibody is KM114.

Further embodiments of the invention include a kit for the production of enhanced transgene expression comprising high molecular weight hyaluron or vitreous and components for adenoviral vector transfection.

Even further embodiments comprise methods of screening for forms of hyaluron. As will be appreciated by those of skill in the art, forms of hyaluron may be classified by their chemical structure, chemical properties, physical properties, and within the context of the invention, their effects upon adenoviral-mediated transgene expression. Non-limiting examples of such forms are high molecular weight hyaluron, low molecular weight hyaluron, hyaluron effective in inhibiting adenoviral-mediated transgene expression, and hyaluron effective in enhancing adenoviral-mediated transgene expression, and hyaluron effective in the treatment of adenoviral disease.

Therefore, certain embodiments comprise obtaining a first sample of hyaluron (a); obtaining a second sample of hyaluron of known form (b); and comparing the effects of the sample obtained in (a) on adenoviral-mediated transgene expression with the effects of the sample obtained in (b) on adenoviral-mediated transgene expression, wherein enhanced transgene expression indicates hyaluron of expression enhancing form and inhibited transgene expression indicates hyaluron of expression inhibiting form. Similarly, certain embodiments comprise the steps of comparing the samples of (a) and (b) by gel electrophoresis and correlating the electrophoretic mobility of the samples of (a) and (b) with their effects on adenoviral-mediated transgene expression. Particularly preferred embodiments comprise agarose gel electrophoresis.

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Vitreous enhances adenoviral-mediated transgene expression. Bars represent standard error from the mean.

FIG. 2. Hyaluronic acid lyase abrogates vitreous enhanced adenoviral-mediated transgene expression.

FIG. 3. Enhanced transgene expression is not dependent on adenoviral binding or internalization.

FIGS. 4A, 4B, 4C, and 4D. Vitreous enhances adenoviral-mediated transgene expression in cells transduced using an alternate adenoviral receptor. WERI-Rb cells (1.times.10.sup.4 cells/well) were incubated in serum-free media with (C,D) or without (A,B) 0.5% vitreous. The cells were transduced at a ratio of 5 vp/cell with a chimeric adenovirus (AdV5/F35) in which the fiber/knob domains of AdV35 replaced the fiber/knob domains of AdV5. Bright field (A,C) and fluorescent (B,D) photographs of representative fields are shown.

FIG. 5. Vitreous-mediated enhancement of transgene expression: Time dependence of vitreous addition. The reference line indicates baseline transgene expression in the absence of vitreous.

FIG. 6. Vitreous-mediated enhancement of transgene expression: Time dependence of vitreous addition. The reference line represents transgene expression in cells continuously exposed to 0.5% vitreous for the entire 20 hour incubation period.

FIG. 7. Activation of adenoviral-mediated transgene expression by hyaluron. Bars represent standard error from the mean.

FIG. 8. Inhibition of adenoviral-mediated transgene expression by "outdated" hyaluron. Bars represent standard error from the mean.

FIG. 9. PMA-activated CD44 is important for enhancement of adenoviral-mediated transgene expression. Bars represent standard error from the mean and significance was determined using the paired Student's t-test.

FIG. 10. Anti-CD44 inhibits adenoviral-mediated transgene expression. Bars represent standard error from the mean.

FIG. 11. Effect of boiling Vitreous on enhancement of adenoviral-mediated transgene expression.

FIG. 12. Effect of combined vitreous and low molecular weight hyaluron on enhancement of adenoviral-mediated transgene expression.

FIG. 13. Effect of Vitreous on Adenoviral mediated gene expression in human conjunctival explants.

FIG. 14. Lyase Digestion of High Molecular Weight (HMW) Hyaluron.

FIG. 15. Effect of Lyase-digested hyaluron on Adenoviral-mediated transgene expression.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have made the surprising discovery that an important component of the vitreous of the eye, hyaluron, modulates the expression of transgenes introduced into cells by adenoviral vectors and infectivity of wild type adenovirus. Surprisingly, the inventors have discovered that high molecular weight hyaluron, either on its own or as a component of vitreous, acts to enhance the expression of functional transgenes introduced into cells by adenoviral vectors outside of the ocular environment both in vivo and in vitro. This enhancement of expression is independent of attachment and entry of the adenoviral vector into the cell.

Even more surprising and significant is the inventors' discovery that low molecular weight hyaluron acts to inhibit adenoviral infection and adenovector transduction. Most significantly, hyaluron of low molecular weight, or that which has been modified or degraded by enzymatic or other treatment may be used to treat adenoviral infection and disease. Thus, low molecular weight or modified hyaluron or vitreous may serve as a prophylactic or therapeutic composition for the treatment of adenoviral infection.

By acting to inhibit is meant that an agent acts to partially or completely hinder, restrain, slow, diminish, retard, reduce, suppress, repress or interfere with a biological process to any extent, partially or completely. Thus, an inhibitor is an agent that is capable of partially or completely hindering, restraining, slowing, diminishing, retarding, curbing, restraining, reducing, suppressing, repressing or interfering with a biological process such as a biochemical reaction, viral or cellular growth or other physiological process, including, but not limited to infection or disease processes, disease or life cycle progress, organ function or performance and the like.

The inventors have further discovered that hyaluron activates a series of intracellular events through its interaction with CD44, a member of a family of adhesion molecules commonly found on many cell types. Thus, an antibody that specifically binds to the hyaluron binding domain and blocks hyaluron activation of CD44 inhibits both vitreous-enhanced and base line adenoviral-mediated transgene expression. Jurkat cells that have been engineered to express CD44, but not wild-type Jurkat cells, which are known not to express CD44 or any other hyaluron-binding receptor, can be shown to exhibit enhanced transgene expression delivered by adenoviral vectors in the presence of vitreous or high molecular weight hyaluron. Phorbol ester greatly enhanced the effect caused by vitreous. Previous reports have shown that PMA causes oligomerization and activation of CD44. Low molecular weight hyaluron, which can be produced by incubation with lyase (or by using outdated or degraded hyaluron), not only failed to enhance adenoviral-mediated transgene expression but surprisingly and unexpectedly inhibited base-line adenoviral vector-mediated transgene expression.

The enhancement effect is independent of the promoter or the transgene used but is specific for adenoviral vectors even if they use different binding and internalization receptors. A time course of this effect suggests that hyaluron enhancement of adenoviral-mediated transgene expression occurs after viral binding and internalization.

Previous reports have demonstrated that high molecular weight hyaluron can bind and activate CD44 signal transduction but low molecular weight hyaluron can bind but not activate the same mechanism. Thus low molecular weight hyaluron serves as an inhibitor of CD44 function. High molecular weight hyaluron enhances adenoviral-mediated transgene expression while low molecular weight hyaluron inhibits base line adenoviral transduction. The glucuronic acid--N-acetyl glucosamine disaccharide that forms the base unit of hyaluron can activate CD44 but only when injected intracellularly. CD44 has been implicated in many cellular functions including serving as a cell trafficking protein, controlling cytoskeleton structure and motility, and regulating intracellular protein trafficking by controlling microtubule and actin assembly. A CD44 regulatory cascade mediated by low molecular weight G-proteins may affect many of these functions.

1. Adenoviruses

Adenoviruses comprise linear double stranded DNA, with a genome ranging from 30 to 35 kb in size (Reddy et al., 1998; Morrison et al., 1997; Chillon et al., 1999). There are over 50 serotypes of human adenovirus, and over 80 related forms which are divided into six families based on immunological, molecular, and functional criteria (Wadell et al, 1980). Physically, adenovirus is a medium-sized icosahedral virus containing a double-stranded, linear DNA genome which, for adenovirus type 5, is 35,935 base pairs (Chroboczek et al., 1992). Adenoviruses require entry into the host cell and transport of the viral genome to the nucleus for infection of the cell and replication of the virus.

Salient features of the adenovirus genome are an early region (E1, E2, E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene), a late region (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP), inverted-terminal-repeats (ITRs) and a y sequence (Zheng, et al., 1999; Robbins et al., 1998; Graham and Prevec, 1995). The early genes E1, E2, E3 and E4 are expressed from the virus after infection and encode polypeptides that regulate viral gene expression, cellular gene expression, viral replication, and inhibition of cellular apoptosis. Further on during viral infection, the MLP is activated, resulting in the expression of the late (L) genes, encoding polypeptides required for adenovirus encapsidation. The intermediate region encodes components of the adenoviral capsid. Adenoviral inverted terminal repeats (ITRs; 100 200 bp in length), are cis elements, function as origins of replication and are necessary for viral DNA replication. The .psi. sequence is required for the packaging of the adenoviral genome.

The mechanism of infection by adenoviruses, particularly adenovirus serotypes 2 and 5, has been extensively studied. A host cell surface protein designated CAR (Coxsackie Adenoviral Receptor) has been identified as the primary binding receptor for these adenoviruses. The endogenous cellular function of CAR has not yet been elucidated. Interaction between the fiber knob and CAR is sufficient for binding of the adenovirus to the cell surface. However, subsequent interactions between the penton base and additional cell surface proteins, members of the .alpha..sub.v integrin family, are necessary for efficient viral internalization. Disassembly of the adenovirus begins during internalization; the fiber proteins remain on the cell surface bound to CAR. The remainder of the adenovirus is dissembled in a stepwise manner as the viral particle is transported through the cytoplasm to a pore complex at the nuclear membrane. The viral DNA is extruded through the nuclear membrane into the nucleus where viral DNA is replicated, viral proteins are expressed, and new viral particles are assembled. Specific steps in this mechanism of adenoviral infection may be potential targets to modulate viral infection and gene expression.

2. Engineered Adenoviruses and Adenoviral Vectors

In particular embodiments, an adenoviral expression vector is contemplated for the delivery of expression constructs. "Adenovirus expression vector" or "Adenoviral vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein. Thus, an Adenoviral vector may include any of the engineered vectors that comprise Adenoviral sequences.

An adenovirus expression vector according to the present invention comprises a genetically engineered form of the adenovirus. The nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the known serotypes and/or subgroups A F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain one adenovirus vector for use in the present invention. This is because adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

Advantages of adenoviral gene transfer include the ability to infect a wide variety of cell types, including non-dividing cells, a mid-sized genome, ease of manipulation, high infectivity and they can be grown to high titers (Wilson, 1996). Further, adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner, without potential genotoxicity associated with other viral vectors. Adenoviruses also are structurally stable (Marienfeld et al., 1999) and no genome rearrangement has been detected after extensive amplification (Parks et al., 1997; Bett et al., 1993).

Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo (U.S. Pat. Nos. 5,670,488; 5,932,210; 5,824,544). This group of viruses can be obtained in high titers, e.g., 10.sup.9 to 10.sup.11 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells.

Although adenovirus based vectors offer several unique advantages over other vector systems, they often are limited by vector immunogenicity, size constraints for insertion of recombinant genes, low levels of replication, and low levels of transgene expression. A major concern in using adenoviral vectors is the generation of a replication-competent virus during vector production in a packaging cell line or during gene therapy treatment of an individual. The generation of a replication-competent virus could pose serious threat of an unintended viral infection and pathological consequences for the patient. Armentano et al., describe the preparation of a replication-defective adenovirus vector, claimed to eliminate the potential for the inadvertent generation of a replication-competent adenovirus (U.S. Pat. No. 5,824,544). The replication-defective adenovirus method comprises a deleted E1 region and a relocated protein IX gene, wherein the vector expresses a heterologous, mammalian gene.

A common approach for generating adenoviruses for use as a gene transfer vector is the deletion of the E1 gene (E1.sup.-), which is involved in the induction of the E2, E3 and E4 promoters (Graham and Prevec, 1995). Subsequently, a therapeutic gene or genes can be inserted recombinantly in place of the E1 gene, wherein expression of the therapeutic gene(s) is driven by the E1 promoter or a heterologous promoter. The E1.sup.-, replication-deficient virus is then proliferated in a "helper" cell line that provides the E1 polypeptides in trans (e.g., the human embryonic kidney cell line 293). Alternatively, the E3 region, portions of the E4 region or both may be deleted, wherein a heterologous nucleic acid sequence under the control of a promoter operable in eukaryotic cells is inserted into the adenovirus genome for use in gene transfer (U.S. Pat. Nos. 5,670,488; 5,932,210).

Of course, in particular embodiments of the present invention, it is contemplated that low molecular weight or modified hyaluron or vitreous may be used in treatment of such inadvertently produced replication-competent adenovirus. As stated above, vectors contemplated for use in the present invention are replication defective. However, approaches involving replication competent adenoviral vectors, leading to so-called amplification are also contemplated. Thus, particular embodiments are contemplated in which the extent and rate of amplification of replication competent adenoviral vectors is modulated through the application of low molecular weight or modified hyaluron or vitreous.

A class of chimeric adenoviral vector (AdV5/F35, for example) has been created that is capable of delivery of transgenes to hematopoietic progenitors cells. AdV5/F35 vectors are highly effective at transfering transgenes to primitive progenitor cells (Yotnda et al., 2001). These vectors are also capable of infecting the hoechst negative `side population` of marrow cells. Immunomodulatory genes delivered by these vectors are capable of transduction and some levels of expression over about 5 days. In a preferred embodiment of the present invention cells wherein a transgene has been introduced by such chimeric adenoviral vectors, exemplified but not limited to AdV5/F35, are treated with hyaluron sufficient to enhance transgene expression.

3. Gene Therapy with Adenovirus and Adenoviral and Related Vectors.

In certain preferred embodiments, the inventors contemplate the use of hyaluron or vitreous and related compounds to enhance the expression of transgenes introduced into cells for the purposes of gene therapy.

Gene therapy generally involves the introduction into cells of transgenes whose expression results in amelioration or treatment of disease or genetic disorders. The transgenes involved may be those that encode proteins, structural or enzymatic RNAs, inhibitory products such as antisense RNA or DNA, or any other gene product. Expression is the generation of such a gene product or the resultant effects of the generation of such a gene product. Thus, enhanced expression includes the greater production of any transgene product or the augmentation of that product's role in determining the condition of the cell, tissue, organ or organism. The delivery of transgenes by adenoviral vectors involves what may be termed transduction of cells. As used here, transduction is defined as the introduction into a cell a transgene or transgene construct by an adenoviral or related vector.

Many experiments, innovations, preclinical studies and clinical trials are currently under investigation for the use of adenoviruses as gene delivery vectors. For example, adenoviral gene delivery-based gene therapies are being developed for liver diseases (Han et al., 1999), psychiatric diseases (Lesch, 1999), neurological diseases (Smith, 1998; Hermens and Verhaagen, 1998), coronary diseases (Feldman et al., 1996), muscular diseases (Petrof, 1998), and various cancers such as colorectal (Dorai et al., 1999), bladder (Irie et al., 1999), prostate (Mincheff et al., 2000), head and neck (Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra et al., 1999) and ovarian (Vanderkwaak et al., 1999). In particular embodiments of the present invention, high molecular weight hyaluron or vitreous is employed in the enhancement of expression of the transgenes delivered by adenoviral vectors.

Thus, the objects of this invention may be accomplished by enhanced expression of a therapeutic gene contained within a gene delivery system, such as a recombinant adenoviral vector delivery system, formulated such that a transduced cell is exposed to the expression-enhancing agent, such as high molecular weight hyaluron or vitreous, and results in the enhanced expression of the therapeutic gene.

The transduced cell may exist in cell culture or in vivo. In vivo, cells as contemplated in the present invention may be located in any tissue or organ of the relevant organism. A tissue may comprise a host cell or cells to be transformed or contacted with a nucleic acid delivery composition and/or an additional agent. The tissue may be part or separated from an organism. In certain embodiments, a tissue and its constituent cells may comprise, but is not limited to, blood (e.g., hematopoietic cells (such as human hematopoictic progenitor cells, human hematopoietic stem cells, CD34.sup.+ cells CD4.sup.+ cells), lymphocytes and other blood lineage cells), bone marrow, brain, stem cells, blood vessel, liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stomach, testes.

In certain embodiments, the host cell or tissue may be comprised in at least one organism. In certain embodiments, the organism may be, human, primate or murine. In other embodiments the organism may be any eukaryote or eukaryotic cell susceptible to infection or transduction by adenovirus and related viruses or adenoviral vectors. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit their division to form progeny.

Gene therapy strategies have been developed for cancer therapy. Distinct approaches have been developed to treat neoplasms based on gene transfer methods. Methods have been developed to correct specific lesions at defined genetic loci which give rise to neoplastic transformation and progression (Spandidos et al., 1990; Banerjee et al., 1992). Overexpression of dominant oncogenes may be addressed using techniques to inhibit the transforming gene or gene product. Loss of tumor suppressor gene function may be approached using methods to reconstitute wild-type tumor suppressor gene function. Besides these methods to achieve mutation compensation, genetic techniques have been developed to specifically and selectively eradicate tumor cells. These approaches of molecular chemotherapy rely on specific expression of toxin genes in neoplastic cells (Abe et al., 1993). Finally, gene transfer methods have been used to achieve antitumor immunization. These methods of genetic immunopotentiation use techniques of genetic immunoregulation to enhance immune recognition of tumors. Consequently, a variety of distinct approaches have been developed to accomplish gene therapy of cancer, all of which may make use of adenoviral vectors for the tranduction of cells.

Hurwitz, et al. (1999) have described an adenoviral vector containing the herpes simplex thymidine kinase gene that, when transduced into Y79Rb human retinoblastoma cells in vitro, is effective in facilitating the killing of those retinoblastoma cells when treated with the prodrug ganciclovir. A murine model of retinoblastoma, created by the intravitreal injection of Y79Rb cells also responded to trandsduction and treatment. Therefore, gene therapy can effectively reduce the tumor burden in the murine model of human retinoblastoma. Clearly, enhanced expression of the transgene delivered by the adenoviral vector would further enhance the killing effects of the treatment. In particular, the enhancement of adenoviral vector expression within the environment of the eye, i.e. the vitreous would be advantageous. More generally, AdV-TK/ganciclovir suicide gene therapy has been shown to be effective in treating a wide variety of tumors in animal models. See, e.g. Eastham et al., 1996 (prostate); Chen et al., 1994 (brain); Chen et al., 1995 (hepatic); O'Malley et al., 1995 and Goebel et al., 1996 (neck); Behbakht et al., 1996 and Rosenfeld et al., 1996 (ovarian); Esandi et al., 1997 (mesothelioma). Particular embodiments of the present invention include the use of hyaluron in the enhancement or augmentation of AdV-TK/gancilovir gene therapy.

The particular transgene delivered by the adenoviral vector is not limiting and includes those useful for various therapeutic and research purposes, as well as reporter genes and reporter gene systems and contructs useful in tracking the expression of transgenes and the effectiveness of adenoviral and adenoviral vector transduction. Thus, by way of example, the following are classes of possible genes whose expression may be enhanced by using the compositions and methods of the present invention: developmental genes (e.g. adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth or differentiation factors and their receptors, neurotransmitters and their receptors), oncogenes (e.g. ABLI, BLC1, BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOX, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES), tumor suppresser genes (e.g. APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT1), enzymes (e.g. ACP desaturases and hycroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehycrogenases, amylases, amyloglucosidases, catalases, cellulases, cyclooxygenases, decarboxylases, dextrinases, esterases, DNA and RNA polymerases, hyaluron synthases, galactosidases, glucanases, glucose oxidases, GTPases, helicases, hemicellulases, hyaluronidases, integrases, invertases, isomersases, kinases, lactases, lipases, lipoxygenases, lyases, lysozymes, pectinesterases, peroxidases, phosphatases, phospholipases, phophorylases, polygalacturonases, proteinases and peptideases, pullanases, recombinases, reverse transcriptases, topoisomerases, xylanases), and reporter genes (e.g. Green fluorescent protein and its many color variants, luciferase, CAT reporter systems, Beta-galactosidase, etc.).

The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors p53, p16 and C-CAM are described below.

p53 currently is recognized as a tumor suppressor gene. High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses, including SV40. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently-mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum of other tumors.

The p53 gene encodes a 393-amino-acid phophoprotein that can form complexes with host proteins such as large-T antigen and E1B. The protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue. Interestingly, wild-type p53 appears to be important in regulating cell growth and division. Overexpression of wild-type p53 has been shown in some cases to be anti-proliferative in human tumor cell lines. Thus, p53 can act as a negative regulator of cell growth (Weinberg, 1991) and may directly suppress uncontrolled cell growth or indirectly activate genes that suppress this growth. Thus, absence or inactivation of wild-type p53 may contribute to transformation. However, some studies indicate that the presence of mutant p53 may be necessary for full expression of the transforming potential of the gene.

Wild-type p53 is recognized as an important growth regulator in many cell types. Missense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene. A single genetic change prompted by point mutations can create carcinogenic p53. Unlike other oncogenes, however, p53 point mutations are know to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity. Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line. Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encoding wild-type p53 into two human breast cancer cell lines restores growth suppression control in such cells (Casey et al., 1991). A similar effect has also been demonstrated on transfection of wild-type, but not mutant, p53 into human lung cancer cell lines (Takahasi et al., 1992). p53 appears dominant over the mutant gene and will select against proliferation when transfected into cells with the mutant gene. Normal expression of the transfected p53 does not affect the growth of cells with endogenous p53. Thus, such constructs might be taken up by normal cells without adverse effects. It is thus proposed that the treatment of p53-associated cancers with wild-type p53 will reduce the number of malignant cells or their growth rate.

The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kina