WO2001070175A2

WO2001070175A2 - Osteocalcin promoter directed adenovirus replicaton for therapy

Info

Publication number: WO2001070175A2
Application number: PCT/US2001/009101
Authority: WO
Inventors: Leland W. K. Chung; Chinghai Kao; Thomas A. Gardner; Song-Chu Ko
Original assignee: The University Of Virginia Patent Foundation
Priority date: 2000-03-21
Filing date: 2001-03-21
Publication date: 2001-09-27
Also published as: IL151818A0; JP2003527128A; WO2001070175A3; AU2001252941A1; CA2403795A1; EP1272615A2

Abstract

The present invention relates to replication-competent adenovirus vectors which are specific for cells which allow an osteocalcin transcriptional regulatory sequence to function, such as prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR), and methods of use of such viruses are provided. These viruses comprise an adenoviral gene under control of an osteocalcin transcriptional regulatory sequence. The gene can be, for example, a gene required for adenoviral replication. The viruses can also comprise at least one additional adenoviral gene under control of at least one additional tissue-specific transcriptional regulatory sequence. Thus, virus replication can be restricted to target cells exhibiting osteocalcin gene expression. Such replication-competent adenovirus vectors are useful in the treatment of prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, occular melanoma, lung cancer, or breast cancer.

Description

OSTEOCALCIN PROMOTER DIRECTED ADENOVIRUS REPLICATION FOR

THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. section 119(e) of co- pending U.S. provisional application 60/191,063, filed March 21, 2000, the entire text of which is herein incorporated by reference without disclaimer.

I. INTRODUCTION

The invention generally relates to targeted therapy using reconibinant vectors and particularly adenovirus vectors. The invention specifically relates to replication- conditional adenovirus vectors and methods for using them. Such adenovirus vectors are able to selectively replicate in a tissue-specific and tumor-restrictive manner to provide a therapeutic benefit from the presence of the adenovirus vector per se and/or from heterologous gene products expressed from the vector. More particularly, the present invention relates to methods and compositions related to novel viral vectors which can be used as therapeutic agents for treating metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, breast cancer. Additionally, this invention finds application in the treatment of benign but nonetheless serious and life threatening conditions and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis. These applications of this invention are discussed below, first in terms of treatment of metastatic cancers and then in terms of treatment of other tissues. There remains, however, a single modality of treatment common to all these applications—the systemic administration of an adenovirus with a gene essential for replication under the control of an osteocalcin transcriptional regulatory sequence.

The transcriptional regulatory sequences utilized with the adenoviral vectors of the present invention are capable of selectively driving expression of an adenovirus gene essential for replication in a tissue-specific and tumor-restrictive manner. Thus, due to the tissue specificity of the transcriptional regulatory sequences used with the viral vectors, the viral vectors of the present invention are effective therapeutic agents not only when administered via direct application, such as by injection into the target tissue, but also when administered systemically to the body via intravenous administration, oral administration or the like, because gene expression will be limited and localized to specific cell types.

π. BACKGROUND OF THE INVENTION

A. CANCER THERAPY

Osteosarcoma, a bone cancer occurring primarily in teenagers and young adults, affects approximately 2100 individuals yearly in the United States (Boring, C. C, Squires, T. S., Tong, T., and Montgomery. S. Cancer statistics, 1994, CA Cancer J. Clin.,

44;7-26, 1994). This malignancy accounts for as many as 5% of all childhood malignancies and 60% of all malignant childhood bone tumors (Hudson, M., Jaffe, M. R., and Jaffe, N. Pediatric osteosarcoma: therapeutic strategies, results, and prognostic actors derived from a 10-year experience. J. Clin. Oncol., 8: 1988-1997, 1990). Despite radical surgical resection of the primary tumor and aggressive adjuvant chemotherapy, the overall 2-year metastasis-free survival rate approaches only 66%. More than 30% of patients with this disease develop lung metastasis within the first year (Link, M. P., Goorin, A. M., Mixer, A. W., Link, M. P., Goorin, A. M., Miser, A. W., Green, A. A., Pratt, C. H., Belasco, J. B., Pritchard, J., Malpas, J. S., Baker, A. R., Kirkpatrick, J. A., Ayala, A. O., Schuster, J. J., Abelson, H. T., Simone, J. V., and Vietti, T. J. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity. N. Engl. J. Med, 314: 1600-1602, 1991. Goorin, A. M., Perez- Atayde, A., Gebbhardt, M., et al. Weekly high-dose methotrexate and doxorubicin for osteosarcoma: the Dunn-Farber Cancer Institute/The Children's Hospital-Study HI. J. Clin. Oncol., 5: 1178-1184, 1987). The survival rate among those affected with osteosarcoma has not changed significantly over the past 10 years, despite changes in adjuvant chemotherapy,

Kane, M. J. Chemotherapy of advanced soft tissue and osteosarcoma. Semin. Oncol., 16:297- 304, 1989.

Prostate adenocarcinoma is the second leading cause of cancer death in North American men, with frequent metastases to the lymph nodes and bone (Landis, S. H., Murray, T., Bolden, S. and Wingo, P. A. Cancer statictics, 1998. CA Cancer J. Clin., 48: 6, 1998). A standard first-line treatment for prostate cancer metastasis is androgen ablation therapy, which delays disease progression, though recurrence with limited response to chemotherapy invariably occurs (Kantoff, P. W., Halabi, S., Conaway, M., Picus, J., Kirshner, J., Hars, N., Trump, D., Winer, E. P. and Nogelzang, Ν. J. Hydrocortisone with or without mitoxantrone in men with hormone-refractory prostate cancer: results of the Cancer and Leukemia Group B 9182 study. J. Clin. Oncol., 17: 2506, 1999). Patients who develop androgen-independent progression will die of this disease in about 12 months (Kantoff, P. W., Halabi, S., Conaway, M., Picus, J., Kirshner, J., Hars, N., Trump, D., Winer, E. P. and Nogelzang, Ν. J. Hydrocortisone with or without mitoxantrone in men with hormone-refractory prostate cancer: results of the Cancer and Leukemia Group B 9182 study. J. Clin. Oncol., 17: 2506, 1999). Among the various therapeutic options for the treatment of androgen-refractory prostate cancer are gene therapy with tumor suppressors (Ko, S. C, Gotoh, A., Thalmann, G. Ν., Zhau, H. E., Johnston, D. A., Zhang, W. W., Kao, C. and Chung, L. W. K. Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model. Hum. Gene Ther., 7: 1683, 1996), immune modulators (Sanda, M. G., Ayyagari, S. R., Jaffee, E. M., Epstein, J. L, Clif , S. L., Cohen, L. K., Dranoff, G., Pardoll,

D. M., Mulligan, R. C. and Simons, J. W. Demonstration of a rational strategy for human prostate cancer gene therapy. J. Urol., 151: 622, 1994, and toxic genes (Gotoh, A., Ko, S. C, Shirakawa, T., Cheon, J., Kao, C, Miyamoto, T., Gardner, T. A., Ho, L. J., Cleutjens, C. B., Trapman, J., Graham, F. L. and Chung, L. W. K. Development of prostate-specific antigen promoter-based gene therapy for androgen-independent human prostate cancer. J. Urol, 160: 220, 1998; Eastham, J. A., Chen, S. H., Sehgal, L, Yang, G., Timme, T. L, Hall, S. J., Woo, S. L. and Thompson, T. C. 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: 515, 1996; Koeneman, K. S., Kao, C, Ko, S. C, Yang, L., Wada, Y., Kallmes, D. A., Gillenwater, J. Y., Zhau, H. E., Chung, L. W. K. and Gardner, T.

A. Osteocalcin directed gene therapy for prostate cancer bone metastasis. World J. Urol., 18: 102, 2000; Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon, J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal- epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998). With respect to toxic gene therapy, several therapeutic genes, such as p53 and herpes simplex thymidine kinase, have been applied in cancer gene therapy with limited success. The major problem relates to the inefficient gene transduction rate that can be achieved with the current viral vector and delivery methodology. This difficulty can be overcome by granting the viral vector delivery vehicle the ability to propagate and infect other target cells. Thus by propagation of the viral vector, the desired recombinant gene construct will be delivered from the limited transduced cells to neighboring cells. Theoretically, so as long as one cell within a tumor nodule infected, the virus will replicate in the cell to produce more virus to infect neighboring tumor cells. This propagation will be continued until the entire tumor nodule is eradicated. However, virus replication needs to be controlled so that normal tissue will not be damaged.

The concept of delivery and expression of therapeutic toxic genes to tumor cells through the use of tissue-specific promoters has been well recognized. This approach could decrease the toxic effect of therapeutic genes on neighboring normal cells when virus- mediated gene delivery results in the infection of the normal cells. Examples include the uses of the albumin or α-fetoprotein promoter to target hepatoma cells (Kuriyama, S., Yoshikawa, M., Ishizaka, S., Taujli, T., Ikenaka, K., Kagawa, T., Morita, N., and Mikoshiba, K. A. potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector, Cell Struct. Punct, 16: 503-510, 1991), the bone morphogenic protein promoter for brain to target glioma cells (Shimizu, K. Selective gene therapy of malignant glioma using brain-specific promoters; its efficacy and basic investigation, Nippon Rinsbo, 52: 3053-3058, 1994), the tyrosinase promoter to kill melanoma cells (Vile, R. G., Nelson, J. A., Castleden, S., Chong, H., and Hart, I. R. Systemic gene therapy of murine melanoma using tissue specific expression of the HSVtk gene involves an immune component. Cancer Res., 54:6228-6234, 1994), and the carcinoembryonic antigen promoter for gastric carcinoma cells (Tanaka, T., Kanai. F., Okabe, S., Yoshida, Y., Wakimoto, H., Hamada, H., Shiratori, Y., Lan, K-H., Ishitobi, M., and Omata, M. Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro. Cancer Res., 46: 1341-1345, 1996). To date, the best studied therapeutic gene is herpes simplex virus TK gene. Herpes simplex virus-TK converts the pro-drug ACV to a phosphorylated form that is cytotoxic to dividing cells (Moolten, F. L., Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes; paradigm for a prospective cancer control strategy. Cancer Res., 46:5276-5281, 1986). Critical to successful results is the "bystander" effect, which confers cytotoxicity on neighboring nontransduced cells; effective tumor cell kill can be achieved without the delivery to and expression of suicide genes in every tumor cell in vivo. This approach has been demonstrated recently to be efficacious in causing regression of many solid tumors, including metastatic colon carcinoma in the rat liver, (Chen, S. JJ., Chen, X.H.L., Wang, Y., Kosal, K. E., Finegold, J. J., Rich, S. S., and Woo, S.L.C., Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc. Natl. Acad. Sci. USA. 92:2577-2581, 1995), gastric carcinoma, (Tanaka, T., Kanai. F., Okabβ, S., Yoshida, Y., Wakimoto, H., Hamada, H., Shiratori, Y., Lan, K-H., Ishitobi, M., and Omata, M.

Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigenproducing human gastric carcinoma cells in vitro. Cjancer Res., 46: 1341-1345, 1996), and malignant mesothelioma (Smythe, W. R., Hwang, B. S., Elshami, A. A., Amin, K. M., Eck, S., Davidson, B. L., Wilson, J. M., Kaiser, L. R., and Albelda, S. M. Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

Ann. Surg., 222:78-86, 1995).

With respect to replication competent vectors, based on the hypothesis that virus replication needs to be controlled in gene therapy so that normal tissue will not be damaged, several conditional replication-competent adenoviruses have been developed. For example, adenovirus ONYX-015 only replicates in p53 deficient tumor cells (Heise, C. C, et al. Cancer Gene Ther 6:499-504 (1999), the entire disclosure of which is incorporated herein by reference). Thus, another recent therapeutic option for the treatment of androgen- refractory prostate cancer is gene therapy with replication-competent adenovirus (Ad) which have their replication conditioned by a tissue-specific promoter, such as prostate-specific antigen (PSA) (Rodrigez, R., Schuur, E. R., Lim, H. Y., Henderson, G. A., Simons, J. W. and

Henderson, D. R. Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res., 57: 2559, 1997; Yu, D-. C, Sakamoto, G. T. and Henderson, D. R. Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon virus 764, an attenuated replication competent adenovirus for prostate cancer therapy. Cancer Res., 59: 1498, 1999; Yu, D-. C, Chen, Y., Seng, M., Dilley, J. and Henderson, D. R. The addition of adenovirus type 5 region E3 enables calydon virus 787 to eliminate distant prostate tumor xenografts. Cancer Res., 59: 4200, 1999.), (the entire disclosures of each of which are incorporated herein by reference). These replication- competent adenoviruses, however, are only capable of replicating in those cells expressing PSA.

Osteocalcin (OC), a noncollagenous Gla protein produced specifically in osteoblasts, is synthesized, secreted, and deposited at the time of bone mineralization (Price, P. A. Nitamin-K dependent formation of bone GLA protein (onteocalcin) and its function. Nitam. Horm., 42:65-108, 1985). A recent study showed that immunohistochemical staining of OC was positive in primary osteoblastic osteosarcoma and chondroblastic osteosarcoma specimens as well as in five of seven fibroblastic osteosarcomas (Park, Y. K., Yung, M. H., Kim, Y. W., and Park, H. R. Osteocalcin expression in primary bone tumors: in situ hybridization and immunohistochemical study. J. Korean Med. Sci., 10:268-273, 1995). In addition, OC activity was detected in a wide spectrum of human tumors. This is consistent with the clinical observations that many human tumors exhibited calcification characteristics both in the primary and at distant metastases. Thus, osteocalcin expression is also observed in disease tissues involving calcification, such as, for example, without limitation, benign prostate hyperplasia (BPH), cancers and artheroscierosis (see published International Application No WO 98/313376, the entire disclosure of which is incorporated herein by reference).

The osteocalcin promoter (OC) has been shown to be highly effective in directing the transcription of reporter genes in both rat and human osteosarcoma cell lines Ward W et al, J. Clin. Oncol. 1994; 12:1849-1858; Ducy P, et al, Molecular and Cellular Biology. 1995; 15:1858-1869, the entire disclosures of which are incorporated herein by reference). The activity of osteocalcin promoter has also been demonstrated to be osteoblast- specific in a transgenic mouse study. The OC promoter contains several species-specific and overlapping regulatory elements (Heinrichs, A. A., Banerjee, C, Bortell, R., Owen, T. A., Stein, J. L., Stein, G.S. and Lian, J. B. Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species-specific regulation. J. Cell. Biochem., 53: 240, 1993). The "osteocalcin-box" contains sites to bind factors such as homeobox MSX proteins, API, AP2, NF-1, viral core enhancer, c-AMP, and vitamin D and glucocorticoid receptors (Heinrichs, A. A., Banerjee, C, Bortell, R., Owen, T. A., Stein, J. L., Stein, G.S. and Lian, J. B. Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species-specific regulation. J. Cell. Biochem., 53: 240, 1993; Hoffmann, H.M., Beumer, T.L., Rahman, S., McCabe, L.R., Banerjee. C, Aslam, F., Tiro, J.A., Wijnen, A.F.N., Stein, J.L., Stein, G.S. and Lian, J.B. Bone tissue specific transcription of the osteocalcin gene: role of an activator osteoblast-specific complex, and suppressor Hox proteins that bind the OC box. J. Cell Biochem., 61: 310, 1996; Towler, D. A„ Rutledge, S J. and Rodan, G. A. Msx-2/Hox 8.1: a transcriptional regulator of the rat osteocalcin promoter. Mol. Endocrinol., 8: 1484, 1994; Liu, M. and Freedman, L. P. Transcriptional synergism between the vitamin D3 receptor and other nonreceptor transcription factors. Mol. Endocrinol., 8: 1593, 1994; Sneddon, W. B., Bogado, C. E., Kiernan, M. S. and Demay, M. B. DΝA sequences downstream from the vitamin D response element of the rat osteocalcin gene are required for ligand-dependent transactivation. Mol. Endocrinol., 11: 210, 1997; Sneddon, W. B. and Demay, M. B. Characterization of an enhancer required for 1 ,25-dihydroxyvitamin D3 -dependent transactivation of the rat osteocalcin gene. J. Cell. Biochem., 73: 400, 1999). The osteoblast-specific cis-acting element OSE2 binds to transcriptional activator of osteoblast differentiation, Osf2/Cbfal (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L. and Karsenty, G. Osf2/Cbfal: a transcriptional activation of osteoblast differentiation. Cell, 89: 747, 1997). Li YP, et al. demonstrated that sequences located in the first exon of the human osteocalcin gene possess a differentiation-related osteocalcin silencer element (OSE). Osteocalcin was rendered transcribable in UMR-106 cells and proliferating normal osteoblasts after deletion of the -3 to +51 region. Site-specific mutagenesis of this region revealed that a 7 bp sequence (TGGCCCT) (+29 to +35) is critical for silencing function (Li YP, et al, Nucleic Acids Res 1995 Dec 25 ;23 (24): 5064-72, the entire contents of which are incorporated herein by reference in their entirety)

Mouse OC promoter contains an additional OSE1 cis-acting element (Ducy, P. and Karsenty, G. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol. Cell. Biol., 15: 1858, 1995), but has anon-functional vitamin D responsive element (Heinrichs, A. A., Banerjee, C, Bortell, R., Owen, T. A., Stein, J. L.,

Stein, G.S. and Lian, J. B. Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species-specific regulation. J. Cell. Biochem., 53: 240, 1993). With respect to the rat osteocalcin regulatory sequence, previous studies have demonstrated that an intronic sequence, TTTCTTT (+118 to +124) is capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in cells of the osteoblast lineage, by interacting with a specific nuclear protein. Further analyses of intronic sequences have identified a second silencer motif in this region. Two copies of a CCTCCT motif are present within the first intron of the rat osteocalcin gene (+106 to +111 and +135 to +140) and are capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in rat osteosarcoma cells (Kearns, A.E., et al, Endocrinology 1999 Sep;140(9):4120-6, the entire contents of which are incorporated herein by reference). The sequences in the rat osteocalcin gene that lie 3' to the vitamin D response element (NDRE) contain a GGTTTGG motif (-420 to -414) that is essential for transcriptional activation of osteocalcin-CAT (OC- CAT) fusion genes by l,25(OH)2D3. Sneddon, W.B. et al, demonstrated that VDR- dependent transactivation of the rat osteocalcin gene requires not only the NDRE (-456 to - 442) but also sequences between -430 and -414. The protein(s) that interacts with these sequences is capable of enhancing transcription in both a position and orientation- independent fashion (Sneddon, W.B. et al., JCell Biochem 1999 Jun l;73(3):400-7, the entire contents of which are incorporated herein by reference in their entirety).

In the case of arteriosclerosis, while this calcification disease process is accompanied by the formation of arteriosclerotic plaques surrounding effective blood vessels, osteocalcin expression is increased in these plaques. Frequently, the over-expression of OC in these tissues is associated with increased calcium deposition. Jie et al., Calcified Tissue Intl. 59:352-356 (1996) and Jie et al., Atherosclerosis 116:117-123 (1995). See also Balica et al., Circulation 95:1954-1960 (1997). In U.S. Pat. No. 5,772,993 (the entire contents of which are incorporated herein by reference) and concurrent publications it was shown that a recombinant adenovirus containing TK gene under the control of the OC promoter, when supplemented with a prodrug ACV, could suppress osteosarcoma growth through intralesional injection in both rat and human osteosarcoma models (Cheon J, et al, Cancer Gene Therapy. 1997;4:359-365; and Ko S C, et al, Cancer Research. 1996; 56:4614-4619). OC promoter-directed toxic gene, herpes simplex virus thymidine kinase (HSV-TK), was found to be expressed in human prostate cancer cells when injected intratumorally in the prostate and the site of prostate cancer skeletal metastasis in patients (unpublished observations) (Koeneman, K. S., Kao, C, Ko, S. C, Yang, L., Wada, Y., Kallmes, D. A., Gillenwater, J. Y., Zhau, H. E., Chung, L. W. K. and Gardner, T. A. Osteocalcin directed gene therapy for prostate cancer bone metastasis. World J. Urol., 18: 102, 2000). Moreover, with respect to treatment of arteriosclerosis, the over-expression of OC, and accompanying calcium deposition, around the arteriosclerotic plaques formed lends itself to Ad-OC-TK-mediated gene therapy. The combined administration of this therapeutic agent, optionally coupled with the administration of ACN, offers gene therapy for actual regression of arteriosclerotic plaque, and effective treatment of arteriosclerosis. Ad-OC-TK can be administered safely to animals by intravenous route for the treatment osteosarcoma lung metastases (Shirakawa, T., Ko, S. C, Gardner, T. A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L. W. K. and Kao, C. In vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapy. Cancer Gene Ther., 5: 274, 1998) and to primary and metastatic prostate cancers (e.g. bone and lymph node) through intratumoral administration, despite the detection of adenoviruses in serum of the treated patients (Koeneman, K. S., Kao, C, Ko, S. C, Yang, L., Wada, Y., Kallmes, D. A., Gillenwater, J. Y., Zhau, H. E., Chung, L. W. K. and Gardner, T. A. Osteocalcin directed gene therapy for prostate cancer bone metastasis. World J. Urol., 18: 102, 2000). Indeed, the safety of Ad-OC-TK is far superior to that of another adenoviral toxic gene, Ad-CMN-TK, where an universal promoter, cytomegalovirus (CMN), was used to replace OC; upon intravenous administration of Ad-CMN-TK plus the pro-drug, gancyclovir (GCN) killed 75% of the animals whereas none of the Ad-OC-TK/GCN treated animals died from this treatment (Shirakawa, T., Ko, S. C, Gardner, T. A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L. W. K. and Kao, C. In vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapy. Cancer Gene

Ther., 5: 274, 1998; Brand, K., Arnold, W., Bartels, T., Lieber, A., Kay, M. A., Strauss, M. and Dorken, B. Liver-associated toxicity of the HSN-tk/GCN approach and adenoviral vectors. Cancer Gene Ther., 4: 9, 1997). Despite the superiority of Ad-OC-TK to that of Ad- CMN-TK, there is a need for a more effective therapeutically effective adenovirus vector for cancer therapy. Thus, in view of the above, the development of a novel tissue-specific and tumor-restrictive adenovirus vector for cancer therapy, which would be replication-competent in prostate tumor cells regardless of their androgen receptor and PSA status, would represent significant progress in the area of cancer therapy. The present invention is directed to another conditional replication-competent adenovirus, Ad-OC-Ela, based on the activity of osteocalcin promoter and the Ela gene product. The AdOC-Ela adenovirus can replicate in and destroy only those cells that support osteocalcin promoter activity, such as the cancerous cells of prostate cancer, brain cancer, ovarian cancer, thyroid cancer, osteosarcoma, ocular melanoma, lung cancer, breast cancer. This invention also finds application for the treatment of those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis.

m. SUMMARY OF THE INVENTION

The present invention relates to compositions related to novel viral vectors which can be used as therapeutic agents for treating metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, breast cancer. Additionally, this invention finds application for the treatment of benign but nonetheless serious and life threatening conditions and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis. The present invention further relates to novel methods for using the therapeutic compositions.

The invention is based, in part, on the fact that adenoviral vectors constructed with an osteocalcin transcriptional regulatory sequence described herein are capable of selectively driving expression of an adenovirus gene essential for replication in a tissue specific and tumor-restrictive manner. The invention is further based, in part, on the discovery that such adenoviral vectors can be used as therapeutic agents for treating prostate cancer, brain cancer, ovarian cancer, thyroid cancer, osteosarcoma, ocular melanoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH), and arteriosclerosis. Thus, due to the tissue- specificity and tumor-restrictiveness of the osteocalcin transcriptional regulatory sequence used with the adenoviral vectors, the adenovirus can be administered in a tumor-restrictive and tissue-specific manner, with the use of osteocalcin transcriptional regulatory sequence which allows for tissue specific expression of the adenovirus gene essential for replication and/or heterologous nucleotide sequence. An example of such an osteocalcin transcriptional regulatory sequence is the osteocalcin promoter which is activated only within cells of osteoblastic lineage and those disease tissues involving calcification, such as, for example, without limitation, benign prostate hyperplasia (BPH), and arteriosclerosis. Thus, an adenovirus vector constructed with an essential gene under the control of an osteocalcin transcriptional regulatory sequence can be expressed effectively and specifically in targeted tumor cells and tissues, thereby minimizing the side effects of expression of the adenovirus vector in non-osteoblastic cells and non-cancerous or non-arteriosclerotic cells.

In addition, due to the tissue specificity of the osteocalcin transcriptional regulatory sequence used with the adenoviral vectors, the viral vectors of the present invention are effective therapeutic agents not only when administered via direct application, such as by injection, but also when administered systemically to the body via intravenous administration, oral administration or the like, because gene expression will be limited and localized to specific, osteoblastic cell and disease tissues involving calcification, such as, for example, without limitation, benign prostate hyperplasia (BPH), cancers and arteriosclerosis.

In one embodiment, the invention provides an adenovirus vector comprising an adenovirus with an essential gene under transcriptional control of an osteocalcin transcriptional regulatory sequence. The osteocalcin transcriptional regulatory sequence is capable of mediating gene expression specific to cells which allow an osteocalcin transcriptional regulatory sequence to function, such as for example, and without limitation, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and those prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR). The osteocalcin transcriptional regulatory sequence can comprise a promoter and/or enhancer or enhancer-like sequence from an osteocalcin gene, provided that the osteocalcin transcriptional regulatory sequence is capable of mediating gene expression specific to cells expressing osteocalcin. In one embodiment, an osteocalcin transcriptional regulatory sequence comprises a promoter from an osteocalcin gene. In one embodiment, an osteocalcin transcriptional regulatory sequence comprises an enhancer or enhancer-like sequence from an osteocalcin gene. In one embodiment, an osteocalcin transcriptional regulatory sequence comprises a promoter from an osteocalcin gene and an enhancer or enhancer-like sequence from an osteocalcin gene. In one embodiment, the osteocalcin transcriptional regulatory sequence is transcriptionally active in cells which allow an osteocalcin transcriptional regulatory sequence to function, such as cells expressing osteocalcin.

In certain embodiments, an osteocalcin transcriptional regulatory sequence comprises the 1,370-bp nucleotide sequence of SEQ ID NO:l as shown in Figure 21. In certain embodiments, an osteocalcin transcriptional regulatory sequence comprises a portion of SEQ ID NO:l capable of mediating cell-specific transcription in osteocalcin-producing cells such as for example, without limitation, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR). In another embodiment, an osteocalcin transcriptional regulatory sequence comprises the sequence from about -290 to about +30 relative to the transcriptional start site of an osteocalcin gene (nucleotides about 141 to about 454 of SEQ ID NO:l). In another embodiment, an osteocalcin transcriptional regulatory sequence comprises the sequence from about -250 to about +30 relative to the transcriptional start site of an osteocalcin gene (nucleotide about 1 to about 454 of SEQ ID NO:l). In another embodiment, an osteocalcin transcriptional regulatory sequence comprises the sequence to about -236 to about -223 and/or the sequence to about -140 to about -117 (nucleotides about 191 to about 204 and/or about 286 to about 310, respectively, of SEQ ID NO:l), relative to the transcriptional start site of an osteocalcin gene, combined with a non-osteocalcin promoter. In yet another embodiment, an osteocalcin transcriptional regulatory sequence comprises the nucleotide sequence from nucleotides about 1 to about 100, about 1 to about 150, about 1 to about 200, about 1 to about 250, about 1 to about 300, about 1 to about 350, about 1 to about 400, about

1 to about 450, about 1 to about 500, about 1 to about 550, about 1 to about 600, about 1 to about 650, about 1 to about 700, about 1 to about 750, about 1 to about 800, about 1 to about 850, about 1 to about 900, about 1 to about 950, about 1 to about 1000, about 1 to about 1050, about 1 to about 1100, about 1 to about 1150, about 1 to about 1200, about 1 to about 1250, about 1 to about 1300, about 1 to about 1350, and about 1 to about 1370, respectively, of SEQ ID NO: 1. In each embodiment, an osteocalcin transcriptional regulatory sequence is defined as a transcriptional regulatory sequence or transcriptional regulatory sequence capable of effecting transcription in a cell, which allows an osteocalcin transcriptional regulatory sequence to function, such as a cell expressing osteocalcin, such as for example, without limitation, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR).

In some embodiments, the osteocalcin transcriptional regulatory sequence is human, mouse, or rat in origin. In some embodiments, the mouse or rat osteocalcin transcriptional regulatory sequence is capable of mediating prostate-specific gene expression in humans.

In some embodiments, the adenovirus gene under control of an osteocalcin transcriptional regulatory sequence contributes to cytotoxicity (directly or indirectly), such as a gene essential for viral replication, i one embodiment, the adenovirus gene is an early gene. In another embodiment, the early gene is El A. In another embodiment, the early gene is E1B. In yet another embodiment, both El A and E1B are under transcriptional control of an osteocalcin transcriptional regulatory sequence. In other embodiments, the adenovirus gene essential for replication is a late gene, hi various embodiments, the additional late gene is LI, L2, L3, L4, or L5.

In another embodiment, the adenovirus vector comprising an adenovirus gene under transcriptional control of an osteocalcin transcriptional regulatory sequence further comprises at least one additional adenovirus gene under transcriptional control of at least one additional osteocalcin-specific transcriptional regulatory sequence. In one embodiment, a composition comprises this adenovirus. In one embodiment, this composition further comprises a pharmaceutically acceptable excipient. hi one embodiment, the at least one additional osteocalcin-specific transcriptional regulatory sequence is a second osteocalcin transcriptional regulatory sequence. In one embodiment, the at least one additional osteocalcin transcriptional regulatory sequence can have a sequence different from that of the first osteocalcin transcriptional regulatory sequence. In one embodiment, the at least one additional osteocalcin-specific transcriptional regulatory sequence comprises an osteocalcin transcriptional regulatory sequence.

In other embodiments, the adenovirus vector can further comprise a heterologous gene or transgene, wherein said heterologous gene or transgene is under the transcriptional control of an osteocalcin transcriptional regulatory sequence. In one embodiment, the heterologous gene is a reporter gene such as for example, and without limitation, the luciferase reporter gene or beta-galactosidase reporter gene. In one embodiment, the heterologous gene is conditionally required for cell survival. In some embodiments, the transgene is a cytotoxic gene. hi another embodiment, a method of treating metastatic cancer in an individual is provided, the method comprising the step of administering to the individual an effective amount of an adenovirus vector in which an adenovirus gene is under transcriptional control of an osteocalcin transcriptional regulatory sequence, wherein the metastatic cancer is prostate cancer, brain cancer, ovarian cancer, thyroid cancer, osteosarcoma, ocular melanoma, lung cancer, or breast cancer, hi another embodiment, a method of treating metastatic cancer in an individual is provided, the method comprising the step of administering to the individual an effective amount of an adenovirus vector in which an adenovirus gene is under transcriptional control of an osteocalcin transcriptional regulatory sequence, wherein the metastatic cancer is prostate cancer, and wherein the prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells fail to express PSA or androgen receptor (AR). hi one embodiment, the adenovirus gene is essential for viral replication, hi one embodiment, the adenovirus gene is an early gene. In one embodiment, the adenovirus gene is E1A. In one embodiment, the adenovirus gene is E1B. In one embodiment, the osteocalcin transcriptional regulatory sequence comprises an enhancer or enhancer-like sequence from an osteocalcin gene. In one embodiment, the osteocalcin transcriptional regulatory sequence comprises a promoter from an osteocalcin gene. In one embodiment, the osteocalcin transcriptional regulatory sequence comprises a promoter from an osteocalcin gene and an enhancer or enhancer-like sequence from an osteocalcin gene, h one embodiment, the adenovirus further comprises an additional adenovirus gene under transcriptional control of at least one additional transcriptional regulatory sequence. In one embodiment, the second transcriptional regulatory sequence comprises an osteocalcin transcriptional regulatory sequence, hi one embodiment, the additional adenovirus gene is essential for viral replication. In one embodiment, the additional adenovirus gene is an early gene. In one embodiment, the additional adenovirus gene is El A. In one embodiment, the additional adenovirus early gene is El B. In one embodiment, the additional adenovirus gene is a late gene. In various embodiments, the late gene can be LI, L2, L3, L4, or L5.

In another aspect, the invention provides a host cell transformed with any adenovirus vector(s) described herein.

In another aspect, the invention provides a composition comprising an adenovirus vector comprising an adenovirus gene under transcriptional control of an osteocalcin transcriptional regulatory sequence, hi one embodiment, the composition further comprises a pharmaceutically acceptable excipient. In another aspect, the invention provides kits which contain an adenoviral vector(s) described herein.

In another aspect, a method is provided for propagating an adenovirus vector specific for cells which allow an osteocalcin transcriptional regulatory sequence to function, such cells including, for example, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which express osteocalcin and which fail to express PSA or androgen receptor (AR), said method comprising infecting such cells which allow an osteocalcin transcriptional regulatory sequence to function with any of the adenovirus vector(s) described herein, whereby said adenovirus vector is propagated. In another aspect, a method for modifying the genotype of a target cell is provided, the method comprising contacting a cell which allows an osteocalcm transcriptional regulatory sequence to function, such cells including, for example, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which fail to express PSA or androgen receptor (AR), with any adenovirus described herein, wherein the adenovirus enters the cell.

In another aspect, methods are provided for detecting cells expressing osteocalcin in a biological sample, comprising contacting cells of a biological sample with an adenovirus vector(s) described herein, and detecting replication of the adenovirus vector, if any. In one embodiment, a method is provided for detecting cells which allow an osteocalcin transcriptional regulatory sequence to function, for example, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which express osteocalcin and which fail to express PSA or androgen receptor (AR), in a biological sample, the method comprising the steps of: contacting a biological sample with an adenovirus vector comprising an essential adenoviral early or late gene under transcriptional control of an osteocalcin transcriptional regulatory sequence, under conditions suitable for osteocalcin transcriptional regulatory sequence- mediated gene expression in cells which allow an osteocalcin transcriptional regulatory- sequence to function; and determining if the osteocalcin transcriptional regulatory sequence mediates gene expression in the biological sample, where osteocalcin transcriptional regulatory sequence-mediated gene expression is indicative of the presence of cells which allow an osteocalcin transcriptional regulatory sequence to function. In one embodiment, the gene is a heterologous (non-adeno virus gene). In one embodiment, the heterologous gene is a reporter gene, and production of the product of the reporter gene is detected.

In another embodiment, a method is provided for conferring selective toxicity or cytotoxicity on a target cell, said method comprising contacting a target cell which allows an osteocalcin transcriptional regulatory sequence to function, for example, in prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which express osteocalcin and which fail to express PSA or androgen receptor (AR), with any adenovirus disclosed herein, wherein the adenovirus enters the cell.

In yet another embodiment, an adenovirus is provided which comprises a heterologous gene under transcriptional control of an osteocalcin transcriptional regulatory sequence, hi one embodiment, the heterologous gene is a reporter gene. In one embodiment, the heterologous gene is conditionally required for cell survival, hi one embodiment, a method is provided for detecting cells which allow an osteocalcin transcriptional regulatory sequence to function, such as, for example, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which express osteocalcin and which fail to express PSA or androgen receptor (AR), in a sample comprising the steps of: contacting a biological sample with an adenovirus vector comprising a gene under transcriptional control of an osteocalcin transcriptional regulatory sequence, under conditions suitable for osteocalcm transcriptional regulatory sequence- mediated gene expression in cells which allow an osteocalcin transcriptional regulatory sequence to function; and determining if osteocalcin transcriptional regulatory sequence mediates gene expression in the biological sample, where osteocalcin transcriptional regulatory sequence-medicated gene expression is indicative of the presence of cells expressing osteocalcin.

As described in more detail herein, an osteocalcin transcriptional regulatory sequence can comprise any number of configurations, including, but not limited to, an OC promoter; an OC enhancer or OC enhancer-like sequence; an OC silencer; an OC promoter and an OC enhancer or OC enhancer-like sequence; an OC promoter and a non-OC

(heterologous) enhancer; a non-OC (heterologous) promoter and an OC enhancer or OC enhancer-like sequence; a non-OC promoter and multiple copies of enhancers; and multimers of the foregoing. Methods are described herein for measuring the activity of an osteocalcin transcriptional regulatory sequence and thus for determining whether a given cell allows an osteocalcin transcriptional regulatory sequence to function. The promoter and enhancer or OC enhancer-like sequence of an osteocalcin transcriptional regulatory sequence may be in any orientation and/or distance from the coding sequence of interest, and may comprise multimers of the foregoing, as long as the desired OC cell-specific transcriptional activity is obtained. Transcriptional activation can be measured in a number of ways known in the art (and as described in more detail below), but is generally measured by detection and/or quantitation of mRNA or the protein product of the coding sequence under control of (i.e., operatively linked to) an osteocalcin transcriptional regulatory sequence. As discussed herein, an osteocalcin transcriptional regulatory sequence can be of varying lengths, and of varying sequence composition. By "transcriptional activation" or an "increase in transcription", it is intended that transcription will be increased above basal levels in the target cell (i.e. cells that allow an osteocalcin transcriptional regulatory sequence to function, such as, for example, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which express osteocalcin and which fail to express PSA or androgen receptor (AR)) by at least about 20-fold, more preferably at least about 50-fold, more preferably at least about 100-fold, even more preferably at least about 200-fold, even more preferably at least about 400- to about 500-fold, even more preferably, at least about 1000-fold. Basal levels are generally the level of activity, if any, in a non- osteocalcin-producing cell, or the level of activity (if any) of a reporter construct lacking an osteocalcin transcriptional regulatory sequence as tested in an osteocalcin-producing cell. Optionally, a transcriptional terminator or transcriptional "silencer" can'be placed upstream of the osteocalcin transcriptional regulatory sequence, thereby preventing unwanted read- through transcription of the coding segment under transcriptional control of the osteocalcin transcriptional regulatory sequence. Also, optionally, the endogenous promoter of the coding segment to be placed under transcriptional control of the osteocalcin transcriptional regulatory sequence can be deleted. Another embodiment of the invention is an adenovirus which replicates preferentially in mammalian cells expressing osteocaclin.

IN. 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 understood better by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1. Construction of a replication-competent type 5 adenovirus, Ad-OC-Ela, by homologous recombination of a shuttle vector, pOC-Ela, and a recombinant vector, pJM17, in 293 cells.

Figure 2. Immunohistochemical demonstration of the presence of OC in primary and metastatic human prostate cancer specimens. Note positive OC stain was detected in primary cancer associated stroma (Panel A) and both stroma and tumor epithelium (Panel B). Positive immunostaining of OC was also found in lymph node (Panel D) and bone (Panel E) metastasis. Background immunostaining was found in control primary (Panel C) and bone metastatic (Panel F) prostate cancer.

Figure 3. Inhibition of human prostate cancer and bone and prostate stromal cell growth in vitro by the replication-competent Ad-OC-Ela. Cell growth was assessed in vitro in the presence of Ad-OC-Ela, Ad-CMN-beta-gal or Ad-CMN-PA. The percentage of cell viability was measured on day 3 after infection of the test virus (ranged from 0.01 to 5 MOI or pfu/cell). Results of these studies showed that: (a) Although Ad-CMN-PA and Ad-CMN- beta-gal did not affect the growth of C4-2, Ad-OC-Ela inhibited the cell growth of C4-2 and 293 in a viral concentration-dependent manner. Ad-OC-Ela was not effective in inhibiting the cell growth of WH and Lovo cells because there is a lack of OC promoter activity and OC expression in WH and Lovo cells, (b) Comparison of the efficacy of Ad-OC-Ela-induced cell lysis in C4-2, LΝCaP, PC-3, ARCaP, and DU-145 human prostate cancer cell lines. Note strongly PSA and AR positive (LNCaP, C4-2) and negative (PC-3, DU-145) and marginally PSA and AR positive (ARCaP) human prostate cancer cell lines are susceptible to Ad-OC-

Ela-induced cell lysis, (c) Both human prostate and bone fibroblasts are also sensitive to Ad- OC-Ela-induced cell lysis. Data represent the average of triplicate experiments determined with standard deviation within 15% of the mean.

Figure 4. Differential inhibition of PC-3 and Lovo tumor growth in vivo upon intratumoral injection of Ad-OC-Ela. Four weeks after subcutaneous injection of either 1 x lθ6 PC-3 or Lovo cells into athymic nude mice, a single dose of 2 x 10^ pfu of Ad-OC-Ela or Ad-CMN-beta-gal was administered intratumorally to tumor-bearing mice. Although Ad- CMN-beta-gal did not exhibit the significant inhibition of PC-3 cell growth, Ad-OC-Ela inhibited PC-3 but not Lovo tumor growth in vivo.

Figure 5. Demonstration of intravenous Ad-OC-Ela on serum PSA levels in SCID/bg mice injected intraosseously with C4-2 cells, (a) Serum PSA level of an untreated control mouse after intraosseous injection of 1 x 10^ C4-2 cells. Note exponential rise of serum PSA in the untreated mouse (mouse #1). (b)-(f) (or mouse #2 to #6) serum PSA levels of animals injected with Ad-OC-Ela (2 x 10^ pfu) via tail vein after detecting a rising of serum PSA in animals that received intraosseous injection of 1 x 10°^" C4-2 cells. Arrowheads indicate the time (in week) intravenous administration of Ad-OC-Ela. Systemic Ad-OC-Ela results in rapid decline of serum PSA, and 4/5 (80%) animals responded markedly to repeated Ad-OC-El a treatment. Figure 6. Gross morphology, x-ray and histopathologic evidence of Ad-OC-Ela in eradicating the growth of intraosseous human prostate tumor xenografts. (a) This panel shows that a single administration of Ad-OC-Ela (2 x 10^ pfu) via tail vein induced marked tumor regression in the treated mouse (see left Panels), (b) The regression of prostate tumors by Ad-OC-Ela is supported by the histopathologic evidence of the appearance of prostate tumor cells in the skeleton (see Panel A) and these tumor cells expressed PSA (see Panel B). hi the treated specimen, no tumor cells (see panel C) nor PSA (data not shown) was observed in the skeleton, (c) X-gal staining of normal mouse and human bone infected with Ad- CMN-beta-gal (1 x 10^ pfu). Note whereas the mouse bone marrow cells are susceptible to Ad-CMN-beta-gal infection, the cortical bone of the mouse is resistant to Ad infection (Panel

A). Panel B shows that human PC-3 prostate tumor is susceptible to Ad-CMN-beta-gal infection, when maintained as explants on soft agar. Normal human bone exposed to this virus did not result in detectable beta-gal activity suggesting that normal human bone cells maybe resistant to Ad vector infection (Panel C).

Figure 7. RT-PCR of Vitamin D receptor (VDR) in normal and neoplastic human cancer cell lines. Left track of the Figure represents marker RNA.

Figure 8. Western blot of Vitamin D receptor (VDR) in human prostate cancer (C4-2, PC3), human renal carcinoma (RCC52), human osteosarcoma (MG-63) cell lines.

Figure 9. RT-PCR of human osteocalcin (hOC) mRNA. The effect of Vitamin D on hOC expression was studied in cultured human prostate cancer (C4-2 and PC3), human renal cancer (RC52), human osteosarcoma (MG-63) and human transitional cell carcinoma (WH) cell lines.

Figure 10. Cytotoxicity of a replication-competent Ad-hOC-El plus Vitamin D on a human renal carcinoma RCC52 cell line in vitro.

Figure 11. Cytotoxicity of a replication-competent Ad-sPS A-El plus Vitamin D on PC3 cells. Figure 12. Cytotoxicity of a replication-competent Ad-sPSA-El plus Vitamin D on DU145 cells.

Figure 13. Cytotoxicity of a replication-competent Ad-sPS A-El plus Vitamin D on C4-2 cells.

Figure 14. Cytotoxicity of a replication-competent Ad-hOC-El plus Vitamin D on PC-3 cells.

Figure 15. Cytotoxicity of a replication-competent Ad-hOC-El on DU-145 cells.

Figure 16. Cytotoxicity of Ad-hOC-El plus Vitamin D on C4-2 cells.

Figure 17A-G. (A) Cytotoxicity of Ad-CMV-PA on RCC52 cells. (B) Cytotoxicity of Ad-CMV-PA on PC-3 cells. (C) Cytotoxicity of Ad-CMV-PA on DU145 cells. (D)

Cytotoxicity of Ad-CMV-PA on C4-2 cells. (E) Cytotoxicity of wild-type Ad vector on the growth of PC-3 cells. (F) Cytotoxicity of wild-type Ad vector on the growth of RCC52 cells. (G) Cytotoxicity of wild-type Ad vector on the growth of C4-2 cells.

Figure 18. Cytotoxicity of C4-2 cells when co-cultured with a mouse pluripotent osteogenic Dl stromal cell line transduced with a herpes simplex thymidine kinase (TK) gene in the presence of a prodrug ganciclovir (GCV).

Figure 19. Cytotoxicity of C4-2 cells co inoculated with a mouse pluripotent osteogenic Dl stromal cell line in vivo.

Figure 20. Radiographic and gross morphologic evidence of tumor regression in SCID/bg mice harboring C4-2 tumors intraosseously.

Figure 21. Nucleotide sequence of the 1,370-bp mouse osteocalcin promoter (SEQ JJD

NO:l). V. DEFINITIONS

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. The term "tissue-specific" is intended to mean that the transcriptional regulatory sequence to which the gene essential for viral replication is operably linked functions in that tissue so that replication proceeds in that tissue.

The term "transcriptional regulatory sequence" is used according to its art- recognized meaning. It is intended to mean any DNA sequence which can, by virtue of its sequence, cause the linked gene to be either up- or down-regulated in a particular cell, hi one embodiment of the present invention, the native transcriptional regulatory sequence is completely deleted from the vector and replaced with a heterologous transcriptional regulatory sequence. The transcriptional regulatory sequence may be adjacent to the coding region for the gene that is essential for replication, or may be removed from it. Accordingly, in the case of a promoter, the promoter will generally be adj acent to the coding region. In the case of an enhancer or enhancer-like sequence, however, an enhancer or enhancer-like sequence can be found at some distance from the coding region such that there is an intervening DNA sequence between the enhancer or enhancer-like sequence and the coding region, hi some cases, the native transcriptional regulatory sequence remains on the vector but is non-functional with respect to transcription of the gene essential for replication. In some cases, the native transcriptional regulatory sequence remains on the vector and is augmented by placement of the tissue-specific tumor-restrictive transcriptional regulatory sequence to which the gene essential for viral replication is operably linked.

An "adenovirus vector" or "adenoviral vector" (used interchangeably) is a term well understood in the art and generally comprises a polynucleotide (defined herein) comprising all or a portion of an adenovirus genome. For purposes of the present invention, an adenovirus vector contains an osteocalcin transcriptional regulatory sequence operably linked to a polynucleotide. The operably linked polynucleotide can be adenoviral or heterologous. An adenoviral vector construct of the present invention can be in any of several forms, including, but not limited to, naked DNA, DNA encapsulated in an adenovirus coat,

DNA encapsulated in liposomes, DNA complexed with polylysine, complexed with synthetic polycationic molecules, conjugated with transferrin, and complexed with compounds such as PEG to immunologically "mask" the molecule and/or increase half-life, or conjugated to a non-viral protein. Preferably, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also includes any of their analogs or modified forms of these bases, such as methylated nucleotides, internucleotide modifications such as uncharged linkages and thioates, use of sugar analogs, and modified and/or alternative backbone structures, such as polyamides. For purposes of this invention, adenovirus vectors are replication-competent in a target cell such as a tumor cell.

The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleo tides.

Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be a oligodeoxy- nucleoside phosphoramidate (P— NH₂) or a mixed phosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24:1841-8; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-23; Schultz et al. (1996) Nucleic Acids Res. 24:2966-73. A phosphorothiate linkage can be used in place of a phosphodiester linkage. Braun et al. (1988) J. Immunol. 141:2084-9; Latimer et al. (1995) Mol. Immunol. 32:1057-1064. hi addition, a double- stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a

DNA polymerase with an appropriate primer.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.

A polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases are the same in comparing the two sequences. This alignment and the percent homo logy or sequence identity can be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania).

As used herein, "a cell which allows an osteocalcin transcriptional regulatory sequence to function", a cell in which the function of an osteocalcin transcriptional regulatory- sequence is "sufficiently preserved", "a cell in which an osteocalcin transcriptional regulatory sequence functions" is a cell in which an osteocalcin transcriptional regulatory sequence, when operably linked to, for example, a reporter gene, increases expression of the reporter gene at least about 20-fold, more preferably at least about 50-fold, more preferably at least about 100-fold, more preferably at least about 200-fold, even more preferably at least about 400- to 500-fold, even more preferably at least about 1000-fold, when compared to the expression of the same reporter gene when not operably linked to the osteocalcin transcriptional regulatory sequence. Methods for measuring levels (whether relative or absolute) of expression are known in the art and are described herein.

"Under transcriptional control" is a term well-understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operably (operatively) linked to an element or transcriptional regulatory sequence which contributes to the initiation of, or promotes, transcription. As noted below, "operably linked" refers to a juxtaposition wherein the elements transcriptional regulatory sequences are in an arrangement allowing them to function.

As used herein, "cytotoxicity" is a term well understood in the art and refers to a state in which one or more of a cell's usual biochemical or biological functions are aberrantly compromised (i.e., inhibited or elevated). These activities include, but are not limited to metabolism; cellular replication; DNA replication; transcription; translation; and uptake of molecules. "Cytotoxicity" includes cell death and/or cytolysis. Assays are known in the art which indicate cytotoxicity, such as dye exclusion, ³H-thymidine uptake, and plaque assays. The term "selective cytotoxicity", as used herein, refers to the cytotoxicity conferred by an adenovirus vector of the present invention on a cell which allows an osteocalcin transcriptional regulatory sequence to function when compared to the cytotoxicity conferred by the adenovirus on a cell which does not allows an osteocalcin transcriptional regulatory sequence to function. Such cytotoxicity may be measured, for example, by plaque assays, reduction or stabilization in size of a tumor comprising target cells, or the reduction or stabilization of serum levels of a marker characteristic of the tumor cells or a tissue-specific marker, e.g., a cancer marker such as prostate specific antigen.

"Replication" and "propagation" are used interchangeably and refer to the ability of a adenovirus vector of the invention to reproduce or proliferate. This term is well understood in the art. For purposes of this invention, replication involves production of adenovirus proteins and is generally directed to reproduction of adenovirus. Replication can be measured using assays standard in the art and described herein, such as a burst assay or plaque assay. "Replication" and "propagation" include any activity directly or indirectly involved in the process of virus manufacture, including, but not limited to, viral gene expression; production of viral proteins, nucleic acids or other components; packaging of viral components into complete viruses; and cell lysis. The term "heterologous" means a DNA sequence not found in the native vector genome. With respect to a "heterologous transcriptional regulatory sequence", "heterologous" indicates that the transcriptional regulatory sequence is not naturally ligated to the DNA sequence for the gene essential for replication of the vector.

A "heterologous gene" or "transgene" is any gene that is not present in wild- type adenovirus. Preferably, the transgene will also not be expressed or present in the target cell prior to introduction by the adenovirus vector. Examples of preferred transgenes are provided below.

The term "promoter" is used according to its art-recognized meaning. It is intended to mean the DNA region, usually upstream to the coding sequence of a gene or operon, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site.

The term "enhancer" is used according to its art-recognized meaning. It is intended to mean a sequence found in eukaryotes and certain eukaryotic viruses which can increase transcription from a gene when located (in either orientation) up to several kilobases from the gene being studied. These sequences usually act as enhancers when on the 5' side

(upstream) of the gene in question. However, some enhancers are active when placed on the 3' side (downstream) of the gene. The enhancer may also be an enhancer-like sequence.

The tenn "silencer," used in its art-recognized sense, means a sequence found in eucaryotic viruses and eucaryotes which can decrease or silence transcription of a gene when located within several kilobases of that gene.

A "heterologous" promoter or enhancer is one which is not associated with or derived from an osteocalcin gene 5' flanking sequence. Examples of a heterologous promoter are the α-fetoprotein, PSA, DF3, tyrosinase, CEA, surfactant protein, and ErbB2 promoters. Examples of a heterologous enhancer are the α-fetoprotein, PSA, DF3, tyrosinase, CEA, surfactant protein, ErbB2, and SV40 enhancers.

An "endogenous" promoter, enhancer, or transcriptional regulatory sequence is native to or derived from adenovirus.

The term "operably linked" relates to the orientation of polynucleotide elements in a functional relationship. A transcriptional regulatory sequence is operably linked to a coding segment if the transcriptional regulatory sequence promotes transcription of the coding sequence. Operably linked means that the DNA sequences being linked are generally contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable length, some polynucleotide elements may be operably linked but not contiguous.

A "host cell" includes an individual cell or cell culture which can be or has been a recipient of any vector of this invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completed identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with an adenoviral vector of this invention.

A "target cell" is any cell that allows an osteocalcin transcriptional regulatory sequence to function. Preferably, a target cell is a mammalian cell which allows an osteocalcin transcriptional regulatory sequence to function, such as a cell expressing osteocalcin, preferably, a mammalian cell endogenously expressing osteocalcin, more preferably, a human cell, and more preferably, a human cell capable of allowing an osteocalcin transcriptional regulatory sequence to function and which cell fails to express PSA or androgen receptor (AR).

As used herein, "neoplastic cells", "neoplasia", "tumor", "tumor cells", "cancer", and "cancer cells" refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Neoplastic cells can be benign or malignant.

A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term "biological sample" encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.

An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, and pets.

An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of an adenoviral vector is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.

As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread (i.e., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

"Palliating" a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering adenoviral vectors of the present invention. Various combinations of transcriptional regulatory sequences can be included in a vector. One or more may be heterologous. Further, one or more may have the tissue- specificity. On or more of the transcriptional regulatory sequences may be inducible. For example, a single transcriptional regulatory sequence could be used to drive replication by more than one gene essential for replication. This is the case, for example, when the gene product of one of the genes drives transcription of the further gene(s). An example is a heterologous promoter linked to a cassette containing an Ela coding sequence (Ela promoter deleted) and the entire Elb gene, hi this instance, only one heterologous transcriptional regulatory sequence may be necessary. When genes are individually (separately) controlled, however, more than one transcriptional regulatory sequence can be used if more than one such gene is desired to control replication.

The term "gene essential for replication" refers to a genetic sequence whose transcription is required for the viral vector to replicate in the target cell. The vectors of the present invention, therefore, also include transcriptional regulatory sequence combinations wherein there is more than one heterologous transcriptional regulatory sequence, but wherein one or more of these is not tissue-specific or tumor-restrictive. For example, one transcriptional regulatory sequence can be a basal level constitutive transcriptional regulatory sequence. For example, a tissue-specific enhancer or promoter can be combined with a basal level constitutive promoter. In another example, a tissue-specific enhancer or promoter can be combined with an inducible promoter.

VI. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for the adenovirus cell therapy, hi particular, the compositions of the present invention comprise adenoviral vectors employing an osteocalcin transcriptional regulatory sequence to drive viral replication through the regulation of an adenoviral early gene required for viral replication. The methods of the invention involve use of the adenoviral vectors employing an osteocalcin transcriptional regulatory sequence which drive viral replication through the regulation of an adenoviral early gene required for viral replication to treat metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, breast cancer. Additionally, this invention finds application to the treatment of benign but nonetheless serious and life threatening conditions and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis.

Three lines of evidence prompted the inventors to pursue the development of Ad-OC-Ela as a promising novel therapy for the treatment of prostate cancer skeletal metastasis. First, osteocalcin (OC) protein was found to be uniformly and highly expressed in human prostate cancer skeletal metastasis. By co-targeting both prostate cancer cells and proliferating cancer-associated osteoblasts with Ad-OC-Ela, this form of therapy will inhibit not only prostate cancer cell growth but also interrupt prostate tumor-bone stromal interaction through direct targeting and destruction of proliferating osteoblasts. Second, OC promoter- regulated adenoviral replication may be far more efficient in prostate cancer and associated bone stromal cells that failed to express PSA or androgen receptor (AR). OC promoter can therefore be used to drive adenoviral replication in prostate stromal cells (Koeneman, K. S., Kao, C, Ko, S. C, Yang, L., Wada, Y., Kallmes, D. A., Gillenwater, J. Y., Zhau, H. E., Chung, L. W. K. and Gardner, T. A. Osteocalcin directed gene therapy for prostate cancer bone metastasis. World J. Urol, 18: 102, 2000; Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon, J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998; Koeneman, K. S., Yeung, F. and Chung, L. W. K. Osteomimetic properties of prostate cancer cells: a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment. Prostate, 39: 246, 1999) and in vascular pericytes (Doherty, M. J., Ashton, B. A., Walsh, S., Beresford, J. N., Grant, M. E. and

Canfield, A. E. Vascular pericytes express osteogenic potential in vitro and in vivo. J. Bone Miner. Res., 13: 828, 1998) which were shown to express OC and support tumor epithelial growth and progression. Third, the results of the present study demonstrated for the first time that Ad-OC-Ela, when administered intravenously induced regression of pre-existing human prostate cancer bone xenografts. This study established that Ad-OC-Ela will be an attractive intravenous Ad vector for the treatment of human prostate cancer skeletal metastasis.

The prefened vectors of the present invention are adenoviral vectors. In one preferred embodiment, the adenovirus vector is a human adenovirus. There are a number of different types of adenovirus, such as Ad2, Ad5, and Ad40, which may differ to minor or significant degrees. Particularly, Ad5 and Ad40 differ as to their host cell tropism, as well as the nature of the disease induced by the virus.

In another embodiment, the adenovirus vector for use in the compositions and methods of the invention is canine adenovirus type 1 or canine adenovirus type 2. By way of example, and not by way of limitation, examples of canine adenovirases that may be used are those described in International Patent Application Numbers WO 91/11525 and WO 94/26914, (the entire contents of each of which are incorporated herein by reference).

In another embodiment, the adenovirus vector for use in the compositions and methods of the invention is a bovine adenovirus. By way of example, and not by way of limitation, an example of a bovine adenovirus is that described in International Patent Application Number WO 95/16048 (the entire contents of which are incorporated herein by reference).

In yet another embodiment, the adenovirus vector for use in the compositions and methods of the invention is ovine adenovirus. By way of example, and not by way of limitation, an example of an ovine adenoviral vector suitable for use in the present invention is the ovine adenovirus OAV287 described in U.S. Patent No. 6,020,172 (the entire contents of which are incorporated herein by reference). For the purpose of the subject invention, Ad5 will be exemplified. What follows is a brief description of adenovirus-based vectors in general and replication- competent adenovirus vectors in particular.

A. ADENOVIRUS-BASED VECTORS

Adenovirus is a large, non-enveloped virus consisting of a dense protein capsid and a large linear (36 kb) double stranded DNA genome. Adenovirus infects a variety of both dividing and non-dividing cells, gaining entry by receptor-mediated uptake into endosomes, followed by internalization. After uncoating, the adenovirus genome expresses a large number of different gene products that are involved in viral replication, modification of host cell metabolism and packaging of progeny viral particles. Three adenovirus gene products are essential for replication of viral genomes: (1) the terminal binding protein which primes DNA replication, (2) the viral DNA polymerase and (3) the DNA binding protein (reviewed in Tamanoi and Stillman, 1983, Immunol. 109:75-87). hi addition, processing of the terminal binding protein by the adenovirus 23kDa L3 protease is required to permit subsequent rounds of reinfection (Stillman et al, 1981, Cell, 23:497-508) as well as to process adenovirus structural proteins, permitting completion of self-assembly of capsids (Bhatti and Weber, 1979, Virology, 96:478-485).

Packaging of nascent adenovirus particles takes place in the nucleus, requiring both cis-acting DNA elements and trans-acting viral factors, the latter generally construed to be a number of viral structural polypeptides. Packaging of adenoviral DNA sequences into adenovirus capsids requires the viral genomes to possess functional adenovirus encapsidation signals, which are located in the left and right termini of the linear viral genome (Hearing et al, 1987, J. Virol. 61 :2555-2558). Additionally, the packaging sequence must reside near the ends of the viral genome to function (Hearing et al, 1987, J. Virol. 61 :2555-2558; Grable and

Hearing, 1992, J. Virol, 66:723-731). The El A enhancer, the viral replication origin and the encapsidation signal compose the duplicated inverted terminal repeat (ITR) sequences located at the two ends of adenovirus genomic DNA. The replication origin is defined loosely by a series of conserved nucleotide sequences in the ITR which must be positioned close to the end of the genome to act as a replication-priming element (reviewed in Challberg and Kelly,

1989, Biochem, 58:671-717; Tamanoi and Stillman, 1983, Immunol. 109:75-87). As shown by several groups, the ITRs are sufficient to confer replication to a heterologous DNA in the presence of complementing adenovirus functions. Adenovirus "mini-chromosomes" consisting of the terminal ITRs flanking short linear DNA fragments (in some cases non- viral DNAs) were found to replicate in vivo at low levels in the presence of infecting wild-type adenovirus, or in vitro at low levels in extracts prepared from infected cells (e.g., Hay et al,

1984, J. Mol. Biol. 175:493-510; Tamanoi and Stillman, 1983, Immunol. 109:75-87). The expression of foreign genes in "replication-defective" adenovirases (deleted of region El) has been exploited for a number of years in many labs, and a variety of published reports describe several different approaches often used in constructing these vectors (Vernon et al, 1991, J. Gen. Virol., 72:1243-1251; Wilkinson and Akrigg, 1992,

Nuc. Acids Res., 20:2233-2239; Eloit et al, 1990, J. Gen. Virol, 71:2425-2431; Johnson, 1991; Prevec et al, 1990, J. Infect. Dis., 161:27-30; Haj-Ahmad and Graham, 1986, J. Virol, 57:267-274; Lucito and Schneider, 1992, J. Virol, 66:983-991; reviewed in Graham and Prevec, 1992, Butterworth-Heinemann, 363-393). In general, replication-defective viruses are produced by replacing part, or all, of essential region El with a heterologous gene of interest, either by direct ligation to viral genomes in vitro, or by homologous recombination within cells in vivo (procedures reviewed in Berkner, 1992, Curr. Topics Micro. Immunol, 158:39-66). These procedures all produce adenovirus vectors that replicate in complementing cell lines such as 293 cells which provide the El gene products in trans. Replication competent adenovirus vectors also have been described that have the heterologous gene of interest inserted in place of non-essential region E3 (e.g., Haj-Ahmad and Graham, 1986, J. Virol. 57:267-274), or between the right ITR and region E4 (Saito et al, 1985, J. Virol, 54:711-719). In both, replication defective viruses and replication competent viruses, the heterologous gene of interest is incorporated into viral particles by packaging of the recombinant adenovirus genome.

The El A gene is expressed immediately after viral infection (0-2 hours) and before any other viral genes. El A protein acts as a trans-acting positive-acting transcriptional regulatory factor, and is required for the expression of the other early viral genes E1B, E2, E3, E4, and the promoter-proximal major late genes. Despite the nomenclature, the promoter proximal genes driven by the major late promoter are expressed during early times after Ad5 infection. Flint (1982) Biochem. Biophys. Acta 651:175-208; Flint (1986) Advances Virus Research 31:169-228; Grand (1987) Biochem J. 241:25-38. In the absence of a functional EIA gene, viral infection does not proceed, because the gene products necessary for viral DNA replication are not produced. Nevins (1989) Adv. Virus Res. 31:35-81. The transcription start site of Ad5 EIA is at nt 498 and the ATG start site of the EIA protein is at nt 560 in the virus genome.

The E1B protein functions in trans and is necessary for transport of late mRNA from the nucleus to the cytoplasm. Defects in E1B expression result in poor expression of late viral proteins and an inability to shut off host cell protein synthesis. The promoter of E1B has been implicated as the defining element of difference in the host range of Ad40 and Ad5: clinically Ad40 is an enterovirus, whereas Ad5 causes acute conjunctivitis.

Bailey et al. (1993) Virology 193:631; Bailey et al. (1994) Virology 202:695-706. E1B proteins are also necessary for the virus to overcome restrictions imposed on viral replication by the host cell cycle and also to reduce the apoptotic effects of El A. Goodrum et al. (1997) J. Virology 71:548-561. The E1B promoter of Ad5 consists of a single high-affinity recognition site for Sp 1 and a TATA box.

The E2 region of adenovirus codes for proteins related to replication of the adenoviral genome, including the 72-kDa DNA-binding protein, the 80-kDa precursor terminal protein and the viral DNA polymerase. The E2 region of Ad5 is transcribed in a rightward orientation from two promoters, termed E2 early and E2 late, mapping at 76.0 and 72.0 map units, respectively. While the E2 late promoter is transiently active during late stages of infection and is independent of the EIA transactivator protein, the E2 early promoter is crucial during the early phases of viral replication.

The E2 early promoter, mapping in Ad5 from 27050-27150, consists of a major and a minor transcription initiation site, the latter accounting for about 5% of the E2 transcripts, two non-canonical TATA boxes, two E2F transcription factor binding sites and an

ATF transcription factor binding site. For a detailed review of the E2 promoter architecture see Swaminathan et al, Curr. Topics in Micro, and Imm. (1995) 199 part 3:177-194.

The E2 late promoter overlaps with the coding sequences of a gene encoded by the counterstrand and is therefore not amenable for genetic manipulation. However, the E2 early promoter overlaps only for a few base pairs with sequences coding for a 33 kDa protein on the counterstrand. Notably, the Spel restriction site (Ad5 position 27082) is part of the stop codon for the above mentioned 33 kDa protein and conveniently separates the major E2 early transcription initiation site and TATA-binding protein site from the upstream transcription factor binding sites E2 F and ATF. Therefore, insertion of an osteocalcin transcriptional regulatory sequence having Spel ends into the Spel site in the 1 -strand would disrupt the endogenous E2 early promoter of Ad5 and should allow osteocalcin-restricted expression of E2 transcripts.

The E4 gene produces a number of transcription products. The E4 region codes for two polypeptides which are responsible for stimulating the replication of viral genomic DNA and for stimulating late gene expression. The protein products of open reading frames (ORFs) 3 and 6 can both perform these function by binding the 55-kDa protein from

E1B and heterodimers of E2F-land DP-1. The ORF 6 protein requires interaction with the E1B 55-kDa protein for activity while the ORF 3 protein does not. hi the absence of functional protein from ORF 3 and ORF 6, plaques are produced with an efficiency less than 10^"6 that of wild type virus. To further restrict viral replication to cells that allow an osteocalcin transcriptional regulatory sequence to function, such as osteocalcin-producing cells, E4 ORFs 1-3 can be deleted, making viral DNA replication and late gene synthesis dependent on E4 ORF 6 protein. By combining such a vector with sequences in which the E1B region is regulated by an osteocalcin transcriptional regulatory sequence, a virus can be obtained in which both the E1B function and E4 function are dependent on an osteocalcin transcriptional regulatory sequence driving E1B.

The major late genes relevant to the subject invention are LI, L2, L3, L4, and L5, which encode proteins of the Ad5 virus virion. All of these genes (typically coding for structural proteins) are probably required for adenoviral replication. The late genes are all under the control of the major late promoter (MLP), which is located in Ad5 at about +5986 to about +6048.

In some embodiments, an osteocalcin transcriptional regulatory sequence is used with an adenoviras gene that is essential for propagation, so that replication-competence is preferentially achievable in the target cell that allow an osteocalcin transcriptional regulatory sequence to function, such as a cell expressing osteocalcin. Preferably, the gene is an early gene, such as EIA, E1B, E2, or E4. (As noted supra, E3 is not essential for viral replication.) More preferably, the early gene under an osteocalcin transcriptional regulatory sequence control is EIA and/or EIB. More than one early gene can be placed under control of an osteocalcin transcriptional regulatory sequence. Example 1 provides a more detailed description of such a construct.

In other embodiments, in addition to conferring selective cytotoxic and/or cytolytic activity by virtue of preferential replication competence in cells that allow an osteocalcin transcriptional regulatory sequence to function, such as cells expressing osteocalcin, the adenoviras vectors of this invention can further include a heterologous gene (transgene) under the control of an osteocalcin transcriptional regulatory sequence, hi this way, various genetic capabilities may be introduced into target cells allowing an osteocalcin transcriptional regulatory sequence to function, such as cells expressing osteocalcin, particularly cancer cells of prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, or breast cancer as well as those prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which fail to express PSA or androgen receptor (AR). For example, in certain instances, it may be desirable to enhance the degree and/or rate of cytotoxic activity, due to, for example, the relatively refractory nature or particular aggressiveness of the osteocalcin-producing target cell. This could be accomplished by coupling the cell-specific replicative cytotoxic activity with cell-specific expression of, for example, HSV-tk and/or cytosine deaminase (cd), which renders cells capable of metabolizing 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil (5-FU).

Using these types of heterologous genes or transgenes may also confer a bystander effect.

Other desirable transgenes that may be introduced via an adenovirus vector(s) include genes encoding cytotoxic proteins, such as the A chains of diphtheria toxin, ricin or abrin [Palmiter et al. (1987) Cell 50:435; Maxwell et al. (1987) Mol. Cell. Biol. 7:1576; Behringer et al. (1988) Genes Dev. 2:453; Messing et al. (1992) Neuron 8:507; Piatak et al.

(1988) J. Biol. Chem. 263:4937; Lamb et al. (1985) Eur. J. Biochem. 148:265; Frankel et al.

(1989) Mo. Cell. Biol. 9:415], genes encoding a factor capable of initiating apoptosis, sequences encoding antisense transcripts or ribozymes, which among other capabilities may be directed to rnRNAs encoding proteins essential for proliferation, such as structural proteins, or transcription factors; viral or other pathogenic proteins, where the pathogen proliferates intracellularly, genes that encode an engineered cytoplasmic variant of a nuclease (e.g. RNase A) or protease (e.g. awsin, papain, proteinase K, carboxypeptidase, etc.), or encode the Fas gene, and the like. Other genes of interest include cytokins, antigens, transmembrane proteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-.alpha., -.beta., -.gamma., TNF-. alpha., -.beta., NGF, and the like. The positive effector genes could be used in an early phase, followed by cytotoxic activity due to replication, hi alternative embodiments, adenovirus vectors are provided with any of the other genes essential for replication, such as, for example, but not limited to, E2 or E4, under the control of a heterologous transcriptional regulatory sequence.

With respect to the packaging capacity of the adenoviras vectors of the present invention, if no adenovirus sequences have been deleted, an adenoviral vector can be packaged with extra sequences totaling up to about 5% of the genome size, or approximately 1.8 kb. If non-essential sequences are removed from the adenoviras genome, then an additional 4.6 kb of insert can be accommodated (i.e., a total of about 1.8 kb plus 4.6 kb, which is about 6.4 kb). Examples of non-essential adenoviral sequences that can be deleted are E3 and E4 (as long as the E4 ORF6 is maintained).

Any of the adenoviral vectors described herein can be used in a variety of forms, including, but not limited to, naked polynucleotide (usually DNA) constracts. Adenoviral vectors can, alternatively, comprise polynucleotide constracts that are complexed with agents to facilitate entry into cells, such as cationic liposomes or other compounds such as polylysine; packaged into infectious adenoviras particles (which may render the adenoviral vector(s) more immunogenic); complexed with agents to enhance or dampen an immune response; or complexed with agents that facilitate in vivo transfection, such as DOTMA, DOTAP.TM., and polyamines.

The adenoviral vectors maybe delivered to the target cell in a variety of ways, including, but not limited to, liposomes, general transfection methods that are well known in the art, such as calcium phosphate precipitation, electroporation, direct injection, and intravenous infusion. The means of delivery will depend in large part on the particular adenoviral vector (including its form) as well as the type and location of the target cells (i.e., whether the cells are in vitro or in vivo). If used in packaged adenovirases, adenoviras vectors may be administered in an appropriate physiologically acceptable carrier at a dose of about 10⁴ PFU to about 10¹⁴ PFU. The multiplicity of infection will generally be in the range of about 0.001 PFU to 100 PFU. If administered as a polynucleotide construct (i.e., not packaged as a virus) about 0.01 μg to 1000 μg of an adenoviral vector can be administered. The adenoviral vector(s) may be administered one or more times, depending upon the intended use and the immune response potential of the host or may be administered as multiple simultaneous injections. If an immune response is undesirable, the immune response may be diminished by employing a variety of immunosuppressants, so as to permit repetitive administration, without a strong immune response.

The present invention also includes compositions, including pharmaceutical compositions, containing the adenoviral vectors described herein. Such compositions are useful for administration in vivo, for example, when measuring the degree of transduction and/or effectiveness of cell killing in an individual. Preferably, these compositions further comprise a pharmaceutically acceptable excipient. These compositions, which can comprise an effective amount of an adenoviral vector of this invention in a pharmaceutically acceptable excipient, are suitable for systemic administration to individuals in unit dosage forms, sterile parenteral solutions or suspension, sterile non-parenteral solutions or oral solutions or suspensions, oil in water or water in oil emulsions and the like. Formulations for parenteral and nonparenteral drug delivery are known in the art and are set forth in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990). Compositions also include lyophilized and/or reconstituted forms of the adenoviral vectors (including those packaged as a viras, such as adenoviras) of the invention.

The present invention also encompasses kits containing an adenoviral vector of this invention. These kits can be used for diagnostic and/or monitoring purposes, preferably monitoring. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. Kits embodied by this invention allow for the detection of the presence of cells that allow an osteocalcin transcriptional regulatory sequence to function, such as osteocalcin-producing cells in a suitable biological sample, such as biopsy specimens.

The kits of the invention comprise an adenoviral vector described herein in suitable packaging. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.

VII. METHODS USING THE ADENOVIRUS VECTORS OF THE INVENTION

The subject vectors can be used for a wide variety of purposes, which will vary with the desired or intended result. Accordingly, the present invention includes methods using the adenoviral vectors described above. hi one embodiment, methods are provided for conferring selective cytotoxicity in cells which allow an osteocalcin transcriptional regulatory sequence to function, such as cells expressing osteocalcin comprising contacting the cells with an adenoviras vector described herein. Cytotoxicity can be measured using standard assays in the art, such as dye exclusion, H-thymidine incorporation, and/or lysis. hi another embodiment, methods are provided for propagating an adenoviras specific for cells that allow an osteocalcin transcriptional regulatory sequence to function, such as those cells expressing osteocalcin. These methods entail infecting cells with an adenoviras vector whereby said adenoviras is propagated.

Another embodiment provides methods of killing cells that allow an osteocalcin transcriptional regulatory sequence to function, such as cells expressing osteocalcin in a mixture of cells, comprising infecting a mixture of cells with an adenoviras vector of the present invention. The mixture of cells is generally a mixture of normal cells and cancerous cells producing osteocalcin, and can be an in vivo mixture or in vitro mixture. The invention also includes methods for detecting cells which allow an osteocalcin transcriptional regulatory sequence to function, such as cells expressing osteocalcin in a biological sample. These methods are particularly useful for monitoring the clinical and/or physiological condition of an individual (i.e., mammal), whether in an experimental or clinical setting. In one method, cells of a biological sample are contacted with an adenoviras vector, and replication of the adenoviral vector is detected. Alternatively, the sample can be contacted with an adenoviras in which a reporter gene is under control of an osteocalcin transcriptional regulatory sequence. Expression of the reporter gene indicates the presence of cells that allow the osteocalcin transcriptional regulatory sequence to function, such as osteocalcin-producing cells. Non-limiting examples of reporter genes for use in the methods of the invention include luciferase, and beta-galactosidase.

The transcriptional activation or increase in transcription that is observed in such osteocalcin-producing cells, is that transcription which will be increased above basal levels in the target cell (i.e. cells that allow an osteocalcin transcriptional regulatory sequence to function, such as, for example, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which express osteocalcin and which fail to express PSA or androgen receptor (AR)) by at least about 20-fold, more preferably at least about 50-fold, more preferably at least about 100-fold, even more preferably at least about 200-fold, even more preferably at least about 400- to about 500-fold, even more preferably, at least about 1000-fold. In some cases the increase in transcription may be about 2-fold, about 5-fold, or about 10-fold over basal levels in the target cell. Alternatively, an adenovirus can be constructed in which a gene conditionally required for cell survival is placed under control of an osteocalcin transcriptional regulatory sequence. This gene may encode, for example, antibiotic resistance. The adenovirus is introduced into the biological sample, and at a later time interval the sample is treated with an antibiotic. The presence of surviving cells expressing antibiotic resistance indicates the presence of cells that allow an osteocalcin transcriptional regulatory sequence to function. A suitable biological sample is one in which osteocalcin-producing cells may be or are suspected to be present. Generally, in mammals, a suitable clinical sample is one in which cancerous cells producing osteocalcin, such as prostate cancer cells, are suspected to be present. Such cells can be obtained, for example, by needle biopsy or by any other suitable surgical procedure. Cells to be contacted may be treated to promote assay conditions such as selective enrichment and/or solubilization. hi these methods, osteocalcin-producing cells can be detected using in vitro assays that detect proliferation, which are standard in the art. Examples of such standard assays include, but are not limited to, burst assays (which measure viras yields) and plaque assays (which measure infectious particles per cell). Also, propagation can be detected by measuring specific adenoviral DNA replication, which are also standard assays. The invention also provides methods of modifying the genotype of a target cell, comprising contacting the target cell with an adenoviras vector described herein, wherein the adenoviral vector enters the cell.

The invention further provides methods of suppressing tumor cell growth, preferably a tumor cell that expresses osteocalcin, comprising contacting tumor cells and non tumor cells with an adenoviral vector of the invention such that the adenoviral vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a ³H-thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor cell growth means any or all of the following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage. "Suppressing" tumor growth indicates a growth state that is curtailed when compared to growth without contact with, i.e., transfection by, an adenoviral vector described herein. The invention also provides methods of lowering the levels of a tumor cell marker in an individual, comprising administering to the individual an adenoviral vector of the present invention, wherein the adenoviral vector is selectively cytotoxic toward cells producing the tumor cell marker. Tumor cell markers include, but are not limited to, PSA, hK2, and carcinoembryonic antigen. Methods of measuring the levels of a tumor cell marker are known to those of ordinary skill in the art and include, but are not limited, to, immunological assays, such as enzyme-linked immunosorbent assay (ELISA), using antibodies specific for the tumor cell marker, hi general, a biological sample is obtained from the individual to be tested, and a suitable assay, such as an ELISA, is performed on the biological sample.

The invention also provides methods of treatment, in which an effective amount of an adenoviral vector(s) described herein is administered to an individual.

Treatment using an adenoviral vector(s) is indicated in individuals with metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, or breast cancer. Also indicated are individuals who are considered to be at risk for developing metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, and breast cancer-associated diseases, such as those who have had disease which has been resected and those who have had a family history of metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, and breast cancer-associated diseases. Detennination of suitability of administering adenoviral vector(s) of the invention will depend, inter alia, on assessable clinical parameters such as serological indications and histological examination of tissue biopsies. Generally, a pharmaceutical composition comprising an adenoviral vector(s) in a pharmaceutically acceptable excipient is administered. Pharmaceutical compositions are described above.

The amount of adenoviral vector(s) to be administered will depend on several factors, such as route of administration, the condition of the individual, the degree of aggressiveness of the disease, the particular osteocalcin transcriptional regulatory sequence employed, and the particular vector construct (i.e., which adenovirus gene(s) is under osteocalcin transcriptional regulatory sequence control).

If administered as a packaged adenovirus, from about 10⁴PFU to about 10¹⁴ PFU, preferably from about 10⁴ PFU to about 10¹² PFU, more preferably from about 10⁴ PFU to about 10¹⁰ PFU. If administered as a polynucleotide construct (i.e., not packaged as a virus), about 0.01 μg to about 100 μg can be administered, preferably 0.1 μg to about 500 μg, more preferably about 0.5 μg to about 200 μg. More than one adenoviral vector can be administered, either simultaneously or sequentially, Administrations are typically given periodically, while monitoring any response. Administration can be given, for example, intratumorally, intravenously or intraperitoneally.

The adenoviral vectors of the invention can be used alone or in conjunction with other active agents, such as chemotherapeutics, that promote the desired objective. In accordance with the present invention, the agent which is capable of providing for the inhibition, prevention, or destruction of the growth of the target tissue or tumor cells upon expression of such agent can thus also be a negative selective marker which is provided as a heterologous gene or transgene; i.e., a material which in combination with a chemotherapeutic or interaction agent inhibits, prevents or destroys the growth of the target cells. Thus, upon introduction to the cells of the negative selective marker, an interaction agent is administered to the host. The interaction agent interacts with the negative selective marker td^'prevent, inhibit, or destroy the growth of the target cells.

Negative selective markers which may be used in the methods of the present invention include, but are not limited to, 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 viras thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase. When 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 l-2-deoxy-2- fluoro-.beta.-D-arabinofuranosil-5-iodouracil (FIAU). Such interaction agents are utilized efficiently by the viral thymidine kinases as substrates, and such interaction agents thus are incorporated lethally into the DNA of the tumor cells expressing the viral thymidine kinases, thereby resulting in the death of the target cells.

When cytosine deaminase is the negative selective marker, a preferred interaction agent is 5-fluorocytosine. Cytosine deaminase converts 5-fluorocytosine to 5- fluorouracil, which is highly cytotoxic. Thus, the target 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 target cells. For example, the interaction agent is administered in an amount based on body weight and 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 administration.

When the vectors of the present invention induce a negative selective marker and are administered to a tissue or tumor in vivo, a "bystander effect" may result, i.e., cells which were not originally transduced with the nucleic acid sequence encoding the negative selective marker may be killed upon administration of the interaction agent. Although the scope of the present invention is not intended to be limited by any theoretical reasoning, the transduced cells may be producing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent, or is taken up by adjacent, non-target 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 target cells.

In one embodiment, the agent which provides for the inhibition, prevention, or destruction of the growth of the tumor cells is a cytokine. In one embodiment, the cytokine is an interleukin. Other cytokines which may be employed include interferons and colony- stimulating factors, such as GM-CSF. friterleukins include, but are not limited to, interleukin-

1, interleukin- lβ , and interleukins-2-15. In one embodiment, the interleukin is interleukin-2.

In a prefened embodiment of the invention, the target tissue is that of metastatic cancers, including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, breast cancer. The virus is distributed throughout the tissue or tumor mass. In another preferred embodiment the target tissue comprises cells which allow an osteocalcin transcriptional regulatory sequence to function, such as for example, but not limited to, prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and those prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR). The viras is distributed throughout the tissue or tumor mass.

VIII ADDITIONAL EMBODIMENTS OF THE INVENTION

In another embodiment, the invention additionally comprises using the adenoviral compositions and methods of the present invention in combination with a gene therapy method for treating prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis. Tissue specific and tumor-restrictive promoters such as the osteocalcin promoter sequence depicted in SEQ ID NO: 1 or any other tissue specific promoter described supra are used to drive tissue-specific and tumor-restrictive expression of therapeutic molecules and introduced in the cells of the cancer. The method comprises introducing an adenoviral vector constructed with an essential gene under the control of a tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l, wherein the adenoviral vector additionally contains another tissue-specific promoter operatively associated with a nucleic acid encoding a therapeutic molecule, into cells of the cancer, including, for example, without limitation, such cancers as prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis. Specifically provided are expression vectors comprising the osteocalcin transcriptional regulatory sequence, and transcriptionally active fragments thereof, operably associated to a heterologous reporter gene, e.g., LacZ, and host cells and transgenic animals containing such vectors. The invention also provides methods for using such vectors, cells and animals for screemng candidate molecules for agonists and antagonists of prostate-related disorders and diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH), cancers and arteriosclerosis. Methods for using molecules and compounds identified by the screening assays for therapeutic treatments also are provided.

For example, and not by way of limitation, a composition comprising a reporter gene is operatively linked to an osteocalcin transcriptional regulatory sequence. The tissue specific promoter such as osteocalcin promoter driven reporter gene is expressed as a transgene in animals. The transgenic animal, and cells derived from the prostate of such a transgenic animal, can be used to screen compounds for candidates useful for modulating prostate-related disorders and diseases involving calcification. Without being bound by any particular theory, such compounds are likely to interfere with the function of trans-acting factors, such as transcription factors, cis-acting elements, such as promoters and enhancers, as well as any class of post-transcriptional, translational or post-translational compounds involved in prostate-related disorders and diseases involving calcification. As such, they are powerful candidates for treatment of such cancers and disorders.

In one embodiment, the invention provides methods for high throughput screening of compounds that modulate specific expression of genes within cells of prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis. In this aspect of the invention, cells from the prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis, are removed from the transgenic animal and cultured in vitro. The expression of the reporter gene is used to monitor osteocalcin-specific gene activity. In a specific embodiment, LacZ is the reporter gene. In another specific embodiment, luciferase is the reporter gene. Compounds identified by this method can be tested further for their effect on prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification in normal animals, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis.

In another embodiment, the transgenic animal models of the invention can be used for in vivo screening to test the mechanism of action of candidate drugs for their effect on prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis.

Specifically, the effects of the drugs on prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, lung cancer, breast cancer, and those diseases involving calcification, including, without limitation, benign prostate hyperplasia (BPH) and arteriosclerosis, can be assayed.

A POLYNUCLEOTIDES AND NUCLEIC ACIDS OF THE INVENTION

The present invention encompasses polynucleotide sequences comprising

5' regulatory regions, and transcriptionally active fragments thereof, of the osteocalcin transcriptional regulatory sequence. In particular, the present invention provides a polynucleotide comprising the osteocalcin promoter sequence depicted in SEQ ID NO:l, and transcriptionally active fragments thereof. The invention further provides probes, primers and fragments of a tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO: 1. In one embodiment, purified nucleic acids consisting of at least 8 nucleotides (i.e., a hybridizable portion) of a tissue specific promoter such as an osteocalcin regulatory sequence are provided; in other embodiments, the nucleic acids consist of at least 20 (contiguous) nucleotides, 25 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides or 500 nucleotides of a tissue specific promoter such as osteocalcm promoter sequence depicted in SEQ ID NO: 1. Methods which are well known to those skilled in the art can be used to construct these sequences, either in isolated form or contained in expression vectors. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. See, e.g., the techniques described in Sambrook et al, 1989, supra, and Ausabel et al, 1989, supra; also see the techniques described in

"Oligonucleotide Synthesis", 1984, Gait M.J. ed., IRL Press, Oxford, which is incorporated herein by reference in its entirety.

In another embodiment, the nucleic acids are smaller than 20, 25, 35, 200 or 500 nucleotides in length. Nucleic acids can be single or double stranded. The invention also encompasses nucleic acids hybridizable to or complementary to the foregoing sequences, hi specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 10, 20, 25, 50, 100, 200, 500 nucleotides or the entire regulatory region of a tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l.

The probes, primers and fragments of the tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO: 1 provided by the present invention can be used by the research community for various purposes. They can be used as molecular weight markers on Southern gels; as chromosome markers or tags (when appropriately labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; and as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include, without limitation, "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The nucleotide sequences of the invention also include nucleotide sequences that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more nucleotide sequence identity to the osteocalcin promoter sequence depicted in SEQ ID NO: 1 , and/or transcriptionally active fragments thereof. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.

When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (t.e., % identity = # of identical overlapping positions/total # of positions x 100). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences also can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl Acad. Sci. USA 87:2264-226%, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 0:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al (1990) J. Mol. Biol. 2 5:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the

XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped

BLAST and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see http://www.ncbi.nhn.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-11. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used. In an alternate embodiment, alignments can be obtained using the NA- MULTIPLE-ALIGNMENT 1.0 program, using a Gap Weight of 5 and a Gap Length Weight of l. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps, i calculating percent identity, typically only exact matches are counted. The invention also encompasses:

(a) DNA vectors that contain any of the foregoing tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l and/or their complements (i.e., antisense);

(b) DNA expression vectors that contain any of the foregoing tissue specific promoter such as the osteocalcin promoter sequences depicted in SEQ ID NO:l operatively associated with a heterologous gene, such as a reporter gene; and (c) genetically engineered host cells that contain any of the foregoing tissue specific promoter such as the osteocalcin promoter sequences depicted in SEQ ID NO:l operatively associated with a heterologous gene such that the tissue specific promoter such as osteocalcin promoter element directs the expression of the heterologous gene in the host cell. Also encompassed within the scope of the invention are various transcriptionally active fragments of this regulatory region. A "transcriptionally active" or "transcriptionally functional" fragment of a tissue specific promoter such as the osteocalcin regulatory sequences depicted in SEQ ID NO:l according to the present invention refers to a polynucleotide comprising a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. For the purpose of the invention, a nucleic acid or polynucleotide is "transcriptionally active" as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional information, and such sequences are operably associated to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide. In particular, the transcriptionally active fragments of the tissue specific promoter such as the osteocalcin regulatory sequences depicted in SEQ JD NO:l of the present invention encompass those fragments that are of sufficient length to promote transcription of a heterologous gene, such as a reporter gene, when operatively linked to the tissue specific promoter such as the osteocalcin promoter sequences depicted in SEQ ID

NO: 1 and transfected into a prostate cell line. Typically, the regulatory region is placed immediately 5' to, and is operatively associated with the coding sequence. As used herein, the term "operatively associated" refers to the placement of the regulatory sequence immediately 5' (upstream) of the reporter gene, such that trans-acting factors required for initiation of transcription, such as transcription factors, polymerase subunits and accessory proteins, can assemble at this region to allow RNA polymerase dependent transcription initiation of the reporter gene.

In one embodiment, the polynucleotide sequence chosen to serve as the tissue- specific transcriptional regulatory sequence may further comprise other nucleotide sequences in addition to those of the osteocalcin regulatory sequences depicted in SEQ ID NO: 1. Such tissue-specific transcriptional regulatory sequence may include, for example, without limitation, the α-fetoprotein, PSA, DF3, tyrosinase, CEA, surfactant protein, and ErbB2 promoters, hi particularly preferred embodiments, the additional tissue-specific transcriptional regulatory sequence may include for example, without limitation, the PSA promoter, the prostate specific enhancer (PSE), superPSE promoter, the modified artificial α- fetoprotein promoter sequence described in U.S. Patent No. 5,998,205, the entire contents of which are incorporated by reference), the modified artificial α-fetoprotein promoter sequence described in Hallebbeck et al. (Hallenbeck et al 1999 Human Gene Therapy 10: 1721-1733, the entire contents of which are incorporated by reference) and the modified artificial α- fetoprotein promoter described in akabayashi et al (Mol. Cell Biol. 1991 11(12):5885-93, the entire contents of which are incorporated by reference). hi yet another embodiment, multiple copies of the osteocalcin promoter sequence depicted in SEQ ID NO:l, or a fragment thereof, maybe linked to each other. For example, the osteocalcin promoter sequence depicted in SEQ ID NO:l, or a fragment thereof, may be linked to another copy of the promoter sequence, or another fragment thereof, in a head to tail, head to head, or tail to tail orientation. Thus, in another embodiment, by way of example, and not by way of limitation, a prostate cell-specific enhancer may be operatively linked to the tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l, or fragment thereof, and used to enhance transcription from the construct containing the tissue specific osteocalcin promoter sequence depicted in SEQ ID NO:l. Also encompassed within the scope of the invention are modifications of osteocalcin promoter sequence depicted in SEQ JD NO:l without substantially affecting its transcriptional activities. Such modifications include additions, deletions and substitutions. In addition, any nucleotide sequence that selectively hybridizes to the complement of osteocalcin promoter sequence depicted in SEQ ID NO:l under stringent conditions, and is capable of activating the expression of a gene essential for replication of the adenoviras is encompassed by the invention. Exemplary moderately stringent hybridization conditions are as follows: prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/m£ denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65 °C in prehybridization mixture containing 100 μg/ml! denatured salmon sperm DNA and 5-20 X 10⁶ cpm of ³²P -labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50 °C for 45 min before autoradiography. Alternatively, exemplary conditions of high stringency are as follows: e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at

65°C, and washing in O.lxSSC/0.1% SDS at 68°C (Ausubel F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3). Other conditions of high stringency which may be used are well known in the art. In general, for probes between 14 and 70 nucleotides in length the melting temperature (TM) is calculated using the formula:

Tm(°C)=81.5+16.6(log [monovalent cations (molar)]) +0.41 (% G+C)-(500/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature is calculated using the equation Tm(°C)=81.5+16.6(log[monovalent cations (molar)])+0.41(% G+C)-(0.61% formamide)-(500/N) where N is the length of the probe. In general, hybridization is carried out at about 20-25 degrees below Tm (for DNA- DNA hybrids) or 10-15 degrees below Tm (for RNA-DNA hybrids). The tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l, or transcriptionally functional fragments thereof, is preferably derived from a mammalian organism. In one embodiment the osteocalcin promoter sequence may be human, mouse or rat-derived, hi another embodiment, the osteocalcin promoter sequence may be that depicted in SEQ ID NO: 1 , or transcriptionally functional fragments thereof. Screening procedures which rely on nucleic acid hybridization make it possible to isolate gene sequences from various organisms. The isolated polynucleotide sequence disclosed herein, or fragments thereof, may be labeled and used to screen a cDNA library constructed from mRNA obtained from appropriate cells or tissues (e.g., prostate tissue) derived from the organism of interest. The hybridization conditions used should be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived. Low stringency conditions are well know to those of skill in the art, and will vary depending on the specific organisms from which the library and the labeled sequence are derived. For guidance regarding such conditions see, for example, Sambrook et al, 1989, Molecular Cloning, A Laboratory

Manual, Second Edition, Cold Spring Harbor Press, N.Y., and Ausabel et al, 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley friterscience, N.Y., each of which is incorporated herein by reference in its entirety.

Further, mammalian osteocalcin transcriptional regulatory sequence homologues may be isolated from, for example, bovine or other non-human nucleic acid, by performing polymerase chain reaction (PCR) amplification using two primer pools designed on the basis of the nucleotide sequence of the osteocalcm promoter sequence depicted in SEQ ID NO: 1 region disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of the mRNA prepared from, for example, bovine or other non-human cell lines, or tissue known to express osteocalcin. For guidance regarding such conditions, see, e.g., frinis et al (Eds.) 1995, PCR Strategies, Academic Press Inc., San Diego; and Erlich (ed) 1992, PCR Technology, Oxford University Press, New York, each of which is incorporated herein by reference in its entirety.

Promoter sequences within the 5' non-coding regions of the tissue specific promoter such as the osteocalcin promoter sequence may be that depicted in SEQ ID NO: 1 may be further defined by constructing nested 5' and/or 3' deletions using conventional techniques such as exonuclease III or appropriate restriction endonuclease digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity, such as described, for example, by Coles et al. (Hum. Mol. Genet., 7:791-800, 1998). In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors. These types of assays are well known to those skilled in the art (WO 97/17359, US 5,374,544, EP 582 796, US 5,698,389, US 5,643,746, US5,502,176, and US 5,266,488).

The tissue specific promoter such as the osteocalcin promoter sequence may be that depicted in SEQ ID NO:l and transcriptionally functional fragments thereof, and the fragments and probes described herein which serve to identify the tissue specific promoter such the osteocalcin promoter sequence may be that depicted in SEQ ID NO: 1 and fragments thereof, may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct these sequences, either in isolated form or contained in expression vectors. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination. See, e.g., the techniques described in Sambrook et al, 1989, supra, and Ausabel et al, 1989, supra; also see the techniques described in "Oligonucleotide Synthesis", 1984, Gait M.J. ed., IRL Press, Oxford, which is incorporated herein by reference in its entirety.

Alterations in the regulatory sequences can be generated using a variety of chemical and enzymatic methods which are well known to those skilled in the art. For example, regions of the sequences defined by restriction sites can be deleted. Oligonucleotide-directed mutagenesis can be employed to alter the sequence in a defined way and/or to introduce restriction sites in specific regions within the sequence. Additionally, deletion mutants can be generated using DNA nucleases such as Bal31, Exoiπ, or SI nuclease. Progressively larger deletions in the regulatory sequences are generated by incubating the DNA with nucleases for increased periods of time (see, e.g., Ausubel et al, 1989, supra).

The altered sequences are evaluated for their ability to direct expression of heterologous coding sequences in appropriate host cells. It is within the scope of the present invention that any altered regulatory sequences which retain their ability to direct expression of a coding sequence be incorporated into recombinant expression vectors for further use.

IX ANALYSIS OF OSTEOCALCIN-SPECIFIC PROMOTER ACTIVITY

The tissue specific promoter such as the osteocalcm promoter sequence depicted in SEQ ID NO:l shows selective tissue and cell-type specificity; i.e., it induces gene expression in prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which fail to express PSA or androgen receptor (AR). Thus, the osteocalcin promoter sequence depicted in SEQ ID NO:l, and transcriptionally active fragments thereof, of the present invention may be used to induce expression of a heterologous coding sequence in prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which fail to express PSA or androgen receptor (AR). In one embodiment, the present invention provides for the use of the tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO: 1 to achieve tissue specific expression of a target gene.

The activity and the specificity of the tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO: 1 can further be assessed by monitoring the expression level of a detectable polynucleotide operably associated with the tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l in different types of cells and tissues. As discussed hereinbelow, the detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein. X THE OSTEOCALCIN TRANSCRIPTIONAL REGULATORY

SEQUENCE DRIVEN REPORTER CONSTRUCTS

The osteocalcin transcriptional regulatory sequences according to the invention may also be advantageously part of a recombinant expression vector that may be used to express a coding sequence, or reporter gene, in a desired host cell or host organism. The osteocalcin transcriptional regulatory sequence of the present invention, and transcriptionally active fragments thereof, may be used to direct the expression of a heterologous coding sequence, hi particular, the present invention encompasses mammalian osteocalcin transcriptional regulatory sequences, hi accordance with the present invention, transcriptionally active fragments of the osteocalcin transcriptional regulatory sequence encompass those fragments of the region which are of sufficient length to promote transcription of a reporter coding sequence to which the fragment is operatively linked.

A variety of reporter gene sequences well known to those of skill in the art can be utilized, including, but not limited to, genes encoding fluorescent proteins such as green fluorescent protein (GFP), enzymes (e.g. CAT, beta-galactosidase, luciferase) or antigenic markers. For convenience, enzymatic reporters and light-emitting reporters analyzed by colorometric or fluorometric assays are preferred for the screening assays of the invention, hi one embodiment, for example, a bioluminescent, chemiluminescent or fluorescent protein can be used as a light-emitting reporter in the invention. Types of light- emitting reporters, which do not require substrates or cofactors, include, but are not limited to the wild-type green fluorescent protein (GFP) of Victoria aequoria (Chalfie et al, 1994, Science 263:802-805), and modified GFPs (Heim et al, 1995, Nature 373:663-4; PCT publication WO 96/23810). Transcription and translation of this type of reporter gene leads to the accumulation of the fluorescent protein in test cells, which can be measured by a fluorimeter, or a flow cytometer, for example, by methods that are well known in the art (see, e.g., Lackowicz, 1983, Principles of Fluorescence Spectroscopy, Plenum Press, New York).

Another type of reporter gene that maybe used are enzymes that require cofactor(s) to emit light, including but not limited to, Renilla luciferase. Other sources of luciferase also are well known in the art, including, but not limited to, the bacterial luciferase

(luxAB gene product) of Vibrio harveyi (Karp, 1989, Biochim. Biophys. Acta 1007:84-90; Stewart et al. 1992, J. Gen. Microbiol, 138:1289-1300), and the luciferase from firefly, Photinus pyralis ( De Wet et al. 1987, Mol. Cell. Biol. 7:725-737), which can be assayed by light production (Miyamoto et al, 1987, J. Bacteriol. 169:247-253; Loessner et al. 1996, Environ. Microbiol 62:1133-1140; and Schultz & Yarus, 1990, J. Bacteriol. 172:595-602). Reporter genes that can be analyzed using colorimetric analysis include, but are not limited to, β-galactosidase (Nolan et al 1988, Proc. Natl Acad. Sci. USA 85:2603- 07), β-glucuronidase (Roberts et al. 1989, Curr. Genet. 15:177-180), luciferase (Miyamoto et al, 1987, J. Bacteriol. 169:247-253), or β-lactamase. In one embodiment, the reporter gene sequence comprises a nucleotide sequence which encodes a LacZ gene product, β- galactosidase. The enzyme is very stable and has a broad specificity so as to allow the use of different histochemical, chromogenic or fluorogenic substrates, such as, but not limited to, 5- bromo-4-chloro-3-indoyl-β-D-galactoside (X-gal), lactose 2,3,5-triphenyl-2H-tetrazolium (lactose-tetrazolium) and fluorescein galactopyranoside (see Nolan et al, 1988, supra).

In another embodiment, the product of the E. coli β-glucuronidase gene (GUS) can be used as a reporter gene (Roberts et al. 1989, OUT. Genet. 15:177-180). GUS activity can be detected by various histochemical and fluorogenic substrates, such as X-glucuronide (Xgluc) and 4-methylumbelliferyl glucuronide.

In addition to reporter gene sequences such as those described above, which provide convenient colorimetric responses, other reporter gene sequences, such as, for example, selectable reporter gene sequences, can routinely be employed. For example, the coding sequence for chloramphenicol acetyl transferase (CAT) can be utilized, leading to osteocalcin transcriptional regulatory sequence-dependent expression of chloramphenicol resistant cell growth. The use of CAT and the advantages of a selectable reporter gene are well known to those skilled in the art (Eikmanns et al. 1991, Gene 102:93-98). Other selectable reporter gene sequences also can be utilized and include, but are not limited to, gene sequences encoding polypeptides which confer zeocin (Hegedus et al. 1998, Gene 207:241-249) or kanamycin resistance (Friedrich & Soriano, 1991, Genes. Dev. 5:1513- 1523).

Other reporter genes, such as toxic gene products, potentially toxic gene products, and antiproliferation or cytostatic gene products, also can be used, hi another embodiment, the detectable reporter polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein. This type of assay is well known to those skilled in the art (US 5,502,176 and US 5,266,488).

Osteocalcin transcriptional regulatory sequence driven reporter constracts can be constructed according to standard recombinant DNA techniques (see, e.g., Methods in

Enzymology, 1987, volume 154, Academic Press; Sambrook et al 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York; and Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley friterscience, New York, each of which is incorporated herein by reference in its entirety). Methods for assaying promoter activity are well-known to those skilled in the art (see, e.g., Sambrook et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). An example of a typical method that can be used involves a recombinant vector carrying a reporter gene and sequences from an osteocalcin transcriptional regulatory sequence. Briefly, the expression of the reporter gene (for example, green fluorescent protein, luciferase, β-galactosidase or chloramphenicol acetyl transferase) is detected when placed under the control of a biologically active polynucleotide fragment. Genomic sequences located upstream of the first exon of the osteocalcin gene may be cloned into any suitable promoter reporter vector. For example, a number of commercially available vectors can be engineered to insert the osteocalcin transcriptional regulatory sequence of the invention for expression in mammalian host cells. Non-limiting examples of such vectors are pSAPBasic, pSEAP-Enhancer, pβgal-Basic, pβ gal-Enhancer, or pEGFP-1 Promoter Reporter vectors (Clontech, Palo Alto, CA) or pGL2-basic or pGL3 -basic promoterless luciferase reporter gene vector (Promega, Madison, WT). Each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, green fluorescent protein, luciferase or β-galactosidase. The osteocalcin transcriptional regulatory sequences of the osteocalcin gene are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained with a vector lacking an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect the control vector indicates the presence of a promoter or a functional fragment thereof in the insert.

Expression vectors that comprise an osteocalcin transcriptional regulatory sequence may further contain a gene encoding a selectable marker. A number of selection systems may be used, including but not limited to, the herpes simplex viras thymidine kinase

(Wigler et al, 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026) and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:817) genes, which can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Proc.

Natl Acad. Sci. USA 77:3567; O'Hare et al, 1981, Proc. Natl Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colbene- Garapin et al, 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al, 1984, Gene 30:147) genes. Additional selectable genes include trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) and glutamine synthetase (Bebbington et al, 1992, Biotech 10:169).

XI CHARACTERIZATION OF TRANSCRIPTIONALLY ACTIVE REGULATORY FRAGMENTS OF THE OSTEOCALCIN TRANSCRIPTIONAL REGULATORY SEQUENCE

A fusion construct comprising an osteocalcin transcriptional regulatory sequence, or a fragment thereof, can be assayed for transcriptional activity. As a first step in promoter analysis of the osteocalcin transcriptional regulatory sequence, the transcriptional start point (+1 site) of the osteocalcin transcriptional regulatory sequence under study has to be determined using primer extension assay and/or RNAase protection assay, following standard methods (Sambrook et /.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Press). The DNA sequence upstream of the +1 site is generally considered as the promoter region responsible for gene regulation. However, downstream sequences, including sequences within introns, also may be involved in gene regulation. To begin testing for promoter activity, a -3 kb to +3 kb region (where +1 is the transcriptional start point) may be cloned upstream of the reporter gene coding region. Two or more additional reporter gene constracts also maybe made which contain 5' and/or 3' truncated versions of the regulatory region to aid in identification of the region responsible for prostate-specific expression. The choice of the type of reporter gene is made based on the application. hi a preferred embodiment, a GFP reporter gene construct is used. The application of green fluorescent protein (GFP) as a reporter is particularly useful in the study of prostate-specific gene promoters. A major advantage of using GFP as a reporter lies in the fact that GFP can be detected in freshly isolated prostate cells without the need for substrates. hi another embodiment of the invention, a Lac Z reporter construct is used.

The Lac Z gene product, β-galactosidase, is extremely stable and has a broad specificity so as to allow the use of different histochemical, chromogenic or fluorogenic substrates, such as, but not limited to, 5-bromo-4-chloro-3-indoyl-β-D-galactoside (X-gal), lactose 2,3,5- triphenyl-2H-tetrazolium (lactose-tetrazolium) and fluorescein galactopyranoside (see Nolan et al, 1988, supra).

For promoter analysis in transgenic mice, GFP that has been optimized for expression in mammalian cells is preferred. The promoterless cloning vector pEGFPl (Clontech, Palo Alto, CA) encodes a red shifted variant of the wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells (Cormack et al, 1996, Gene 173:33; Haas et al, 1996, Curr. Biol. 6: 315). Moreover, since the maximal excitation peak of this enhanced GFP (EGFP) is at 488 nm, commonly used filter sets such as fluorescein isothiocyanate (FITC) optics which illuminate at 450-500 nm can be used to visualize GFP fluorescence. pEGFPl proved to be useful as a reporter vector for promoter analysis in transgenic mice (Okabe et al, 1997, FEBS Lett. 407: 313). In an alternate embodiment, transgenic mice containing transgenes with an osteocalcin transcriptional regulatory sequence upstream of the Lac Z or luciferase reporter genes are utilized. Putative promoter fragments can be prepared (usually from a parent phage clone containing 8-10 kb genomic DNA including the promoter region) for cloning using methods known in the art. However, the feasibility of this method depends on the availability of proper restriction endonuclease sites in the regulatory fragment, hi a preferred embodiment, the required promoter fragment is amplified by polymerase chain reaction

(PCR; Saiki et al, 1988, Science 239:487) using oligonucleotide primers bearing the appropriate sites for restriction endonuclease cleavage. The sequence necessary for restriction cleavage is included at the 5' end of the forward and reverse primers which flank the regulatory fragment to be amplified. After PCR amplification, the appropriate ends are generated by restriction digestion of the PCR product. The promoter fragments, generated by either method, are then ligated into the multiple cloning site of the reporter vector following standard cloning procedures (Sambrook et α .,1989, supra). It is recommended that the DNA sequence of the PCR generated promoter fragments in the constracts be verified prior to generation of transgenic animals. The resulting reporter gene construct will contain the putative promoter fragment located upstream of the reporter gene open reading frame, e.g. , GFP orJαc Z cDNA.

XII OSTEOCALCIN TRANSCRIPTIONAL REGULATORY SEQUENCE ANALYSIS USING TRANSGENIC MICE

The mammalian osteocalcin transcriptional regulatory sequence can be used to direct expression of, inter alia, a reporter coding sequence, a homologous gene or a heterologous gene in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, sheep, and non-human primates, e.g., baboons, monkeys and chimpanzees may be used to generate transgenic animals. The term "transgenic," as used herein, refers to non-human animals expressing osteocalcin transcriptional regulatory sequence from a different species (e.g., mice expressing osteocalcin transcriptional regulatory sequence from either the rat or human osteocalcin gene), as well as animals that have been genetically engineered to over-express endogenous (i.e., same species) osteocalcin transcriptional regulatory sequence or animals that have been genetically engineered to knock-out specific sequences. hi one embodiment, the present invention provides for transgenic animals that carry a transgene such as a reporter gene under the control of the osteocalcin transcriptional regulatory sequence or transcriptionally active fragments thereof in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al (1992, Proc. Natl Acad. Sci. USA 89:6232-6236). When it is desired that the transgene be integrated into the chromosomal site of the endogenous corresponding gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.

Any technique known in the art may be used to introduce a transgene under the control of the osteocalcin transcriptional regulatory sequence into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Hoppe & Wagner, 1989, U.S. Patent No. 4,873,191); nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal or adult cells induced to quiescence (Campbell et al, 1996, Nature 380:64-66; Wilmut et al, Nature 385:810-813); retroviras gene transfer into germ lines (Van der Putten et al, 1985, Proc.

Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al, 1989, Cell 65:313-321); elecfroporation of embryos (Lo, 1983, Mol. Cell. Biol. 31:1803- 1814); and sperm-mediated gene transfer (Lavitrano et al, 1989, Cell 57:717-723; see, Gordon, 1989, Transgenic Animals, fritl. Rev. Cytol 115:171-229).

XIII SCREENING ASSAYS

Compounds that interfere with the abnormal function and/or growth of prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and associated bone stromal cells which fail to express PSA or androgen receptor

(AR) can provide therapies targeting defects in prostate-related disorders. Such compounds maybe used to interfere with the onset or the progression of prostate-related disorders, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, rumors, osteosarcoma, ocular melanoma, lung cancer, or breast cancer. Compounds that stimulate or inhibit promoter activity may be used to. ameliorate symptoms of prostate-related disorders. Transgenic animals or cells containing an osteocalcin transcriptional regulatory sequence, or fragment thereof, operably linked to a reporter gene, can be used as systems for the screening of agents that modulate osteocalcin transcriptional regulatory sequence activity. Such agents that modulate osteocalcin transcriptional regulatory sequence activity can then be used to develop new methods of treatment of prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR). In addition, osteocalcin transcriptional regulatory sequence containing transgenic mice provide an experimental model both in vivo and in vitro to develop new methods of treating prostate-related disorders, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, or breast cancer by targeting drags to cause arrest in the progression of such disorders.

The present invention encompasses screening assays designed to identify compounds that modulate activity of the osteocalcin transcriptional regulatory sequence. The present invention encompasses in vitro and cell-based assays, as well as in vivo assays in transgenic animals. As described hereinbelow, compounds to be tested may include, but are not limited to, oligonucleotides, peptides, proteins, small organic or inorganic compounds, antibodies, etc.

Examples of compounds may include, but are not limited to, peptides, such as, for example, soluble peptides, including, but not limited to, Ig-tailed fusion peptides, and members of random peptide libraries; (see, e.g., Lam, et al, 1991, Nature 354:82-84;

Houghten, et al, 1991, Nature 354:84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, et al, 1993, Cell 12:161-11 ), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Such compounds may further comprise compounds, in particular drugs or members of classes or families of drugs, known to ameliorate the symptoms of aprostate- related disorder.

Such compounds include, but are not limited to, families of antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), p- chlorophenylalanine,/?-propyldopacetamide dithiocarbamate derivatives e.g., FLA 63; anti- anxiety drugs, e.g., diazepam; monoamine oxidase (MAO) inhibitors, e.g., iproniazid, clorgyline, phenelzine and isocarboxazid; biogenic amine uptake blockers, e.g., tricyclic . antidepressants such as desipramine, imipramine and amitriptyline; serotonin reuptake inhibitors e.g., fluoxetine; antipsychotic drugs such as phenothiazine derivatives (e.g., chlorpromazine (thorazine) and trifluopromazine)), butyrophenones (e.g., haloperidol (Haldol)), thioxanthene derivatives (e.g., chlorprothixene), and dibenzodiazepines (e.g., clozapine); benzodiazepines; dopaminergic agonists and antagonists e.g., L-DOPA, cocaine, amphetamine, α-methyl-tyrosine, reserpine, tetrabenazine, benzotropine, pargyline; noradrenergic agonists and antagonists e.g., clonidine, phenoxybenzamine, phentolamine, tropolone; nitrovasodilators (e.g., nitroglycerine, nitroprusside as well as NO synthase enzymes); and growth factors (e.g., VEGF, FGF, angiopoetins and endostatin). hi one preferred embodiment, primary cultures of germ cells containing a mammalian osteocalcin transcriptional regulatory sequence operatively linked to a heterologous gene are used to develop assay systems to screen for compounds which can inhibit or enhance sequence-specific DNA-protein interactions. Such methods comprise contacting a compound to a cell that expresses a gene under the control of a osteocalcin franscriptional regulatory sequence, or a transcriptionally active fragment thereof, measuring the level of the gene expression or gene product activity and comparing this level to the level of gene expression or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the mammalian osteocalcin transcriptional regulatory sequence has been identified. Alterations in gene expression levels may be by any number of methods known to those of skill in the art e.g., by assaying for reporter gene activity, assaying cell lysates for mRNA transcripts, e.g. by Northern analysis or using other methods known in the art for assaying for gene products expressed by the cell.

Once a compound has been identified that inhibits or enhances osteocalcin transcriptional regulatory sequence activity, it may then be tested in an animal-based assay to determine if the compound exhibits the ability to act as a drug to ameliorate and/or prevent symptoms of prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, or breast cancer and/or prevent the proliferation of prostate cancer cells, prostate stromal cells, vascular pericytes, proliferating cancer- associated osteoblasts, and prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR)

The invention is illustrated by way of the following non-limiting examples.

XJN EXAMPLE 1: SYSTEMIC OSTEOCALCIN (OC) PROMOTER- DRIVEN REPLICATION-COMPETENT ADENOVIRUS FOR THE

TREATMENT OF ANDROGEN-INDEPENDENT PROSTATE CANCER BONE METASTASIS

SUMMARY

Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed at high levels by osteoblasts, calcified benign and malignant tumors. In this study, an effective systemic therapy was developed using a conditional OC replication-competent adenoviral (Ad) vector under the control of OC promoter for the treatment of hormone-refractory prostate cancer bone metastasis. A recombinant Ad vector, Ad-OC-Ela, was constructed which contained an OC promoter driven Ela gene. The efficacy of Ad-OC-Ela in inhibiting the growth of human prostate cancer cells lines (LNCaP, C4-2, PC-3, DU145, ARCaP) and human bone (MG-63) and prostate fibroblast (9096F) cell lines was evaluated tin vitro. Ad- OC-Ela was also evaluated by intratumoral and systemic administration to subcutaneous PC- 3 and intraosseous C4-2 human prostate cancer xenograft models in athymic and SCID/bg mice, respectively. Immunohistochemistry studies demonstrated that OC is prevalently expressed in both primary and metastatic prostate cancers with positive OC stains found in both tumor epithelial and prostate or bone stromal cell compartment. The growth of prostate cancer cell lines, either PSA-secreting (LNCaP, C4-2, ARCaP) or non-secreting (PC-3, DU145), and bone (MG-63) and prostate (9096F) stromal cell lines were markedly inhibited by Ad-OC-El a through viral lytic activity, hi athymic nude mice bearing subcutaneous androgen receptor-negative PC-3 xenografts, a single intratumoral injection of Ad-OC-Ela (2 x lθ9 PFU) inhibited tumor growth. In SCTD/bg mice bearing intraosseous androgen receptor-positive C4-2 xenografts, a single intravenous administration of Ad-OC-Ela (2 x lθ9 PFU) eliminated PSA elevation in 100% of the treated mice. Serum PSA rebound was suppressed in 80% (4/5) of mice through subsequent systemic Ad-OC-Ela administration.

Forty percent (2/5) of the mice were considered as "cured" without subsequent PSA rebound nor tumor cells found in the skeleton. Thus, systemic OC promoter-driven conditional replication-competent adenoviras is highly effective in inducing tumor regression in previously established hormone-refractory primary prostate cancer and its skeletal metastasis in experimental models through cell lysis in both tumor epithelium and its supporting stroma.

MATERIALS AND METHODS

CELLS AND CELL CULTURE

LNCaP, an androgen-responsive, androgen receptor-positive, and PSA- secreting human prostate cancer cell line, was derived from a cervical lymph node metastasis by Horosewicz et al.26 From this parental cell line, we derived a series of androgen- independent (defined as cells that are capable of forming PSA-secreting solid tumors when inoculated in castrated athymic male mice without the supporting stromal cells or extracellular matrices) and lineage-related LNCaP sublines.27, 28 Qne of the sublines, C4-2, remains androgen receptor and PSA positive and acquires osseous metastatic potential when inoculated either subcutaneously or orthotopically.27, 28 ARCaP is an androgen-repressed, low androgen receptor and PSA-expressing human prostate cancer cell line established by our laboratory. This cell line is highly tumorigenic and metastatic and represents an advanced form of human prostate cancer model.29 PC-3 is an androgen-independent, androgen receptor and PSA-negative human prostate cancer cell line established by Kaighn et al (Kaighn, M. E., Narayan, K. S., Ohnuki, Y., Lechner, J. F. and Jones, L. W. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest. Urol, 17: 16, 1979) from the bone marrow aspirates of a patient with confirmed metastatic disease. DU- 145 is an androgen-independent, androgen receptor and PSA-negative human prostate cancer cell line established by Stone et al (Stone, K. R., Mickey, D. D., Wunderli, H., Mickey, G. H. and Paulson, D. F. Isolation of a human prostate carcinoma cell line (DU145). Int. J. Cancer, 21: 274, 1978) from a patient with prostate cancer brain metastasis. Lovo, a colon cancer cell line, was established by Dre inko et al from localized colon tumor tissue specimen, and was kindly provided by Dr. L. Y. Yang, University of Texas M. D. Anderson Cancer Center, Houston, TX (Drewinko, B., Romsdahl, M. M., Yang, L. Y., Ahearn, M. J. and Trujillo, J. M. Establishment of human carcinoembryonic antigen-producing colon adenocarcinoma cell line. Cancer Res., 36: 467, 1976). WH, a cell line derived from a human bladder transitional cell carcinoma specimen, was established by Zhau et al. (Zhau, H. E., Hong, S. J. and Chung, L. W. K. A fetal rat uro genital sinus mesenchymal cell line (rUGM): accelerated growth and conferral of androgen-induced growth responsiveness upon a human bladder cancer epithelial cell line in vivo. hit. J, Cancer., 56: 706, 1994) 293 is a transformed human embryonic kidney cell line established by Graham et al with a complementing adenoviral El region that supports adenoviral replication (Graham, F. L. Growth of 293 cells in suspension culture. J. Gen. Virol, 68: 937, 1987). A human prostate fibroblast cell line, 9096F, was established by our laboratory from a surgical prostate biopsy specimen(Ozen, M., Multani, A. S., Kuniyasu, H., Chung, L. W. K., von Eschenbach, A. C. and Pathak, S. Specific histologic and cyto genetic evidence for in vivo malignant transformation of murine host cells by three human prostate cancer cell lines. Oncol. Res., 9: 433, 1997). A human bone stromal cell line, MG-63 was established from an osteosarcoma specimen and was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The PC-3, DU-145 and 293 cell lines were also obtained from ATCC. In this study, C4-2 and 9096F cells were maintained in T medium (Life Technologies, hie.) containing 10% FBS as described previously (Gotoh, A., Ko, S. C, Shirakawa, T., Cheon, J., Kao, C, Miyamoto, T., Gardner, T. A., Ho, L. J., Cleutjens, C. B., Trapman, J., Graham, F. L. and Chung, L. W. K. Development of prostate- specific antigen promoter-based gene therapy for androgen-independent human prostate cancer. J. Urol, 160: 220, 1998; Ozen, M., Multani, A. S., Kuniyasu, H., Chung, L. W. K., von Eschenbach, A. C. and Pathak, S. Specific histologic and cytogenetic evidence for in vivo malignant transformation of murine host cells by three human prostate cancer cell lines. Oncol. Res., 9: 433, 1997). LNCaP, PC-3, DU-145, ARCaP, WH and MG-63 cells were all maintained in T medium (Life Technologies, hie.) containing 5% FBS (Gotoh, A., Ko, S. C,

Shirakawa, T., Cheon, J., Kao, C, Miyamoto, T., Gardner, T. A., Ho, L. J., Cleutjens, C. B., Trapman, J., Graham, F. L. and Chung, L. W. K. Development of prostate-specific antigen promoter-based gene therapy for androgen-independent human prostate cancer. J. Urol, 160: 220, 1998; Zhau, H. Y., Chang, S. M., Chen, B. Q., Wang, Y., Zhang, H., Kao, C, Sang, Q. A., Pathak, S. J. and Chung, L. W. K. Androgen-repressed phenotype in human prostate cancer. Proc. Natl. Acad. Sci. U S A., 93: 15152, 1996; Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcin promoter- based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996). Lovo cells were maintained in F-12 Nutrient Mixture (Life Technologies, hie.) containing 10% FBS. 293 cells were maintained in MEM (Life

Technologies, Inc.) containing 10% FBS. The cells were fed three times a week with fresh growth medium and maintained at 37 yC in 5%> CO2-

CONSTRUCTION AND PRODUCTION OF THE REPLICATION-COMPETENT AD-OC-El A

All plasmids were constracted according to standard published protocols (Bett, A. J., Haddara, W., -Prevec, L. and Graham, F. L. An efficient and flexible system for construction of adenoviras vectors with insertions or deletions in early regions 1 and 3. Proc. Natl. Acad. Sci. U S A., 91 : 8802, 1994). Briefly, a Bam Hl-Xca I fragment containing the backbone of an Ad5 vector from bp 549 to bp 5792 was digested from pXC 548C, a derivative of plasmid pXCl (McKinnon, R. D., Bacchetti, S. and Graham, F. L. Tn5 mutagenesis of the transforming genes of human adenovirus type 5. Gene, 19: 33, 1982), and inserted into pyElsplB (obtained as gifts from Dr. Frank Graham, MacMaster University, Hamilton, Ontario, Canada) between Bam HI and Xca I site to create a pyBPAEϋ shuttle vector. A pOCEla was constructed by inserting a 1370 bp fragment of murine OC promoter which was cut from pill.5 TK using Xho I and Sal I Enzymes into the Xho I site of pyBPAEπ to drive the Ad5 Ela gene. The shuttle pOCEla vector was co-transfected with a replication-defective recombinant Ad vector, pJM17, into 293 cells by the N-[l-(2,3- dioleoyloxyl)propyl]-N,N,N-trhnethylammoniummethyl sulfate (Boehringer Mannheim Biochemicals)-mediated transfection method (Zhang, W-W., Fang, X., Branch, C. D., Mazur,

W., French, B. A. and Roth, J. A. Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis. Biotechniques, 15: 868, 1993) to generate a partially E3-deleted replication-competent adenoviras, Ad-OC-Ela, as shown in Figure 1. The resulting Ad-OC-Ela was demonstrated to replicate in a restricted manner only in cells that expressed OC promoter activity. The culture medium of the 293 cells showing complete cytopathic effect was collected and centrifuged at 1,000 x g for 10 min. The pooled supernatants were aliquoted and stored at -80 yC as primary viral stock. Viral stocks were propagated in 293 cells, and selected clones of Ad-OC-Ela virus were obtained by plaque purification according to the method of Graham and Pervec (Graham, F. L. and Prevec, L. Manipulation of adenovirus vectors. Vol. 7, pp. 109-128. Clifton, NJ: The Humana Press, hie, 1991). One of the viral clones was selected, propagated in 293 cells, and harvested 36 to 40 h after infection, pelleted, resuspended in PBS, and lysed. Cell debris was removed by subjecting the cells to centrifugation, and the viras in the cell lysate was purified by CsCl2 gradient centrifugation. Concentrated viras was dialyzed, aliquoted, and stored at -80 yC. The viral titer was determined by plaque assay as described previously (Gotoh, A., Ko, S. C,

Shirakawa, T., Cheon, J., Kao, C, Miyamoto, T., Gardner, T. A., Ho, L. J., Cleutjens, C. B., Trapman, J., Graham, F. L. and Chung, L. W. K. Development of prostate-specific antigen promoter-based gene therapy for androgen-independent human prostate cancer. J. Urol, 160: 220, 1998). The control viruses used in this study, Ad-CMV-pA and Ad-CMV-beta-gal, were constracted, plaque purified and propagated in 293 cells using a similar procedure as described previously (Ko, S. C, Gotoh, A., Thalmarm, G. N., Zhau, H. E., Johnston, D. A., Zhang, W. W., Kao, C. and Chung, L. W. K. Molecular therapy with recombinant p53 adenoviras in an androgen-independent, metastatic human prostate cancer model. Hum. Gene Ther., 7: 1683, 1996). IMMUNOHISTOCHEMICAL STAINING OF PRIMARY AND METASTATIC HUMAN PROSTATE TUMOR SPECIMENS

De-paraffinized primary human prostate cancer specimens and lymph node and bone metastatic specimens were obtained from the Department of Urology and Pathology at the University of Virginia School of Medicine, Charlottesville, VA and McGill University, Montreal, Quebec, Canada. Tissues were treated with 3% H2O2, blocked with SuperBlock (Scytek Laboratories, Logan, Utah), reacted with a monoclonal OC antibody (OC 4-30: Takara Shuzo, Otsu, Japan), and the antibody staining signals were amplified by a biotinylated-peroxidase-conjugated streptavidin system (Bio-Genex Laboratories, San

Ramen, CA). OC stain was visualized after reacting the conjugated peroxidase with an AEC Chromogen, 3-amino-9-ethylcarbazole.

IN VITRO CELL GROWTH ASSAY

5 x 10³ LNCaP, C4-2, PC-3, DU-145, ARCaP, 293, WH, Lovo, MG-63 or 9096F cells were plated in 24-well plates. After 24 hours, the cells were infected with Ad- OC-Ela with a range of concentrations from 0.01 to 5 MOI (or pfu/cell which was estimated to be 0.2 to 100 virus particles/cell) for 2 hours; cells infected with Ad-CMV-pA or Ad- CMV-beta-gal were served as negative controls (Fig. 17A-D). Cell numbers were measured

3 days later by the crystal violet assay using an automated E max spectrophotometric plate reader (Molecular Devices Corp., Sunnyvale, CA) as described previously (Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996). ASSESSMENT OF VIRUS YIELD IN PROSTATE AND BLADDER CANCER CELL LINES INFECTED WITH AD-OC- E1A

2 x 10^ C4-2 or 293 cells were plated in duplicate into six-well plates. 24 hours later, medium was aspirated and replaced with 0.5 ml of T medium or MEM medium containing either Ad-OC-Ela, Ad-CMV-pA or wild-type Ad vector at a MOI of 2 pfu/cell As a negative control, WH cells cultured in T-medium were infected similarly the test viruses. After 2 hours infection by the Ad vectors at 37 yC, cells were washed twice with PBS and added with 2 ml of medium per well. The cell culture media were recovered, diluted, and added to 293 cells for a plaque assay in triplicate at intervals between 0 and 72 hours. The assay involves the addition of 100 μl of the diluted cultured cell mediums to a confluent 293 cell culture that overlay with a 0.75% semi-solid agarose medium. After 5 days, the number of plaques was visualized by staining with 0.5% crystal violet and counted (Goodrum, F. D. and Omelles, D. A. p53 status does not determine outcome of EIB 55- kilodalton mutant adenoviras lyric infection. J. Virol, 72: 9479, 1998).

ASSESSMENT OF ADENOVIRAL INFECTIVITY IN MOUSE AND HUMAN BONES To determine if normal mouse or healthy human bones are susceptible to Ad infection, two studies were performed. First, an Ad-CMV-beta-gal (1 x 10 9 pfu) was injected into the femur of an adult mouse and the bone was harvested three days later for histochemical analysis of beta-gal activity using a previously established method (Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996). Second, a normal bone specimen harvested from a 69 year old man with bone fracture was cultured in T-medium containing 0.6%) soft agar; a human prostate cancer PC-3 xenograft was infected in parallel and secured as a control. The tissue specimens were exposed to Ad-CMV-beta-gal (1 x 10^ pfu) and were processed 3 days after infection. After harvesting bone and prostate tumor specimens, tissues were first washed in PBS and fixed in 0.05 % glutaraldehyde at 4 yC for 24 hours. Bone specimens were put in PBS for 24 hours after fixing and decalcified with 0.25 M EDTA in PBS (pH 7.4) at 4 yC for 5 days. After decalcification, the specimens were stained overnight in a solution of 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2 in PBS. Prostate tumor specimens were processed as described previously and were stained similarly as described above for beta-galactosidase activity (Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996).

IN VIVO ANIMAL EXPERIMENT

To demonstrate tumor specificity of Ad-OC-Ela lytic activity, athymic mice (20 to 25 g) were inoculated subcutaneously with 1 x 10^ PC-3 or Lovo cells suspended in 100 μl T-medium containing 5%> FBS. When the tumor became palpable (4-5 mm in , diameter), the animals were randomly assigned to two experimental groups: group 1, Ad-OC-

Ela; group 2, Ad-CMV-beta-gal. A single dose of viras (2 x 10^ pfu) was injected intratumorally to the mice. After 4 weeks of administration of the test viruses, tumor size was measured and recorded.

For the evaluation of Ad-OC-Ela in the intraosseous prostate tumor model, 1 x 10 6 C4-2 cells were injected into the bone marrow space of the right tibial bone in castrated male SCID/bg mice according to previously published procedures (Wu, T. T., Sikes, R. A., Cui, Q., Thalmann, G. N., Kao, C, Murphy, C. .F., Yang, H., Zhau, H. E., Balian, G. and Chung, L. W. K. Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines. Int. J. Cancer, 77: 887, 1998).

Blood specimens (approximately 100 μl) were obtained from the tail vein for PSA assay once a week. Serum PSA was determined by microparticle enzyme-linked immunosorbent assay (MEIA) using an Abbott EVIx machine (Abbott Park, IL). After the detection of serum PSA elevation, a single dose of 25 μl Ad-OC-Ela, 2 x 10^ pfu (or 4 x lO¹^ viras particles) per animal, was administered intravenously to mice. When serum PSA rebound had occurred, animals were treated with the second or third intravenous injection of the same dose of the test virus at the specified time points as indicated. Serum PSA was monitored weekly and histopathology and X-ray of the tumors were routinely assessed when the animals were sacrificed.

RESULTS

IMMUNOHISTOCHEMICAL STAINING OF OC IN PRIMARY AND METASTATIC PROSTATE TUMOR SPECIMENS

OC has been shown to be a specific marker indicative of differentiation of osteoblast-lineaged cells (Hoffmann, H. M., Catron, K. M., Wijnen, A. F. V., McCabe, L. R., Lian, J. B., Stein, G. S. and Stein, J. L. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins. Proc. Natl. Acad. Sci. U S A., 91: 12887, 1994). OC was also detected in calcified normal tissues, (Bini, A., Mann, K. G., Kudryk, B. J. and Schoen, F. J.

Noncollagenous bone matrix proteins, calcification, and thrombosis in carotid artery atherosclerosis. Arterioscler Thromb. Vase. Biol, 19: 1852, 1999), pericytes (Reilly, T. M., Seldes, R., Luchetti, W. and Brighton, C. T. Similarities in the phenotypic expression of pericytes and bone cells. Clin. Orthop., 346: 95, 1998), and benign tumors (Lantuejoul, S., Isaac, S., Pinel, N., Negoescu, A., Guibert, B. and Brambilla, E. Clear cell tumor of the lung: an immunohistochemical and ultrastractural study supporting a pericytic differentiation. Mod. Pathol, 10: 1001, 1997). Using an immunohistochemical method, OC expression was observed by primary prostate tumor stroma (Figure 2a) and tumor epithelium and stroma (Figure 2b). Positive OC stain was found in 85% (23/27) of the primary prostate cancer specimens and in 100% of lymph node (12/12) and bone (10/10) metastatic specimens. A representative immunohistochemical staining profile of lymph node and bone is shown in Figure 2d and 2e, respectively. Background immunohistochemical staining of OC in primary prostate cancer and bone metastasis is demonstrated in Figure 2c and 2f, respectively. CYTOTOXICITY OF AD-OC-E1 A TO PROSTATE CANCER CELL LINES IN VITRO: INDEPENDENT OF ENDOGENOUS PSA AND AR

STATUS

To assess the cytotoxicity of Ad-OC-Ela, a number of human prostate cancer cell lines, LNCaP, C4-2, PC-3, DU-145 and ARCaP, were exposed in vitro to a wide range of Ad-OC-Ela vector from 0.01 to 5 MOI. Human 293, or WH and Lovo cells were employed as positive or negative controls, respectively (Graham, F. L. Growth of 293 cells in suspension culture. J. Gen. Virol, 68: 937, 1987; Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcm promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996). Whereas exposure of LNCaP and C4-2 cells to 5 MOI of Ad-OC-Ela inhibited the growth of these cells by 70%, it was observed that this same dose of Ad-OC-Ela was found to be ineffective in blocking the growth of WH and Lovo cells which exhibit low or no OC promoter activity (Figure 3a). Cells infected similarly by the control viruses, either without an insert (Ad-CMV-PA) or Ad-CMV-beta-gal, were also found to be unaffected even when exposed to 5 MOI of the viras (Figure 3a). Next, the efficacy of Ad-OC-Ela was evaluated in several other human prostate cancer cell lines that either expressed very low levels (e.g. ARCaP) or non-detectable (e.g. PC-3 and DU-145) levels of PSA and AR. Figure 3b demonstrated that all of the tested human prostate cancer cell lines were sensitive to Ad-

OC-Ela-induced cell lysis in vitro irrespective of their intrinsic levels of PSA and AR expression, hi addition, the effects of Ad-OC-Ela were also evaluated on the growth of human prostate and bone fibroblast cell lines in vitro. As demonstrated in Figure 3c, Ad-OC- Ela infection induced significant cell lysis in both cultured human prostate (e.g. 9096F) and bone (MG-63) fibroblasts.

AD-OC-EIA REPLICATION IN ANDROGEN-INDEPENDENT HUMAN PROSTATE CANCER CELL LINES

To determine whether OC promoter drives El a transgene expression which could result in adenoviral replication in competent human prostate cancer cell lines, viras titers were evaluated in the supernatant of C4-2 cell line, an androgen-independent human prostate cancer cell line, after infection with either Ad-OC-Ela or Ad-CMV-PA. The 293 and WH cells served as positive and negative controls, respectively (Graham, F. L. Growth of 293 cells in suspension culture. J. Gen. Virol, 68: 937, 1987; Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996). Results of these studies demonstrated that the virus titers in the supernatant of all of the tested cell lines were 0 pfu/cell, 0 pfu/cell, and 170 + 60 pfu/cell, respectively for C4-2, WH and 293 cells when infected with Ad-CMV-PA. When infected with Ad-OC-Ela, adenoviras replication was restricted to C4-2 and 293 cells (15 ± 8 and 7.7

± 3.2 pfu/cell, respectively) with an insignificant level of replication detected in WH cells (0.047 ± 0.021 pfu/cell). Ad-OC-Ela was found to be highly efficient, replicating in C4-2 cells with an infectious particle count parallel to that observed in the viral replication- competent 293 cell line.

SPECIFICITY OF INTRATUMORAL AD-OC-E1 A IN ABOLISHING SUBCUTANEOUS PC-3 TUMOR GROWTH IN VIVO

To assess the specificity of Ad-OC-Ela in inhibiting prostate tumor growth in vivo, we compared the activity of this viras on the growth of previously established subcutaneous PC-3 human prostate tumors with that of the control Lovo human colon tumors. Figure 4 shows that Ad-OC-Ela effectively inhibited the growth of PC-3 but not Lovo tumors when injected intratumorally. These data are consistent with the observation that OC promoter activity is present in PC-3 but not in Lovo cells (data not shown).

INTRAVENOUS ADMINISTRATION OF AD-OC-El A TO MICE WITH

PREVIOUSLY ESTABLISHED C4-2 HUMAN PROSTATE TUMORS IN

THE SKELETON

An androgen-independent and PSA-secreting C4-2 human prostate cancer cell line was chosen as the model to evaluate the efficacy of systemic Ad-OC-Ela in previously established prostate tumor xenografts in the skeleton (Wu, T. T., Sikes, R. A., Cui, Q., Thalmann, G. N., Kao, C, Mu hy, C. .F., Yang, H., Zhau, H. E., Balian, G. and Chung, L. W. K. Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen-producing tumors in athymic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines. hit. J. Cancer, 77: 887, 1998). The standard protocol was that once tumor growth in the tibia was established as evidenced by rising serum PSA, intravenous Ad-OC-El a therapy was initiated. Serum PSA was followed weekly and upon PSA rebound animals were subjected to repeated Ad-OC-Ela treatment. A total of six animals were evaluated in this study. Figure 5 a shows that in one control untreated mouse (#1), serum PSA underwent marked elevation from the basal level to more than 10 ng/ml within 6 weeks and increased exponentially to 630 ng/ml at 15 weeks. This profile of rapid PSA rise was consistent with our previous reports (Wu, H. C, Hsieh, J. T., Gleave, M. E., Brown, N. M., Pathak, S. and Chung, L. W. K. Delivation of androgen-independent human LNCaP prostatic cancer cell subline: role of bone stromal cells. Int. J. Cancer, 57: 406, 1994; Thalmann, G. N., Anezinis, P. E., Chang, S. M., Zhau, H. E., Kim, E. E., Hopwood, V. L.,

Pathak, S., von Eschenbach, A. C. and Chung, L. W. K. Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res., 54: 2577, 1994; Wu, T. T., Sikes, R. A., Cui, Q., Thalmann, G. N., Kao, C, Murphy, C. .F., Yang, H., Zhau, H. E., Balian, G. and Chung, L. W. K. Establishing human prostate cancer cell xenografts in bone: induction of osteoblastic reaction by prostate-specific antigen- producing tumors in athymic and SCID bg mice using LNCaP and lineage-derived metastatic sublines. Int. J. Cancer, 77: 887, 1998). Serum PS A profiles of Ad-OC-El a-treated mice are shown in Figure 5b to 5f. Several variants of the PSA responses were noted: 1) Mouse #2 and #3 responded to two intravenous Ad-OC-Ela treatment with a complete regression of the skeletal tumors (Figure 6a, 6b) and a PSA nadir > 15 weeks (Figure 5b, 5c). These two mice are considered as "cured" by systemic Ad-OC-Ela. 2) Mouse #4 and #5 responded to systemic Ad-OC-Ela with a marked and rapid PSA decline. PSA nadir in these mice was maintained for variable period, ranging from 1 to 6 weeks (Figure 5d, 5e). 3) Mouse #6 responded favorably to systemic Ad-OC-Ela initially (PSA nadir lasted for 5 weeks) but gradually escaped systemic Ad-OC-Ela growth inhibitory effect at the 2nd and 3rd dose of systemic Ad-OC-Ela treatment (Figure 5f). GROSS MORPHOLOGY, HISTOPATHOLOGY AND IMMUNOHISTOCHEMISTRY OF PROSTATE TUMOR XENOGRAFTS GROWN IN THE MOUSE TIBIA: EFFECTS OF AD- OC-E1A

Figure 6a demonstrated the gross anatomical difference between a control and a responder mouse to Ad-OC-Ela therapy. As shown by X-ray and gross anatomy, when compared to the untreated control, systemic Ad-OC-Ela caused marked regression of prostate tumor growth in the tibia. This improvement was confirmed by examining the histopathologic sections of tumors obtained from the control and Ad-OC-El a-treated animals (Figure 6b, comparison between Panels A and C). While positive PSA staining was noted in the control specimens (Figure 6b, Panel B), no PSA staining was detected in the Ad-OC-Ela treated specimens (data not shown). The adenoviral infectivity was also compared in mouse bone in situ and human bone and human prostate PC-3 xenograft in vitro. Results of this study showed that a single injection of Ad-CMV-beta-gal efficiently infected the mouse bone cells without affecting the cortical bone (Figure 6c, Panel A). In vitro Ad-CMV-beta-gal efficiently infected the PC-3 tumors but not human bone maintained as explants in soft agar (Figure 6c, comparison of Panels B and C, respectively).

DISCUSSION

Cancer therapies using adenoviral vectors can be divided into two broad categories, the replication-defective and replication-competent (Heise, C. and Kirn, D. H. Replication-selective adenovirases as oncolytic agents. J. Clin. Invest., 105: 847, 2000). Because of the difficulties of infecting all cancer cells with adenoviral vectors, numerous laboratories have designed various versions of viral constracts with the primary goal of achieving increased efficiency of viral gene expression/replication in competent tumor cells without damaging the normal tissues. One of such approach relies on the ability of "bystander" genes, such as HSV-TK or cytosine deaminase, incorporated into replication-defective adenoviral vectors which convert pro-drags to biologically active growth-inhibitory products and elicit efficient cell-kill even in cells that were not transduced with viral-mediated gene (Gotoh, A., Ko, S. C, Shirakawa, T., Cheon, J., Kao, C, Miyamoto, T., Gardner, T. A., Ho, L. J., Cleutjens, C. B., Trapman, J., Graham, F. L. and Chung, L. W. K. Development of prostate- specific antigen promoter-based gene therapy for androgen-independent human prostate cancer. J. Urol, 160: 220, 1998; Eastham, J. A., Chen, S. H., Sehgal, I., Yang, G., Timme, T. L., Hall, S. J., Woo, S. L. and Thompson, T. C. Prostate cancer gene therapy: herpes simplex viras thymidine kinase gene fransduction followed by ganciclovir in mouse and human prostate cancer models. Hum. Gene Ther., 7: 515, 1996; Koeneman, K. S., Kao, C, Ko, S. C, Yang, L., Wada, Y., Kallmes, D. A., Gillenwater, J. Y., Zhau, H. E., Chung, L. W. K. and Gardner, T. A. Osteocalcin directed gene therapy for prostate cancer bone metastasis. World J. Urol, 18: 102, 2000; Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon, J., Gotoh, A., Wu, T. T.,

Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal- epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998; Blackburn, R. V., Galoforo, S. S., Corry, P. M. and Lee, Y. J. Adenoviral-mediated transfer of a heat-inducible double suicide gene into prostate carcinoma cells. Cancer Res., 58: 1358, 1998). The construction of replication-competent ONYX-015 which lacks Elb, a 55 kDa protein, can conceptually replicate in tumor cells that lack functional p53 protein(Heise, C, Williams, A., Xue, S., Propst, M. and Kirn, D. Intravenous administration of ONYX-015, a selectively- replicating adenoviras, induces antitumoral efficiency. Cancer Res., 59: 2623, 1999). Conditional activation of viral gene expression and replication have been achieved using tissue specific promoters, such as PSA for prostate cancer^- and alpha-fetal protein for liver cancer (Kanai, F., Lan, K. H., Shiratori, Y., Tanaka, T., Ohashi, M., Okudaira, T., Yoshida, Y., Wakimoto, H., Hamada, H., Nakabayashi, H., Tamaoki, T. and Omata, M. hi vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene. Cancer Res., 57: 461, 1997). Modification of viral gene structure in an adenoviral vector shows that the efficiency of viral replication is markedly enhanced upon the introduction of adenoviral death protein (Doronin, K., Toth, K., Kuppuswamy, M., Ward, P., Tollefson, A. E. and Wold, W. S. Tumor-specific, replication-competent adenoviras vectors overexpressing the adenoviras death protein. J. Virol, 74: 6147, 2000). In the present study, the possibility was explored of using a tissue-specific (i.e. osteoblast-specific) and tumor- restrictive (i.e. restricted expression in calcified benign and malignant tumors) OC promoter to drive adenoviras replication in cells that contain OC promoter activity. This version of adenoviral vector is aimed at allowing the viral replication in both tumor epithelium and its supporting stromal cells including the fibromuscular stromal cells (Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon, J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998.; Koeneman, K. S., Yeung, F. and Chung, L. W. K. Osteomimetic properties of prostate cancer cells: a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment. Prostate, 39: 246, 1999) and vascular pericyte (Doherty, M. J., Ashton, B. A., Walsh, S., Beresford, J. N., Grant, M. E. and

Canfield, A. E. Vascular pericytes express osteogenic potential in vitro and in vivo. J. Bone Miner. Res., 13: 828, 1998) which express OC. Thus, Ad-OC-Ela could potentially inflict the maximal cell-kill through primarily viral replication in tumor epithelium and secondarily destruction of intracellular communication between tumor and stroma by inducing cell lysis in prostate fibroblasts and vascular pericytes. hi experimental co-culture studies in vitro and a chimeric tumor model in vivo, induction of osteoblast cell death by HSV-TK/acyclovir (ACV) markedly inhibited the growth of prostate tumor cells (unpublished observations). Since OC expression is highly restricted to maturing osteoblasts ( Hoffmann, H. M., Catron, K. M., Wijnen, A. F. V., McCabe, L. R., Lian, J. B., Stein, G. S. and Stein, J. L. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins. Proc. Natl. Acad. Sci. U S A., 91: 12887, 1994), 43 Ad-OC-Ela may potentially damage bone and alter the rate of bone resorption and deposition. This concern has been addressed and observations are summarized below. First, the cortical bone of both mouse and human is restricted to adenoviral infection. Whereas mouse bone marrow is highly susceptible to adenoviral infection, it was observed that human bone including maturing osteoblasts appeared to be more resistant to adenoviral infection. Therefore, it is possible that Ad-OC-Ela replication may be limited to proliferating and maturing osteoblasts in men which express OC promoter activity. Second, intraosseous administration of Ad-OC-HSVTK plus intraperitoneal ACV in intact adult mice did not result in any abnormalities in the skeleton (Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon,

J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998). In fact, OC has been shown as an inhibitor of bone mineralization by preventing the growth of mineral crystal growth in an in vitro assay (Romberg, R. W., Werness, P. G., Riggs, B. L. and Mann, K. G. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium- binding proteins. Biochemistry, 25: 1176, 1986). This role of OC is consistent with the transgenic OC "knockout" mouse model where the destruction of OC-expressing cells by HSV-TK resulted in increased bone mass and and bone formation (Ducy, P., Desbois, C, Boyce, B., Pinero, G., Story, B., Dunstan, C, Smith, E., Bonadio, J., Goldstein, S., Gundberg, C, Bradley, A. and Karsenty, G. Increased bone formation in osteocalcin-deficient mice.

Nature, 382: 448, 1996).

The concept that OC is a tissue-specific and tumor-restrictive promoter that potentially has an advantage over other prostate-specific promoters such as PSA enhancer (Rodrigez, R., Schuur, E. R., Lim, H. Y., Henderson, G. A., Simons, J. W. and Henderson, D. R. Prostate attenuated replication competent adenoviras (ARC A) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res., 57: 2559, 1997; Yu, D-. C, Chen, Y., Seng, M., Dilley, J. and Henderson, D. R. The addition of adenoviras type 5 region E3 enables calydon virus 787 to eliminate distant prostate tumor xenografts. Cancer Res., 59: 4200, 1999), human kallikrein 2 (hK2) (Yu, D-. C, Sakamoto, G. T. and Henderson, D. R. Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon viras 764, an attenuated replication competent adenoviras for prostate cancer therapy. Cancer Res., 59: 1498, 1999) or prostate-specific membrane antigen (PSMA), is discussed below. OC is expressed prevalently in human primary and metastatic prostate cancers, with expression found in both tumor epithelium and surrounding stromal compartment (see Figure 2) (Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon,

J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998; Koeneman, K. S., Yeung, F. and Chung, L. W. K. Osteomimetic properties of prostate cancer cells: a hypothesis supporting the predilection of prostate cancer metastasis and growth in the bone environment. Prostate, 39: 246, 1999). OC expression is not limited to prostate tumors and was found also expressed by other calcified benign and malignant tissues such as smooth muscle plaques associated with heart valve and blood vessels (Doherty, M. J., Ashton, B. A., Walsh, S., Beresford, J. N., Grant, M. E. and Canfield, A. E. Vascular pericytes express osteogenic potential in vitro and in vivo. J. Bone Miner. Res., 13: 828, 1998), osteosarcoma, brain, thyroid, breast, lung and ovarian tumors (unpublished results) irrespective of their PSA and AR status. This is significant since it was estimated that about 20% of prostate cancer patients do not have elevated PSA despite the detection and progression of the disease (Carter, H. B., Pearson, J. D., Metter, E. J., Brant, L. J., Chan, D. W., Andres, R., Fozard, J. L. and Walsh, P. C. Longitudinal evaluation of prostate-specific antigen levels in men with and without prostate disease. JAMA, 267: 2215, 1992). hi addition, although AR gene amplification and overexpression were detected in almost 30% of the clinical prostate cancer specimens (Nisakorpi, T., Hyytinen, E., Koivisto, P., Tanner, M., Keinanen, R., Palmberg, C, Palotie, A., Tammela, T., Isola, J. and Kallioniemi, O. P. In vivo amplification of the androgen receptor gene and progression of human prostate cancer. Nat. Genet., 9: 401, 1995), AR- mutant or AR-null prostate cancer cells and tissues were nevertheless commonly observed

(Gaddipati, J. P., McLeod, D. G., Heidenberg, H. B., Sesterhenn, I. A., Finger, M. J., Moul, J. W. and Srivastava, S. Frequent detection of codon 877 mutation in the androgen receptor gene in advanced prostate cancers. Cancer Res., 54: 2861, 1994). Based on these observations, it is possible that PSA and/or AR-negative tumors may be responsive to Ad-OC-Ela but not Ad- PSA-El a-induced cell lysis. Several previous publications demonstrated that an Ad vector mediated toxic gene, HSV-TK, the expression of which is driven by OC promoter, inhibited the growth of osteosarcoma (Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon, J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L. W. K. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998;

Ko, S. C, Cheon, J., Kao, C, Gotoh, A., Shirakawa, T., Sikes, R. A., Karsenty, G. and Chung, L. W. K. Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models. Cancer Res., 56: 4614, 1996; Cheon, J., Ko, S. C, Gardner, T. A., Shirakawa, T., Gotoh, A., Kao, C. and Chung, L. W. K. Chemogene therapy: Osteocalcin promoter-based suicide gene therapy in combination with methotrexate in a murine osteosarcoma^" model. Cancer Gene Ther., 4: 359, 1997) and its metastasis (Shirakawa, T., Ko, S. C, Gardner, T. A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L. W. K. and Kao, C. hi vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapy. Cancer Gene Ther., 5: 274, 1998) and inhibited prostate tumor growth both in vitro and in vivo (Gardner, T. A., Ko, S. C, Kao, C, Shirakawa, S., Cheon, J., Gotoh, A., Wu, T. T., Sikes, R. A., Zhau, H. E., Cui, Q., Balian, G. and Chung, L.

W. K. Exploiting stromal-epithelial interaction for model development and new strategies of gene therapy for prostate cancer and osteosarcoma metastasis. Gene Ther. Mol. Biol, 2: 41, 1998). Although intratumoral administration of Ad-OC-TK was used in most of these earlier studies, the inventors observed significant remission of osteosarcoma lung metastasis and improvement of survival by intravenous administration of Ad-OC-TK (Shirakawa, T., Ko, S.

C, Gardner, T. A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L. W. K. and Kao, C. hi vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter- based toxic gene therapy. Cancer Gene Ther., 5: 274, 1998). The ability of intravenous Ad- OC-TK to exert anti-tumor effects on osteosarcoma pulmonary metastasis without causing liver toxicity indicated the importance of considering the selection of tumor- or tissue-specific promoters to drive the expression of therapeutic genes or viral replications for cancer therapy, hi this context, it is clear that replication-selective adenovirus may have the advantage of amplifying the input of oncolytic viras and help the spread of agents to adjacent cells (Rodrigez, R., Schuur, E. R., Lim, H. Y., Henderson, G. A., Simons, J. W. and Henderson, D. R. Prostate attenuated replication competent adenoviras (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res., 57: 2559, 1997; Yu, D-. C, Sakamoto, G. T. and Henderson, D. R. Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon viras 764, an attenuated replication competent adenoviras for prostate cancer therapy. Cancer Res., 59: 1498, 1999; Yu, D-. C, Chen, Y., Seng, M., Dilley, J. and Henderson, D. R. The addition of adenovirus type 5 region E3 enables calydon viras 787 to eliminate distant prostate tumor xenografts. Cancer Res., 59: 4200, 1999; Heise, C. and Kirn, D. H. Replication-selective adenovirases as oncolytic agents. J. Clin. Invest., 105: 847, 2000; Heise, C, Williams, A., Xue, S., Propst, M. and Kirn, D. Intravenous administration of ONYX-015, a selectively- replicating adenovirus, induces antitumoral efficiency. Cancer Res., 59: 2623, 1999). hi this study, a substantial efficacy of systemic Ad-OC-Ela was demonstrated for the treatment of androgen-independent prostate cancer skeletal xenografts. It was demonstrated that in order to eliminate the pre-existing human prostate tumor xenografts in the bone, Ad-OC-Ela administration needs to be repeated. Evidence was obtained that all mice responded initially to Ad-OC-Ela therapy (as judged by serum PSA response) and only one mouse (20%) escaped Ad-OC-El a effects gradually and become an Ad-OC-El a non-responder. Forty percent (2/5) of the Ad-OC-Ela treated mice have undergone complete tumor regression and are considered as "cured" in this present protocol. Reasons why mice may lose their response to Ad-OC-Ela are presently unclear but it is reasonable to suggest that Ad-OC-Ela infectivity may be reduced in these resistant tumors through a decreased adenoviral receptor, CAR, on tumor cell surface or a rapid clearance of Ad vectors from systemic circulation or tumor sites. While the current protocol maybe applicable to the treatment of clinical prostate cancer skeletal metastasis, there are precautions that need to be observed: 1) Ad-OC-Ela replication in normal human tissues requires more extensive testing. Human bone and human prostate cancer chimeric xenografts grown subcutaneous may be ideal for this evaluation. 2) Serum PSA response may be an indication but not be proof of tumor regression (Thalmann G. N., Sikes, R. A., Chang, S-M.,

Johnston, D. A., von Eschenbach, A. S. and Chung, L. W. K. Suramin-induced decrease in prostate-specific antigen expression with no effect on tumor growth in the LNCaP model of human prostate cancer. J. Natl. Cancer ist., 88: 794, 1996). Even if this is the potential pitfall of using altered serum PSA as the indicator for an antitumor effect, it is firmly established that serum PSA response does correlate with improved survival, improved pain, increased hemoglobin level, normalization of bone-derived alkaline phosphatase, weight gain, and improved performance status (Millikan, R. E. Chemotherapy of advanced prostatic carcinoma. Semin. Oncol, 26: 185, 1999). Smith et al found that a decrease in the serum PSA level of at least 50% at 8 weeks was correlated with significantly increased survival (Smith, D. C, Dunn, R. L., Strawderman, M. S. and Pienta, K. J. Change in serum prostate-specific antigen as a marker of response to cytotoxic therapy for hormone-refractory prostate cancer. J. Clin. Oncol, 16: 1835, 1998). Such data validate the use of changes in the serum PSA level as a response parameter in trials of therapy in prostate cancer, hi this study, the PSA response parameter demonstrates the efficacy of systemic OC promoter-driven replication-competent gene therapy. In summary, the inventors have established a novel replication-competent adenoviral therapy using a tissue-specific and tumor-restrictive OC promoter to drive the replication of adenoviras for the treatment of prostate cancer metastasis in an experimental human prostate cancer skeletal xenograft model. Ad-OC-Ela was shown to be effective in eliminating preexisting androgen-independent prostate tumors in the bone, without adverse effects on human bone. This study establishes that co-targeting prostate cancer and bone stroma may be an effective means of destroying human prostate tumors in the bone.

XV EXAMPLE 3: EFFECT OF REPLICATION-COMPETENT AD-

HOC-E1 PLUS VITAMIN D ON RENAL CARCINOMA RCC52 CELLS. PC3 CELLS. C4-2 CELLS, AND DU145 CELLS

MATERIALS AND METHODS

To determine the effect of Vitamin D on the expression of Vitamin D receptor, total RNA was isolated from normal prostate cancer (C4-2 and PC3) and renal carcinoma (RC52) cell lines. A 25 cycle RT-PCR targeted at VDR (1.3 KB) was conducted. The effect of Vitamin D on the expression of VDR was also assessed. The effect of Vitamin D on the expression of human OC (hOC) in various cell lines was also examined.

Western blot analysis of the Vitamin D receptor (VDR) in human prostate cancer (C4-2, PC3), human renal carcinoma (RCC52), human osteosarcoma (MG-63) cell lines was performed. On the le ft track a standard marker protein was loaded onto the gel

(molecular weight ranged from 20.6 to 122 kDa).

In order to assess the cytotoxicity of a replication-competent Ad-hOC-El plus Vitamin D on a human renal carcinoma RCC52 cell line in vitro, the following procedure was used, cells were exposed to Ad-hOC-El infection (dose ranged from 0.01 to 5 MOI or pfu per cell, which was estimated to be 0.2 to 100 viras particles per cell). Cells were exposed to Ad- hOC-El for two hours and viras containing media was removed and replaced with T-Media containing 5% fetal calf serum in the presence or absence of Vitamin D. A standard protocol was implemented in this study and others described below, which used concentrations of Vitamin D ranging from 5 nM (C4-2 cells) to 10 nM (DU145, PC3 and RCC52 cells), with medium changes at Day 3 containing fresh Vitamin D. Cell numbers were measured at Day

1,3, 5 and 7 by crystal violet assay using an automated E max spectrophotometric plate reader. Data are presented as relative cell numbers (uninfected control specimens = 1.0) at Day 1,3,5 and 7 after exposure to Ad-hOC-El and Vitamin D. Note that Vitamin D enhanced RCC52 cell kill at 5 MOI of Ad-OC-El. The same procedure was used for determination of the cytotoxicity of a replication-competent Ad-sPSA-El plus Vitamin D on PC3 cells, the determination of the cytotoxicity of a replication-competent Ad-sPSA-El plus Vitamin D on

DU145 cells, the determination of the cytotoxicity of a replication-competent Ad-sPSA-El plus Vitamin D on C4-2 cells, the determination of the cytotoxicity of a replication-competent Ad-hOC-El plus Vitamin D on PC-3 cells, the determination of the cytotoxicity of a replication-competent Ad-hOC-El on DU-145 cells, and for the determination of the cytotoxicity of Ad-hOC-El plus Vitamin D on C4-2 cells.

RESULTS AND DISCUSSION

hi this experiment, the comparative effect of the PSA promoter and the osteocalcin promoter and Vitamin D on OC and PSA activation was examined. The PC3 and

DU145 cell lines do not express PSA nor the androgen receptor, whereas C4-2 cells express both PSA and the androgen receptor. As shown in Figure 1, Vitamin D appears to inhibit VDR mRNA expression in prostate cancer but not renal cancer cell lines. Western blot analysis of the Vitamin D receptor (VDR) in human prostate cancer (C4-2, PC3), human renal carcinoma (RCC52), human osteosarcoma (MG-63) cell lines revealed that, consistent with the decrease in mRNA by Vitamin D treatment in prostate cancer cell lines, VDR protein expression was also slightly reduced by treatment with Vitamin D (Figure 2).

When the effect of Vitamin D on hOC expression was studied in cultured human prostate cancer (C4-2 and PC3), human renal cancer (RC52), human osteosarcoma (MG-63) and human transitional cell carcinoma (WH) cell lines, Vitamin D treatment enhanced hOC mRNA expression in PC3, RC52, MG-63 and WH, but not C4-2, cell lines.

When the effect of the cytotoxity of either a replication-competent Ad-hOC-El plus Vitamin D or replication-competent Ad-sPSA-El plus Vitamin D on various cells lines was examined, the following results were obtained. For the cytotoxicity of a replication- competent Ad-hOC-El plus Vitamin D on a human renal carcinoma RCC52 cell line in vitro,

- Vitamin D enhanced RCC52 cell kill at 5 MOI of Ad-OC-El (Fig. 4). For the cytotoxicity of a replication-competent Ad-sPSA-El plus Vitamin D on PC3 cells, Vitamin D was shown to have minimal effect on the growth of PC3 cells (Fig. 5). For the cytotoxicity of a replication- competent Ad-sPSA-El plus Vitamin D on DU145 cells, it was observed that Vitamin D at the highest concentration, 1 MOI, inhibited the growth of DU145 cells in vitro (Fig. 6). For the cytotoxicity of a replication-competent Ad-sPS A-El plus Vitamin D on C4-2 cells, it was observed that Vitamin D had profound growth inhibition affect on C4-2 cells in vitro at doses above 0.01 MOI (Fig. 7). For the cytotoxicity of a replication-competent Ad-hOC-El plus Vitamin D on PC-3 cells, it was observed that in comparison to Ad-sPSA-El, Vitamin D has greater stimulatory effect in enhancing Ad-OC-El cytotoxicity on PC3 cells in vitro, with obvious growth inhibition detected at greater than 1 MOI of Ad-hOC-El (Fig. 8). For the Cytotoxicity of a replication-competent Ad-hOC-El on DU-145 cells, it was observed that whereas Ad-sPSA-El failed to inhibit the growth of DU145 cells in the absence of Vitamin D (data not shown), Ad-hOC-El exhibited greater growth inhibition on DU145 cells in vitro than Ad-sPSA-El when added to DU145 cells in the presence of Vitamin D (Fig. 9). Finally, for the cytotoxicity of Ad-hOC-El plus Vitamin D on C4-2 cells, it was observed that Ad-hOC-El is highly efficient in inhibiting the growth of C4-2 cells in the presence of Vitamin D, even at 0.01 MOI (Fig. 10).

These results conclusively establish that for the majority of human prostate cancers which have cell populations which are PSA⁺ and androgen receptor positive as well as cell populations which are PSA^" and androgen receptor negative, the AdOC-Ela plus Vitamin

D was superior in terms of its cytotoxicity towards various cancer cells compared to that of Ad-sPSA-El plus Vitamin D when tested on the same cells.

The controls produced the following results: Ad-CMV-PA control virus without insert failed to inhibit the growth of RCC52 cells in vitro (Fig 11A). As expected, Ad-CMV- PA control viras without insert failed to inhibit the growth of PC-3 cells (Fig 1 IB). As expected, Ad-CMV-PA control virus without insert plus Vitamin D failed to inhibit the growth of DU145 cells (Fig 11C). As expected, Ad-CMV-PA control viras without insert failed to inhibit the growth of C4-2 cells (Fig 1 ID). As expected, wild-type Ad vector greater than 0.1 MOI inhibited the growth of PC-3 cells in vitro (Fig 1 IE). As expected, even the lowest dose of wild-type Ad vector inhibited the growth of RCC52 cells in vitro (Fig 1 IF). As expected, wild-type Ad vector is highly efficient in inhibiting the growth of C4-2 cells in vitro (Fig 11G). XVI EXAMPLE 4: ABILITY OF REPLICATION-COMPETENT AD-

MOC-E1 TO INDUCE CYTOTOXICITY OF C4-2 CELLS UPON CO- INOCULATION OF OSTEOGENIC Dl STROMAL CELL

MATERIALS AND METHODS

In order to determine the effect on the cytotoxicity of C4-2 cells (which are androgen receptor and PSA positive prostate cancer cells) when co-cultured with a mouse pluripotent osteogenic Dl stromal cell line transduced with a herpes simplex thymidine kinase (TK) gene in the presence of a prodrag ganciclovir (GCV), C4-2 cell-kill was measured in a luciferase-tagged C4-2 cell line (C4-2 luc). Relative luciferase activity correlated linearly with C4-2 cell numbers. C4-2 luc cells were co-cultured with either Dl or Dl-TK in tissue culture dish (total cell number = 4x10^ per well). hi order to determine the effect on the cytotoxicity of C4-2 cells when co inoculated with a mouse pluripotent osteogenic Dl stromal cell line in vivo, C4-2 cells were injected either alone or together with Dl-TK (2 x 10^ cells per site) formed solid tumors subcutaneously.

The effect of intravenous administration of Ad-mouse OC-Ela (Ad-mOC-Ela) on tumor regression in SCID/bg mice harboring C4-2 tumors intraosseously was also determined.

RESULTS AND DISCUSSION

When C4-2 cells were co-cultured with a mouse pluripotent osteogenic Dl stromal cell line transduced with a herpes simplex thymidine kinase (TK) gene in the presence of a prodrag ganciclovir (GCV), it was observed that Dl or Dl-TK cells stimulated the growth of C4-2 cells in vitro. Upon the addition of GCV, marked inhibition of C4-2 cell numbers (top panel) and total cellular protein (bottom panel) was observed (Fig X). These results indicate that the C4-2 prostate cancer cells require the stromal cells for their own survival As an osteogenic cell line, Dl-TK inoculated alone or with C4-2 cells, formed chimeric osteogenic prostate tumors that resemble human prostate cancer skeletal metastasis (see panel A and B, of Fig. X) which represent an X-ray image of subcutaneous tumors in athymic mice and a representative histomorphology of C4-2/D1-TK chirneric tumor, respectively). Upon intraperitoneal administration of ACV, a dramatic reduction in tumor size was noted (see panel A of Fig. X), accompanied by a sharp decrease in serum PSA (see panel C of Fig. X).

Moreover, when Ad-mouse OC-Ela was administered intravenously, both radiographic and gross morphologic evidence of tumor regression in SCID/bg mice harboring C4-2 tumors intraosseously were obtained. Fig. X (Panel A) shows the effect of the Ad-mouse OC-Ela adenoviral replication-competent vector on SCID/bg mice harboring intraosseous C4- 2 tumors. Fig. X (Panel B) shows the control SCID/bg mice harboring intraosseous C4-2 tumors in the absence of the Ad-mouse OC-Ela adenoviral replication-ceomptent vector. Thus, the replication-competent Ad-mouse OC-Ela adenoviral vector was remarkably effective in its ability to kill both bone stromal cells (for example, but not limited to, MG-63 cells) and human prostate C4-2 cancer cells in mice harboring C4-2 tumors intraosseously.

The experiments described above therefore demonstrates that the growth of prostate cancer cell lines, either PSA-secreting (including, but not limited to, LNCaP, C4-2, ARCaP) or non-secreting (including, but not limited to, PC-3, DU145), and bone (MG-63) and prostate (9096F) stromal cell lines are markedly inhibited by Ad-OC-Ela through viral lytic activity.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A replication-competent adenovirus vector comprising an adenoviras gene under transcriptional control of an osteocalcin transcriptional regulatory sequence.

2. The adenovirus vector of claim 1, wherein the adenoviras gene is essential for viral replication.

3. The adenoviras vector of claim 1 , wherein the osteocalcin transcriptional regulatory sequence comprises an enhancer from an osteocalcin gene.

4. The adenovirus vector of claim 1, wherein the osteocalcin transcriptional regulatory sequence comprises a promoter from an osteocalcin gene.

5. The adenoviras vector of claim 1, wherein the osteocalcin transcriptional regulatory sequence comprises a promoter from an osteocalcin gene and an enhancer from an osteocalcin gene.

6. The adenovirus vector of claim 1, wherein the osteocalcin transcriptional regulatory sequence comprises the sequence of SEQ ID NO: 1.

7. A composition comprising an adenoviras vector of claim 1 and a pharmaceutically acceptable excipient.

8. The adenoviras vector of claim 1, further comprising at least one additional adenoviras gene under transcriptional control of at least one additional prostate-specific transcriptional regulatory sequence.

9. An adenoviras vector of claim 1, further comprising a heterologous gene under transcriptional control of a osteocalcin franscriptional regulatory sequence.

10. A host cell transformed with an adenovirus vector of claim 1.

11. A method of detecting cells which allow an osteocalcin transcriptional regulatory sequence to function in a biological sample comprising the steps of: contacting a biological sample with an adenovirus vector of claim 1, under conditions suitable for osteocalcin franscriptional regulatory sequence-mediated gene expression in cells which allow a osteocalcin transcriptional regulatory sequence to function; and determining if osteocalcin franscriptional regulatory sequence mediates gene expression in the biological sample, wherein osteocalcm transcriptional regulatory sequence-mediated gene expression is indicative of the presence of cells which allow an osteocalcin transcriptional regulatory sequence to function.

12. A method of propagating an adenoviras specific for cells which allow a osteocalcin transcriptional regulatory sequence to function, said method comprising: combining an adenoviras vector according to claim 1 with cells which allow an osteocalcin transcriptional regulatory sequence to function, whereby said adenoviras is propagated.

13. A method for modifying the genotype of a target cell, said method comprising contacting a cell which allows an osteocalcin transcriptional regulatory sequence to function with an adenoviras of claim 1, wherein the adenoviras enters the cell

14. A method of suppressing tumor cell growth, said method comprising contacting tumor cells and non-tmnor cells with an adenoviras vector of claim 1 such that the adenovirus vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell.

15. A kit comprising an adenovirus vector of claim 1.

16. A composition comprising an adenovirus vector of claim 1 and a buffer.

17. The adenovirus vector of claim 2, wherein the adenovirus gene is an early gene.

18. The adenovirus vector of claim 2, wherein the adenovirus gene is a late gene.

19. The adenoviras vector of claim 17, wherein the adenovirus early gene is EIA.

20. The adenoviras vector of claim 17, wherein the adenoviras early gene is EIB.

21. The adenoviras vector of claim 8, wherein the at least one additional prostate-specific transcriptional regulatory sequence comprises an osteocalcin transcriptional regulatory sequence.

22. The adenoviras vector of claim 21, wherein the at least one additional prostate-specific transcriptional regulatory sequence comprises an osteocalcin promoter ssequence.set forth in SEQ ID NO:l.

23. The adenoviras vector of claim 8, wherein the at least one additional prostate-specific transcriptional regulatory sequence comprises a prostate-specific antigen (PSA) transcriptional regulatory sequence.

26. A composition comprising an adenovirus vector of claim 8 and a pharmaceutically acceptable excipient.

27. A method of propagating an adenoviras specific for mammalian cells which allow an osteocalcin transcriptional regulatory sequence to function, said method comprising: combining an adenoviras vector according to claim 8 with mammalian cells which allow an osteocalcin transcriptional regulatory sequence to function, whereby said adenovirus is propagated.

28. A method for modifying the genotype of a target cell, said method comprising contacting a cell which allows an osteocalcin transcriptional regulatory sequence to function with an adenoviras of claim 8, wherein the adenoviras enters the cell.

29. A method of suppressing tumor cell growth, said method comprising contacting a tumor cell with an adenoviras vector of claim 8 such that the adenovirus vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell.

30. A kit comprising an adenovirus vector of claim 8.

31. A composition comprising an adenovirus vector of claim 8 and a buffer.

32. The adenoviras vector of claim 8 wherein said additional adenoviras gene is essential for viral replication.

33. A host cell transformed with an adenoviras vector of claim 8.

34. The adenoviras vector of claim 9, wherein the heterologous gene is a reporter gene.

35. The adenoviras vector of claim 9, wherein the heterologous gene is conditionally required for cell survival.

36. The method of claim 11, in which the cells allow an osteocalcin transcriptional regulatory sequence to function and wherein said cells fail to express PSA or androgen receptor..

37. The adenoviras vector of claim 35 wherein said additional adenoviras gene is an early gene.

38. The adenoviras vector of claim 35 wherein said additional adenoviras gene is a late gene.

39. The adenovirus vector of claim 37 wherein said early gene is EIA.

40. The adenoviras vector of claim 37 wherein said early gene is EIB.

41. A method for conferring selective toxicity on a target cell, said method comprising contacting a cell which allows an osteocalcin transcriptional regulatory sequence to function with an adenoviras vector comprising an adenoviras gene under franscriptional control of an osteocalcin transcriptional regulatory sequence, whereby expression of the gene under transcriptional control of an osteocalcin franscriptional regulatory sequence contributes to cytotoxicity.

42. The method of claim 41, wherein the adenoviras gene is an early gene.

43. The method of claim 41 , wherein the adenoviras gene is essential for replication.

44. The method of claim 41, wherein the early gene is EIA.

45. The method of claim 42, wherein the early gene is EIB.

46. The adenovirus vector of claim 1, wherein the adenovirus gene is an early gene.

47. The adenovirus vector of claim 14, wherein the additional adenovirus gene is an early gene.

48. The method of claim 41 , wherein the adenovirus vector further comprises at least one additional adenovirus gene under transcriptional control of at least one additional prostate-specific transcriptional regulatory sequence.

49. The method of claim 14, wherein said adenoviras gene is essential for viral replication.

50. The method of claim 14, wherein said adenoviras gene is an early gene.

51. The method of claim 14, wherein said adenoviras gene is a late gene.

52. A method of treating cancer in an individual, said method comprising the step of administering the conditional replication-competent adenoviras Ad-OC-Ela to said individual.

53. The method of claim 52 wherein the cancer is in the form of a solid tumor.

54. The method of claim 52 wherein Ad-OC-Ela is administered by injection into tumor.

55. The method of claim 52 wherein the administering is by an intravenous route or a direct injection of the conditional replication-competent adenoviras Ad-OC-Ela.

56. A method of treating cancer in an individual, said method comprising the step of administering a conditional tissue-specific and tumor-restrictive replication-competent adenoviras Ad-OC-Ela to said individual, wherein said conditional tissue-specific and tumor- restrictive replication-competent Ad-OC-Ela vector is specific for cells which allow an osteocalcin transcriptional regulatory sequence to function, wherein said cells are prostate cancer cells, prostate sfromal cells, vascular pericytes, proliferating cancer-associated osteoblasts, and prostate cancer and associated bone stromal cells which fail to express PSA or androgen receptor (AR).

57. The method of claim 56 wherein Ad-OC-Ela is administered by injection into a tumor.

58. The method of claim 56 wherein the administering is by an intravenous route or a direct injection of the conditional replication-competent adenovirus Ad-OC-Ela.

59. A method of treating diseases involving calcification in an individual, said method comprising the step of administering a conditional tissue-specific and tumor-restrictive replication-competent adenoviras Ad-OC-Ela to said individual, wherein said conditional tissue-specific and tumor-restrictive replication-competent Ad-OC-Ela vector is specific for cells which allow an osteocalcin transcriptional regulatory sequence to function, and wherein said diseases comprise benign prostate hyperplasia (BPH) or arteriosclerosis.