EP1272615A2 - Replication d'adenovirus sous controle de promoteur d'osteocalcine, utilisee a des fins therapeutiques - Google Patents

Replication d'adenovirus sous controle de promoteur d'osteocalcine, utilisee a des fins therapeutiques

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Publication number
EP1272615A2
EP1272615A2 EP01926403A EP01926403A EP1272615A2 EP 1272615 A2 EP1272615 A2 EP 1272615A2 EP 01926403 A EP01926403 A EP 01926403A EP 01926403 A EP01926403 A EP 01926403A EP 1272615 A2 EP1272615 A2 EP 1272615A2
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European Patent Office
Prior art keywords
gene
osteocalcin
cells
adenoviras
regulatory sequence
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EP01926403A
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German (de)
English (en)
Inventor
Leland W. K. Chung
Chinghai Kao
Thomas A. Gardner
Song-Chu Ko
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University of Virginia UVA
University of Virginia Patent Foundation
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University of Virginia UVA
University of Virginia Patent Foundation
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Publication of EP1272615A2 publication Critical patent/EP1272615A2/fr
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor

Definitions

  • 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.
  • 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.
  • 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.
  • BPH benign prostate hyperplasia
  • arteriosclerosis 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.
  • 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.
  • 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.,
  • 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, ⁇ . 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, ⁇ . 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).
  • Prostate cancer gene therapy herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models.
  • 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.
  • 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.
  • virus replication needs to be controlled so that normal tissue will not be damaged.
  • 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.
  • 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).
  • 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 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).
  • 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.
  • 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 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.
  • 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.
  • Msx-2/Hox 8.1 a transcriptional regulator of the rat osteocalcin promoter.
  • 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.,
  • CCTCCT motif 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.
  • HSV-TK herpes simplex virus thymidine kinase
  • 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.
  • Ad-OC-TK adenoviral toxic gene
  • Ad-CMN-TK an universal promoter, cytomegalovirus (CMN)
  • GCN gancyclovir
  • 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.
  • Ad-OC-Ela another conditional replication-competent adenovirus
  • 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.
  • 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.
  • metastatic cancers including, without limitation, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, tumors, osteosarcoma, ocular melanoma, lung cancer, breast cancer.
  • 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.
  • BPH benign prostate hyperplasia
  • arteriosclerosis arterios
  • 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.
  • BPH benign prostate hyperplasia
  • 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.
  • 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.
  • 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.
  • BPH benign prostate hyperplasia
  • 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.
  • 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.
  • an osteocalcin transcriptional regulatory sequence comprises the 1,370-bp nucleotide sequence of SEQ ID NO:l as shown in Figure 21.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • a transcriptional regulatory sequence or transcriptional regulatory sequence capable of effecting transcription in a cell, which allows an osteocalcin transcriptional regulatory sequence
  • 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.
  • 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.
  • the adenovirus gene essential for replication is a late gene, hi various embodiments, the additional late gene is LI, L2, L3, L4, or L5.
  • 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.
  • a composition comprises this adenovirus. In one embodiment, this composition further comprises a pharmaceutically acceptable excipient.
  • the at least one additional osteocalcin-specific transcriptional regulatory sequence is a second osteocalcin transcriptional regulatory sequence.
  • the at least one additional osteocalcin transcriptional regulatory sequence can have a sequence different from that of the first osteocalcin transcriptional regulatory sequence.
  • the at least one additional osteocalcin-specific transcriptional regulatory sequence comprises an osteocalcin transcriptional regulatory sequence.
  • 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.
  • the heterologous gene is a reporter gene such as for example, and without limitation, the luciferase reporter gene or beta-galactosidase reporter gene.
  • the heterologous gene is conditionally required for cell survival.
  • the transgene is a cytotoxic gene.
  • a method of treating metastatic cancer in an individual 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).
  • AR androgen receptor
  • 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.
  • 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.
  • the second transcriptional regulatory sequence comprises an osteocalcin transcriptional regulatory sequence, hi one embodiment, the additional adenovirus gene is essential for viral replication.
  • the additional adenovirus gene is an early gene.
  • the additional adenovirus gene is El A.
  • the additional adenovirus early gene is El B.
  • the additional adenovirus gene is a late gene. In various embodiments, the late gene can be LI, L2, L3, L4, or L5.
  • the invention provides a host cell transformed with any adenovirus vector(s) described herein.
  • 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.
  • the invention provides kits which contain an adenoviral vector(s) described herein.
  • a method 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.
  • an osteocalcin transcriptional regulatory sequence 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)
  • a method for modifying the genotype of a target cell 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.
  • 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.
  • 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
  • methods 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.
  • a method 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.
  • the gene is a heterologous (non-adeno virus gene). In one embodiment, the heterologous gene
  • a method for conferring selective toxicity or cytotoxicity on a target cell 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.
  • 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.
  • a target cell which allows an osteocalcin transcriptional regulatory sequence to function, for example, in prostate cancer cells, prostate stromal cells
  • an adenovirus which comprises a heterologous gene under transcriptional control of an osteocalcin transcriptional regulatory sequence
  • the heterologous gene is a reporter gene.
  • the heterologous gene is conditionally required for cell survival
  • 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
  • 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
  • osteocalcin transcriptional regulatory sequence 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.
  • an osteocalcin transcriptional regulatory sequence can be of varying lengths, and of varying sequence composition.
  • osteocalcin transcriptional regulatory sequence 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.
  • PSA or androgen receptor AR
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1 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.
  • FIG. 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
  • FIG. 4 Differential inhibition of PC-3 and Lovo tumor growth in vivo upon intratumoral injection of Ad-OC-Ela.
  • Ad-OC-Ela Four weeks after subcutaneous injection of either 1 x l ⁇ 6 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.
  • 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.
  • FIG. 5 Demonstration of intravenous Ad-OC-Ela on serum PSA levels in SCID/bg mice injected intraosseously with C4-2 cells,
  • Arrowheads indicate the time (in week) intravenous administration of Ad-OC-Ela.
  • 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).
  • FIG. 7 RT-PCR of Vitamin D receptor (VDR) in normal and neoplastic human cancer cell lines. Left track of the Figure represents marker RNA.
  • FIG. 1 Western blot of Vitamin D receptor (VDR) in human prostate cancer (C4-2, PC3), human renal carcinoma (RCC52), human osteosarcoma (MG-63) cell lines.
  • VDR Vitamin D receptor
  • FIG. 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.
  • FIG 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.
  • FIG. 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).
  • TK herpes simplex thymidine kinase
  • Figure 20 Radiographic and gross morphologic evidence of tumor regression in SCID/bg mice harboring C4-2 tumors intraosseously.
  • FIG. 21 Nucleotide sequence of the 1,370-bp mouse osteocalcin promoter (SEQ JJD
  • 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.
  • 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.
  • an enhancer or enhancer-like sequence 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” is a term well understood in the art and generally comprises a polynucleotide (defined herein) comprising all or a portion of an adenovirus genome.
  • 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.
  • the polynucleotide is DNA.
  • 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.
  • adenovirus vectors are replication-competent in a target cell such as a tumor cell.
  • polynucleotide or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleo tides.
  • 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.
  • the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be a oligodeoxy- nucleoside phosphoramidate (P— NH 2 ) or a mixed phosphoramidate-phosphodiester oligomer.
  • P— NH 2 oligodeoxy- nucleoside phosphoramidate
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • operably linked refers to a juxtaposition wherein the elements transcriptional regulatory sequences are in an arrangement allowing them to function.
  • 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, 3 H-thymidine uptake, and plaque assays.
  • selective cytotoxicity 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • a heterologous promoter are the ⁇ -fetoprotein, PSA, DF3, tyrosinase, CEA, surfactant protein, and ErbB2 promoters.
  • a heterologous enhancer are the ⁇ -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.
  • 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.
  • 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).
  • 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).
  • a target cell is a mammalian cell which allows an osteocalcin transcriptional regulatory
  • Neoplastic 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.
  • biological sample encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • 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.
  • 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.
  • treatment is an approach for obtaining beneficial or desired clinical results.
  • 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).
  • 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.
  • 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 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.
  • one transcriptional regulatory sequence can be a basal level constitutive transcriptional regulatory sequence.
  • a tissue-specific enhancer or promoter can be combined with a basal level constitutive promoter.
  • a tissue-specific enhancer or promoter can be combined with an inducible promoter.
  • 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.
  • 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.
  • BPH benign prostate hyperplasia
  • osteocalcin (OC) protein was found to be uniformly and highly expressed in human prostate cancer skeletal metastasis.
  • 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.
  • 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.
  • the prefened vectors of the present invention are adenoviral vectors.
  • the adenovirus vector is a human adenovirus.
  • Ad2, Ad5, and Ad40 which may differ to minor or significant degrees.
  • Ad5 and Ad40 differ as to their host cell tropism, as well as the nature of the disease induced by the virus.
  • the adenovirus vector for use in the compositions and methods of the invention is canine adenovirus type 1 or canine adenovirus type 2.
  • canine adenovirases examples 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).
  • the adenovirus vector for use in the compositions and methods of the invention is a bovine adenovirus.
  • a bovine adenovirus is that described in International Patent Application Number WO 95/16048 (the entire contents of which are incorporated herein by reference).
  • the adenovirus vector for use in the compositions and methods of the invention is ovine adenovirus.
  • 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).
  • Ad5 will be exemplified. What follows is a brief description of adenovirus-based vectors in general and replication- competent adenovirus vectors in particular.
  • 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.
  • 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
  • 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,
  • 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,
  • 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.
  • the heterologous gene of interest is incorporated into viral particles by packaging of the recombinant adenovirus genome.
  • 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.
  • 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.
  • 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
  • the E2 late promoter overlaps with the coding sequences of a gene encoded by the counterstrand and is therefore not amenable for genetic manipulation.
  • the E2 early promoter overlaps only for a few base pairs with sequences coding for a 33 kDa protein on the counterstrand.
  • 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 require interaction with the E1B 55-kDa protein for activity while the ORF 3 protein does not.
  • plaques are produced with an efficiency less than 10 "6 that of wild type virus.
  • E4 ORFs 1-3 can be deleted, making viral DNA replication and late gene synthesis dependent on E4 ORF 6 protein.
  • a virus 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.
  • MLP major late promoter
  • 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.
  • 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.
  • 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).
  • transgene heterologous gene
  • cytotoxic activity 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.
  • heterologous genes or transgenes may also confer a bystander effect.
  • 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.
  • cytotoxic proteins such as the A chains of diphtheria toxin, ricin or abrin
  • genes encoding a factor capable of initiating apoptosis 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.
  • a nuclease e.g. RNase A
  • protease e.g. awsin, papain, proteinase K, carboxypeptidase, etc.
  • 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.
  • 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).
  • 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.
  • adenoviral vectors may be 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).
  • adenoviras vectors may be administered in an appropriate physiologically acceptable carrier at a dose of about 10 4 PFU to about 10 14 PFU.
  • the multiplicity of infection will generally be in the range of about 0.001 PFU to 100 PFU.
  • 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.
  • 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.
  • 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.
  • compositions also include lyophilized and/or reconstituted forms of the adenoviral vectors (including those packaged as a viras, such as adenoviras) of the invention.
  • kits containing an adenoviral vector of this invention 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.
  • 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.
  • the present invention includes methods using the adenoviral vectors described above.
  • 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.
  • 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.
  • cells of a biological sample are contacted with an adenoviras vector, and replication of the adenoviral vector is detected.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 3 H-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 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.
  • immunological assays such as enzyme-linked immunosorbent assay (ELISA)
  • ELISA enzyme-linked immunosorbent assay
  • 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.
  • 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.
  • adenoviral vector(s) of the invention will depend, inter alia, on assessable clinical parameters such as serological indications and histological examination of tissue biopsies.
  • a pharmaceutical composition comprising an adenoviral vector(s) in a pharmaceutically acceptable excipient is administered.
  • Pharmaceutical compositions are described above.
  • adenoviral vector(s) 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).
  • adenovirus is administered as a packaged adenovirus, from about 10 4 PFU to about 10 14 PFU, preferably from about 10 4 PFU to about 10 12 PFU, more preferably from about 10 4 PFU to about 10 10 PFU.
  • a polynucleotide construct i.e., not packaged as a virus
  • about 0.01 ⁇ g to about 100 ⁇ g can be administered, preferably 0.1 ⁇ g to about 500 ⁇ g, more preferably about 0.5 ⁇ g to about 200 ⁇ g.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIAU l-2-deoxy-2- fluoro-.beta.-D-arabinofuranosil-5-iodouracil
  • cytosine deaminase 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.
  • 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.
  • 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.
  • 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.
  • the agent which provides for the inhibition, prevention, or destruction of the growth of the tumor cells is a cytokine.
  • 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-
  • interleukin- l ⁇ 1, interleukin- l ⁇ , and interleukins-2-15.
  • the interleukin is interleukin-2.
  • 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.
  • 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.
  • 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.
  • a tissue specific promoter such as the osteocalcin promoter sequence depicted in SEQ ID NO:l
  • the adenoviral vector additionally contains another tissue-specific promoter operatively associated with a nucleic acid encoding a therapeutic molecule
  • 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.
  • 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.
  • 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.
  • 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.
  • BPH benign prostate hyperplasia
  • 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.
  • LacZ is the reporter gene.
  • 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.
  • BPH benign prostate hyperplasia
  • 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.
  • BPH benign prostate hyperplasia
  • 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.
  • the present invention encompasses polynucleotide sequences comprising
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the molecules are identical at that position.
  • 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 protein searches can be performed with the
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389-3402.
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • 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.
  • ALIGN program version 2.0
  • a gap length penalty of 12 and a gap penalty of 4 can be used.
  • 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:
  • 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
  • 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.
  • tissue specific promoter such as osteocalcin promoter element directs the expression of the heterologous gene in the host cell.
  • various transcriptionally active fragments of this regulatory region are also encompassed within the scope of the invention.
  • 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.
  • 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.
  • 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
  • the regulatory region is placed immediately 5' to, and is operatively associated with the coding sequence.
  • 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.
  • 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.
  • tissue-specific transcriptional regulatory sequence may include, for example, without limitation, the ⁇ -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 ⁇ - fetoprotein promoter sequence described in U.S. Patent No.
  • 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.
  • 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.
  • modifications of osteocalcin promoter sequence depicted in SEQ JD NO:l without substantially affecting its transcriptional activities are also encompassed within the scope of the invention.
  • 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 ⁇ g/m£ denatured salmon sperm DNA.
  • Filters are hybridized for 48 hours at 65 °C in prehybridization mixture containing 100 ⁇ g/ml! denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P -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.
  • exemplary conditions of high stringency are as follows: e.g., hybridization to filter-bound DNA in 0.5 M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
  • Tm(°C) 81.5+16.6(log [monovalent cations (molar)]) +0.41 (% G+C)-(500/N) where N is the length of the probe.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, Exoi ⁇ , 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).
  • 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.
  • 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).
  • 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).
  • 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.
  • the detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein.
  • 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.
  • 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.
  • GFP green fluorescent protein
  • enzymes e.g. CAT, beta-galactosidase, luciferase
  • antigenic markers e.g. CAT, beta-galactosidase, luciferase
  • 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).
  • reporter gene 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
  • Reporter genes that can be analyzed using colorimetric analysis include, but are not limited to, ⁇ -galactosidase (Nolan et al 1988, Proc. Natl Acad. Sci. USA 85:2603- 07), ⁇ -glucuronidase (Roberts et al. 1989, Curr. Genet. 15:177-180), luciferase (Miyamoto et al, 1987, J. Bacteriol. 169:247-253), or ⁇ -lactamase.
  • the reporter gene sequence comprises a nucleotide sequence which encodes a LacZ gene product, ⁇ - 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- ⁇ -D-galactoside (X-gal), lactose 2,3,5-triphenyl-2H-tetrazolium (lactose-tetrazolium) and fluorescein galactopyranoside (see Nolan et al, 1988, supra).
  • X-gal 5- bromo-4-chloro-3-indoyl- ⁇ -D-galactoside
  • lactose 2,3,5-triphenyl-2H-tetrazolium lactose-tetrazolium
  • fluorescein galactopyranoside see Nolan et al, 1988, supra.
  • the product of the E. coli ⁇ -glucuronidase gene 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.
  • 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
  • the coding sequence for chloramphenicol acetyl transferase (CAT) can be utilized, leading to osteocalcin transcriptional regulatory sequence-dependent expression of chloramphenicol resistant cell growth.
  • CAT chloramphenicol acetyl transferase
  • 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).
  • 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
  • the expression of the reporter gene (for example, green fluorescent protein, luciferase, ⁇ -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.
  • 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, p ⁇ gal-Basic, p ⁇ 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 ⁇ -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.
  • selection systems including but not limited to, the herpes simplex viras thymidine kinase
  • 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).
  • ODC ornithine decarboxylase
  • DFMO McConlogue L., 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.
  • glutamine synthetase Bebbington et al, 1992, Biotech 10:169
  • a fusion construct comprising an osteocalcin transcriptional regulatory sequence, or a fragment thereof, can be assayed for transcriptional activity.
  • 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.
  • a -3 kb to +3 kb region 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.
  • 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.
  • GFP green fluorescent protein
  • 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.
  • a Lac Z reporter construct is used.
  • the Lac Z gene product, ⁇ -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- ⁇ -D-galactoside (X-gal), lactose 2,3,5- triphenyl-2H-tetrazolium (lactose-tetrazolium) and fluorescein galactopyranoside (see Nolan et al, 1988, supra).
  • X-gal 5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside
  • lactose 2,3,5- triphenyl-2H-tetrazolium lactose-tetrazolium
  • fluorescein galactopyranoside see Nolan et al, 1988, supra.
  • GFP that has been optimized for expression in mammalian cells
  • 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).
  • EGFP enhanced GFP
  • filter sets such as fluorescein isothiocyanate (FITC) optics which illuminate at 450-500 nm can be used to visualize GFP fluorescence.
  • FITC fluorescein isothiocyanate
  • pEGFPl proved to be useful as a reporter vector for promoter analysis in transgenic mice (Okabe et al, 1997, FEBS Lett. 407: 313).
  • 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 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.
  • 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 ⁇ .,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 orJ ⁇ c Z cDNA.
  • 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.
  • transgenic 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.
  • 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).
  • gene targeting is preferred.
  • 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.
  • 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, rum, 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.
  • 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.
  • 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;
  • 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 .
  • families of antidepressants such as lithium salts, carbamazepine, valproic acid, lysergic acid diethylamide (LSD), p- chlorophenylalanine,/?-propyldopacetamide dithiocarbamate derivatives e
  • 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, ⁇ -methyl-tyrosine, reserpine, tetrabenazine, benzotropine, pargyline; noradrenergic agonists and antagonists e.g., clonidine, phenoxybenzamine, phentolamine, tro
  • 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.
  • a compound 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)
  • AR androgen receptor
  • XJN EXAMPLE 1 SYSTEMIC OSTEOCALCIN (OC) PROMOTER- DRIVEN REPLICATION-COMPETENT ADENOVIRUS FOR THE
  • Osteocalcin a noncollagenous bone matrix protein
  • Ad a conditional OC replication-competent adenoviral vector under the control of OC promoter for the treatment of hormone-refractory prostate cancer bone metastasis.
  • Ad-OC-Ela A recombinant Ad vector, Ad-OC-Ela, was constructed which contained an OC promoter driven Ela gene.
  • Ad-OC-Ela 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.
  • 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 l ⁇ 9 PFU) inhibited tumor growth.
  • 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.
  • LNCaP an androgen-responsive, androgen receptor-positive, and PSA- secreting human prostate cancer cell line
  • LNCaP 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.
  • 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.
  • WH a cell line derived from a human bladder transitional cell carcinoma specimen
  • Zhau et al. Zhau, H. E., Hong, S. J. and Chung, L. W. K.
  • rUGM fetal rat uro genital sinus mesenchymal cell line
  • 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.
  • 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.
  • 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.
  • 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,
  • Ad-OC-Ela a partially E3-deleted replication-competent adenoviras
  • 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,
  • 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
  • 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
  • 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 ⁇ l 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).
  • 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.
  • 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.
  • Ad-OC-Ela lytic activity 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 ⁇ l 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.
  • 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.
  • Serum PSA Blood specimens (approximately 100 ⁇ l) 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 ⁇ l Ad-OC-Ela, 2 x 10 ⁇ pfu (or 4 x lO 1 ⁇ 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.
  • MMIA microparticle enzyme-linked immunosorbent assay
  • 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.
  • 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.
  • Ad-OC-Ela-induced cell lysis in vitro irrespective of their intrinsic levels of PSA and AR expression
  • the effects of Ad-OC-Ela were also evaluated on the growth of human prostate and bone fibroblast cell lines in vitro.
  • Ad-OC- Ela infection induced significant cell lysis in both cultured human prostate (e.g. 9096F) and bone (MG-63) fibroblasts.
  • 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.
  • 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.
  • 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.
  • Figure 6a demonstrated the gross anatomical difference between a control and a responder mouse to Ad-OC-Ela therapy.
  • 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).
  • 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.
  • Prostate cancer gene therapy herpes simplex viras thymidine kinase gene fransduction followed by ganciclovir in mouse and human prostate cancer models.
  • 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.
  • 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.
  • 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.
  • tissue-specific i.e. osteoblast-specific
  • tumor- restrictive i.e.
  • 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., Brownan, G. and Chung, L. W. K.
  • Ad-OC-Ela replication may be limited to proliferating and maturing osteoblasts in men which express OC promoter activity.
  • 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,
  • 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.
  • PSA enhancer Rosham, R., Schuur, E. R., Lim, H. Y., Henderson, G. A., Simons, J. W. and Henderson, D. R.
  • 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.
  • PSMA prostate-specific membrane antigen
  • 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,
  • 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.
  • Ad-OC-TK 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).
  • 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.
  • 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).
  • Ad-OC-Ela systemic Ad-OC-Ela
  • 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.
  • 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.,
  • 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.
  • Vitamin D was assessed for the effect of Vitamin D on the expression of Vitamin D receptor.
  • C4-2 and PC3 normal prostate cancer
  • RC52 renal carcinoma
  • a 25 cycle RT-PCR targeted at VDR 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.
  • VDR Vitamin D receptor
  • C4-2, PC3 human prostate cancer
  • RRC52 human renal carcinoma
  • MG-63 human osteosarcoma
  • Ad-hOC-El 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.
  • 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.
  • 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).
  • Vitamin D treatment enhanced hOC mRNA expression in PC3, RC52, MG-63 and WH, but not C4-2, cell lines.
  • Vitamin D enhanced RCC52 cell kill at 5 MOI of Ad-OC-El (Fig. 4).
  • Vitamin D was shown to have minimal effect on the growth of PC3 cells (Fig. 5).
  • Vitamin D was observed that Vitamin D at the highest concentration, 1 MOI, inhibited the growth of DU145 cells in vitro (Fig. 6).
  • Vitamin D had profound growth inhibition affect on C4-2 cells in vitro at doses above 0.01 MOI (Fig. 4).
  • Ad-CMV-PA control virus without insert failed to inhibit the growth of RCC52 cells in vitro (Fig 11A).
  • Ad-CMV- PA control viras without insert failed to inhibit the growth of PC-3 cells (Fig 1 IB).
  • Ad-CMV-PA control virus without insert plus Vitamin D failed to inhibit the growth of DU145 cells (Fig 11C).
  • Ad-CMV-PA control viras without insert failed to inhibit the growth of C4-2 cells (Fig 1 ID).
  • wild-type Ad vector greater than 0.1 MOI inhibited the growth of PC-3 cells in vitro (Fig 1 IE).
  • even the lowest dose of wild-type Ad vector inhibited the growth of RCC52 cells in vitro (Fig 1 IF).
  • wild-type Ad vector is highly efficient in inhibiting the growth of C4-2 cells in vitro (Fig 11G).
  • C4-2 cells which are androgen receptor and PSA positive prostate cancer cells
  • 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 cells were injected either alone or together with Dl-TK (2 x 10 ⁇ cells per site) formed solid tumors subcutaneously.
  • Ad-mouse OC-Ela Ad-mouse OC-Ela
  • Fig. X shows the effect of the Ad-mouse OC-Ela adenoviral replication-competent vector on SCID/bg mice harboring intraosseous C4- 2 tumors.
  • Fig. X shows the control SCID/bg mice harboring intraosseous C4-2 tumors in the absence of the Ad-mouse OC-Ela adenoviral replication-ceomptent vector.
  • 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.
  • 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.

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Abstract

La présente invention concerne des vecteurs d'adénovirus aptes à la réplication, qui sont spécifiques de cellules qui permettent à une séquence de régulation transcriptionnelle d'ostéocalcine de s'exprimer, telles que des cellules de prostate cancéreuses, des cellules de prostate stromatolithiques, des péricytes vasculaires, des ostéoblastes liées au cancer à prolifération, et des cellules de prostates cancéreuses et cellules stromatolithiques osseuses associées, qui ne parviennent pas à exprimer l'antigène prostatique spécifique (PSA) ou un récepteur androgène (AR). Cette invention concerne également des procédés d'utilisation de virus de ce type. Ces virus comprennent un gène adénoviral sous contrôle d'une séquence de régulation transcriptionnelle d'ostéocalcine. Le gène peut être, par exemple, un gène nécessaire à la réplication adénovirale. Les virus peuvent également comprendre au moins un gène adénoviral supplémentaire sous contrôle d'au moins une séquence de régulation transcriptionnelle spécifique du tissu supplémentaire. La réplication virale peut ainsi être limitée à des cellules cibles exprimant le gène d'ostéocalcine. Des vecteurs d'adénovirus aptes à la réplication de ce type peuvent être utilisés dans le cadre du traitement du cancer de la prostate, du cancer du cerveau, du cancer de l'ovaire, du cancer de la thyroïde, des tumeurs, de l'ostéosarcome, du mélanome oculaire, du cancer du poumon, ou du cancer du sein.
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