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Show moreBackground of the invention 1. Field of the Invention This invention is directed to papovavirus-derived episomes that replicate efficiently in mammalian cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. Papovavirus-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells. 2. Review of Related Articles: One approach to gene therapy of human cancer cells is to introduce vectors expressing antisense sequences to block expression of dominant oncogenes and growth factor receptors. However, high-level expression of the oncogenes requires comparable levels of antisense expression, which presents a considerable technical obstacle, particularly when using expression vectors having a limited potential for achieving multiple copies in stable transfectants. Human cells transduced by retroviral vectors have only one or several copies of integrated retrovirus in stable transfectants. In contrast, hundreds of copies of episomal plasmids can accumulate in stable transfectants because these vectors replicate extrachromosomally. One method to express high levels of antisense transcripts is to utilize episomal plasmid vectors than can replicate extrachromosomally in human cells. Attempts to produce episomal vectors that will replicate in some types of human cells are reported by the literature. Episomal plasmids have been developed from several DNA viruses, including bovine papilloma virus (BPV) (Sarver, et al., 1981, Mol. Cell. Biol, 1:486-496; DiMaio, et al., 1982, Proc. Natl. Acad. Sci., U.S.A., 97:4030-4034), SV40 (Tsui, et al., 1982, Cell, 30:49914508), Epstein-Barr virus (EBV) (Yates, et al., 1985, Nature, 313:812-815; Margolskee, et al., 1988, Mol. Cell. Biol, 8:2837-2847; Belt, et al., 1989, Gene, 84:407-417; Chittenden, et al., 1989, J. Virol., 63:3016-3025), and BK virus (BKV) (Milanesi, et al., 1984, Mol. Cell. Biol., 4:1551-1560). Each of these episomal plasmids contains a viral origin of DNA replication and a virally encoded early gene that trans-activates the viral origin and allows the episome to replicate in the transfected host cell. Although EBV-based episomes have been used to efficiently screen cDNA libraries, the EBV system has limited applications to non-lymphoid cell types (Vidal, et al., 1990, Biochim. Biophys. Acta 1048:171-177)), and the EBV replicon is not active in many cell types. Additionally, EBNA-1 is one of several EBV latent genes that immortalize human lymphocytes, and transfection of the EBV-negative BJAB lymphoma cell line by EBNA-1 induces soft agar growth, indicating transformation of the cells. (Konoshita, 1990, Hokkaido Igaku Zasshi, 65:362-375) Furthermore, stable transfection efficiencies for EBNA-1 negative cell lines transduced by EBV episomal plasmids encoding EBNA-1 (transactivator) and ORI-P (EBV DNA origin) are low, not significantly better than non-episomal plasmids (Yates, et al., 1985; Vidal, et al., 1990.
http://www.google.com/patents?vid=USPAT6339065
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Show moreBackground of the invention 1. Field of the Invention This invention is directed to papovavirus-derived episomes that replicate efficiently in mammalian cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. Papovavirus-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells. 2. Review of Related ArtOne approach to gene therapy of human cancer cells is to introduce vectors expressing antisense sequences to block expression of dominant oncogenes and growth factor receptors. However, high-level expression of the oncogenes requires comparable levels of antisense expression, which presents a considerable technical obstacle, particularly when using expression vectors having a limited potential for achieving multiple copies in stable transfectants. Human cells transduced by retroviral vectors have only one or several copies of integrated retrovirus in stable transfectants. In contrast, hundreds of copies of episomal plasmids can accumulate in stable transfectants because these vectors replicate extrachromosomally. One method to express high levels of antisense transcripts is to utilize episomal plasmid vectors than can replicate extrachromosomally in human cells. Attempts to produce episomal vectors that will replicate in some types of human cells are reported by the literature. Episomal plasmids have been developed from several DNA viruses, including bovine papilloma virus (BPV) (Sarver, et al., 1981, Mol. Cell. Biol., 1: 486-496; DiMaio, et al., 1982, Proc. Natl. Acad. Sci., U.S.A., 97: 4030-4034), SV40 (Tsui, et al., 1982, Cell, 30: 499-508), Epstein-Barr virus (EBV) (Yates, et al., 1985, Nature, 313: 812-815; Margolskee, et al., 1988, Mol. Cell. Biol., 8: 2837-2847; Belt, et al., 1989, Gene, 84: 407-417; Chittenden, et al., 1989, J. Virol., 63: 3016-3025), and BK virus (BKV) (Milanesi, et al., 1984, Mol. Cell. Biol., 4: 1551-1560). Each of these episomal plasmids contains a viral origin of DNA replication and a virally encoded early gene that trans-activates the viral origin and allows the episome to replicate in the transfected host cell. Although EBV-based episomes have been used to efficiently screen cDNA libraries, the EBV system has limited applications to non-lymphoid cell types (Vidal, et at., 1990, Biochim. Biophys. Acta 1048: 171-177)), and the EBV replicon is not active in many cell types. Additionally, EBNA-1 is one of several EBV latent genes that immortalize human lymphocytes, and transfection of the EBV-negative BJAB lymphoma cell line by EBNA-1 induces soft agar growth, indicating transformation of the cells. (Konoshita, 1990, Hokkaido Igaku Zasshi, 65: 362-375).
http://www.google.com/patents?vid=USPAT5624820
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Show moreBackground of the invention: 1. Field of the Invention: This invention is directed to papovavirus-derived episomes that replicate efficiently in mammalian cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. Papovavirus-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells. 2. Review of Related Articles: One approach to gene therapy of human cancer cells is to introduce vectors expressing antisense sequences to block expression of dominant oncogenes and growth factor receptors. However, high-level expression of the oncogenes requires comparable levels of antisense expression, which presents a considerable technical obstacle, particularly when using expression vectors having a limited potential for achieving multiple copies in stable transfectants. Human cells transduced by retroviral vectors have only one or several copies of integrated retrovirus in stable transfectants. In contrast, hundreds of copies of episomal plasmids can accumulate in stable transfectants because these vectors replicate extrachromosomally. One method to express high levels of antisense transcripts is to utilize episomal plasmid vectors than can replicate extrachromosomally in human cells. Attempts to produce episomal vectors that will replicate in some types of human cells are reported by the literature. Episomal plasmids have been developed from several DNA viruses, including bovine papilloma virus (BPV) (Sarver, et al., 1981, Mol. Cell. Biol., 1:486-496; DiMaio, et al., 1982, Proc. Natl. Acad. Sci., U.S.A., 97:4030-4034), SV40 (Tsui, et al., 1982, Cell, 30:499-508), Epstein-Barr virus (EBV) (Yates, et al., 1985, Nature, 313:812-815; Margolskee, et al., 1988, Mol. Cell. Biol., 8:2837-2847; Belt, et al., 1989, Gene, 84:407-417; Chittenden, et al., 1989, J. Virol., 63:3016-3025), and BK virus (BKV) (Milanesi, et al., 1984, Mol Cell. Biol., 4:1551-1560). Each of these episomal plasmids contains a viral origin of DNA replication and a virally encoded early gene that trans-activates the viral origin and allows the episome to replicate in the transfected host cell. Although EBV-based episomes have been used to efficiently screen cDNA libraries, the EBV system has limited applications to non-lymphoid cell types (Vidal, et al., 1990, Biochim. Biophys. Acta 1048:171-177)), and the EBV replicon is not active in many cell types. Additionally, EBNA-1 is one of several EBV latent genes that immortalize human lymphocytes, and transfection of the EBV-negative BJAB lymphoma cell line by EBNA-1 induces soft agar growth, indicating transformation of the cells. (Konoshita, 1990, Hokkaido Igaku Zasshi, 65:362-375) Furthermore, stable transfection efficiencies for EBNA-1 negative cell lines transduced by EBV episomal plasmids encoding EBNA-1 (transactivator) and ORI-P (EBV DNA origin) are low, not significantly better than non-episomal plasmids (Yates, et al., 1985; Vidal, et al., 1990.
http://www.google.com/patents?vid=USPAT5770374
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