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Self-rearranging dna vectorsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Self-rearranging dna vectors description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166286, Self-rearranging dna vectors. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is divisional of U.S. application Ser. No. 10/384,136, filed Mar. 7, 2003, which is a continuation of International Application No. PCT/US01/27682, filed Sep. 7, 2001, which claims benefit of U.S. provisional application Nos. 60/246,904, and 60/231,053, filed Nov. 8, 2000, and Sep. 8, 2000, respectively, all of which are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION [0002] The invention relates to DNA vectors. [0003] Mammalian cell expression vectors based on DNA viruses have been widely discussed as gene delivery vehicles for genetic therapy. Among the different DNA viruses proposed for this purpose have been adenoviruses, baculovirus, Epstein Barr virus, and herpes simplex virus. In addition other smaller viruses that have an intranuclear phase in which the viral genome is present as a double stranded DNA, such as retroviruses and parvoviruses, have been proposed as gene delivery vehicles. [0004] Adenoviral vectors (AdV), for example, have a recognized potential for gene delivery, founded in their broad host range, robust growth in culture, and capacity to infect mitotically quiescent cells (Graham and Prevec, Manipulation of adenovirus vectors, p. 109-128, In E. J. Murray (ed.), Methods in Molecular Biology, vol. 7, Humana, Clifton, N.J., 1991; Trapnell and Gorziglia, Curr. Opin. Biotechnol. 5:617-625, 1994). AdV can be propagated in a helper cell line, 293, a human embryonic kidney cell line transformed by adenovirus type 5 (Graham et al., J. Gen. Virol. 36:59-72, 1994). 293 cells express the viral E1 gene products (E1a and E1b) that are the master regulatory proteins for subsequent viral gene expression. E1 deleted viruses can propagate in 293 cells, but not in other cells. Although it would be expected that E1 deleted viruses lack the machinery to express viral genes, several studies have demonstrated that cellular E1-like components can stimulate viral gene expression (Imperiale et al., Mol. Cell. Biol. 4:867-74, 1984; Onclercq et al., J. Virol. 62:4533-7, 1988; Spergel et al., J. Virol. 66:1021-30, 1992). The expression of these viral genes results in the relatively rapid elimination of transduced cells in vivo as a result of cytotoxic T cell responses (Yang et al., Immunity 1:433-42, 1994; Yang et al., Gene Ther. 3:13744, 1996; Yang et al., J. Virol. 69:2004-15, 1995). [0005] Thus attention has been focused on eliminating the remaining vestiges of viral expression. Viral genes that have been deleted for this purpose include the gene for E4 proteins (Armentano et al., Hum. Gene Ther. 6:1343-53, 1995; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-6, 1996; and Yeh et al., J. Virol. 70:559-565, 1996), DNA binding protein (Engelhardt et al., Proc. Natl. Acad. Sci. USA 21:6196-6200, 1994; and Gorziglia et al., J. Virol. 70:4173-8, 1996), DNA polymerase (Amalfitano et al., J. Virol. 72:926-33, 1998), and the preterminal protein (Schaack et al., Proc. Natl. Acad. Sci. USA 93:14686-91, 1996). The most aggressive approach has been the creation of helper virus-dependent vectors that lack all viral genes (Hardy et al., J. Virol. 71:1842-9, 1997; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-6, 1996; Lieber et al., J. Virol. 70:8944-60, 1996; Mitani et al., Proc. Natl. Acad. Sci. USA 92:3854-8, 1995; and Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996). These vectors have high capacity, evoke reduced cellular immune responses and show prolonged expression in vivo (Morsy et al., Proc. Natl. Acad. Sci. USA 95:7866-71, 1998). However to deploy these viruses on the scale required for human clinical application presents major challenges because a cesium chloride (CsCl) gradient is needed to remove the helper virus. SUMMARY OF THE INVENTION [0006] In one aspect, the invention features a replicatable viral DNA vector encoding a site-specific DNA-altering enzyme and a DNA target recognized by the enzyme, the enzyme selectively converting, in a cell expressing the enzyme, the DNA vector to a rearranged form. [0007] In preferred embodiments, the rearranged form includes an autonomously replicating episome and a linear DNA product. In other preferred embodiments, the vector comprises adenoviral DNA. [0008] In yet other preferred embodiments, the vector includes a genetically-engineered recombination site (such as a target of Cre or FLP). Preferably, such a recombination site includes a recognition sequence of a site-specific DNA altering enzyme. [0009] In another preferred embodiment, the site-specific DNA altering enzyme is a recombinase (such as Cre or FLP) or an integrase. Preferably, such an enzyme is functional in a mammalian cell. Preferred embodiments of the vector also include an origin of replication that functions in a mammalian cell (such as an Epstein Barr Virus replicon). Moreover, the vector typically includes a gene of interest (such as a therapeutic gene that encodes a protein or polypeptide or an RNA product). [0010] In another aspect, the invention features a method for assembling a recombinant adenoviral DNA. The method, in general, includes the steps of: (a) providing a first linearized DNA vector comprising a restriction site and a cos site and a second linearized DNA vector comprising the restriction site, an adenoviral nucleic acid molecule, and a cos site; and (b) ligating the first and second linearized DNA vectors, the ligation assembling a recombinant adenoviral DNA. [0011] In preferred embodiments, the first linearized DNA vector comprises a selectable marker (such as a gene encoding a polypeptide that confers, on a host cell expressing such a polypeptide, resistance to an antibiotic). In other preferred embodiments, the first linearized DNA vector includes an adenoviral left-end inverted terminal repeat, a gene of interest, or both. In still other preferred embodiments, the second linearized DNA vector includes a selectable marker. Preferably, the second linearized DNA vector includes an adenoviral right-end inverted terminal repeat. [0012] The method further includes packaging the assembled adenoviral DNA into a phage and infecting a host cell. Typically the first and second linearized DNAs include cosmid vector DNA. In addition, such adenoviral DNA is typically flanked by cleavage sites (such as intron endonuclease cleavage sites). [0013] In another aspect, the invention features an adenovirus producer cell having a nucleic acid molecule that expresses a dominant negative site-specific DNA-altering enzyme. In preferred embodiments, the site-specific DNA altering enzyme is a dominant negative recombinase (for example, a Cre recombinase such as CreY324C or a Flp recombinase). Exemplary adenovirus producer cells include, without limitation, 293 human embryonic kidney cells, per.C6 cells, and N52 cells. [0014] In yet another aspect, the invention features a vector comprising, in the 5' to 3' direction, a first genetically engineered cis-acting target recognized by a site-specific DNA altering enzyme; a gene of interest; a lineage-specific gene promoter; a second genetically engineered cis-acting target recognized by a site-specific DNA altering enzyme; and a nucleic acid molecule encoding a site-specific DNA altering enzyme. [0015] In still another aspect, the invention features a vector including, in the 5' to 3' direction, a first genetically engineered cis-acting target recognized by a site-specific DNA altering enzyme; a gene of interest; a bi-directional promoter, comprising a second genetically engineered cis-acting target recognized by a site-specific DNA altering enzyme; and a nucleic acid molecule encoding a site-specific DNA altering enzyme. [0016] In related aspects, the invention features a method of gene therapy including the administration to a patient in need of gene therapy a therapeutically effective amount of the vector of the invention, which is expressed in the patient. The invention further relates to a population of cells transfected with the vector of the invention. [0017] Accordingly, the invention further relates to the use of a recombinant viral vector or use of a recombinant viral particle for gene therapy. Such vectors and viral particles may be introduced either in vitro into a host cell removed from the patient, or directly in vivo, into the body to be treated, according to standard methods known in the art. [0018] The invention also relates to a pharmaceutical composition that includes a therapeutically effective amount of a recombinant viral vector or viral particle prepared according to the methods disclosed herein, in combination with a vehicle that is acceptable from a pharmaceutical standpoint. Such a pharmaceutical composition may be prepared according to the techniques commonly employed and administered by any known administration route, for example systemically (in particular, by intravenous, intratracheal, intraperitoneal, intramuscular, subcutaneous, intratumoral, or intracranial routes) or by aerosolization or intrapulmonary administration. [0019] One skilled in the art will appreciate that suitable methods of administering a vector (particularly an adenoviral vector) of the present invention to an animal for purposes of gene therapy, chemotherapy, and vaccination are available, and, although more than one route can be used for administration, one particular route may provide a more immediate and more effective reaction than another. Pharmaceutically acceptable excipients also are well known to those who are skilled in the art, and are readily available. The choice of excipient will be determined, in part, by the particular method used to administer the recombinant vector or particle. Accordingly, there are a wide variety of suitable formulations for use in the context of the present invention. [0020] By "recombinant DNA vector" is meant a DNA sequence containing a desired sequence (such as a gene of interest) and an appropriate regulatory element(s) necessary for the expression of the operably linked sequence in a particular host organism (such as a mammal). [0021] By "operably linked" is meant that a gene and a regulatory element(s) are connected to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s). Continue reading about Self-rearranging dna vectors... Full patent description for Self-rearranging dna vectors Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Self-rearranging dna vectors patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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