| Recombinase-based system for expression of foreign proteins using adenovirus vectors -> Monitor Keywords |
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Recombinase-based system for expression of foreign proteins using adenovirus 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.)Recombinase-based system for expression of foreign proteins using adenovirus vectors description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128165, Recombinase-based system for expression of foreign proteins using adenovirus vectors. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of copending parent application Ser. No. 09/909,414, filed Jul. 17, 2001, which is continuation of parent application Ser. No. 09/263,650, filed on Mar. 5, 1999 (U.S. Pat. No. 6,379,943). FIELD OF THE INVENTION [0002] The present invention relates to methods for efficient and reliable construction of adenovirus vectors that contain and express foreign DNA and are useful for gene transfer into mammalian cells, for vaccines and for gene therapy. The vector system described herein is an improvement and modification of the pBHG system, described in U.S. Pat. No. 6,140,087 (Graham et al.), a foreign equivalent of which published as WO95/00655, both of which are hereby incorporated by reference. BACKGROUND OF THE INVENTION [0003] As taught in U.S. Pat. No. 6,140,087, adenoviruses (Ads) can be used as mammalian cell expression vectors, with excellent potential as live recombinant viral vaccines, as transducing vectors for gene therapy, for research, and for production of proteins in mammalian cells. [0004] In the human Ad genome, early region 1 (E1), E3, and a site upstream of E4 have been utilized as sites for introducing foreign DNA sequences to generate adenovirus recombinants. In the absence of compensating deletions in E1 or E3, a maximum of about 2 kb can be inserted into the Ad genome to generate viable virus progeny. The E1 region is not required for viral replication in complementing 293 cells, or other cells known to complement E1, and up to 3.2 kb can be deleted in this region to generate conditional helper independent vectors with a capacity of 5.0-5.2 kb. In the E3 region, which is not required for viral replication in cultured cells, deletions of various sizes have been utilized to generate nonconditional helper independent vectors with a capacity of up to 4.5-4.7 kb. The combination of deletions in E1 and E3 permits the construction and propagation of adenovirus vectors with a capacity for insertions of up to approximately 8 kb of foreign DNA. [0005] The construction of Adenovirus vectors can be performed in many ways. One approach is to cotransfect permissive cells, usually 293 cells, with a shuttle plasmid containing a portion of the left end of the Ad genome and, most commonly, having the E1 sequences replaced by a foreign DNA, and with DNA isolated from virions cleaved near the left end by a suitable restriction enzyme. Homologous recombination between overlapping viral DNA sequences of the shuttle plasmid and the virion DNA results in production of recombinant viruses containing the foreign DNA. A disadvantage of this method is the need to prepare purified viral DNA. In addition, such methods typically result in the presence of contaminating parental virus in the resulting vector preparations, such as when 100% of the viral DNA is not cleaved, or when the two viral DNA fragments produced by restriction cleavage are rejoined. [0006] Another method has recently been described (Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps M L, "Construction of adenovirus vectors through Cre-lox recombination." J Virol 1997 March; 71(3):1842-1849; see also PCT publication WO97/32481 relating to use of site-specific recombination of virus and helper dependent vectors) which involves infection of 293Cre cells (293 cells engineered to express Cre recombinase) with an Adenovirus containing a floxed packaging signal (1) and transfection with a shuttle plasmid containing an ITR, a packaging signal and an expression cassette followed by a lox site, or cotransfection of 293Cre cells with purified deproteinized Adenoviral DNA and a shuttle plasmid. According to that method, Cre-mediated excision of the packaging signal from virus followed by site-specific recombination with the lox site in the shuttle plasmid produces a recombinant vector containing the expression cassette. However, as Cre action is not 100% efficient, the resulting virus preparations remain contaminated with parental virus, and must be passaged in 293Cre cells to eliminate the contaminating parental virus. A further disadvantage of this method is that it requires use of an infectious virus or DNA extracted from a virus as one of the starting materials, and is thus less attractive for commercial distribution than kits containing only bacterial plasmid DNA. Furthermore, the parental virus can recombine with Ad E1 sequences present in 293 cells, resulting in a virus containing a wild-type packaging signal and a wild-type E1 region. Such recombinant virus has the propensity to overgrow the original vector, leading to contamination of subsequent vector preparations with non-attenuated E1 expressing Ads. [0007] One of the most frequently used and most popular methods for construction of adenovirus vectors is based on "the two plasmid method" (see Bett et al., "Packaging capacity and stability of human adenovirus type 5 vectors," J. Virol. 67:5911-5921, 1993), whereby suitable host cells (typically 293 cells) are cotransfected with two plasmids that separately are incapable of generating infectious virus, but which, when recombined within the transfected cell by homologous recombination, can generate replicating virus. The most widely used plasmids of this type are described in U.S. Pat. No. 6,140,087, hereby incorporated by reference. That system has advantages over other methods using viruses or viral DNA as components since only easily-prepared plasmid DNAs are needed, and there is no background of parental virus that could contaminate the final vector isolates. Furthermore, the plasmids are not only easy and inexpensive to produce by those skilled in the art, but can be easily stored and transported, making them convenient for commercial distribution, (i.e. particularly when precipitated with ethanol or when lyophilized, these vectors do not require a cold chain for distribution). However, although this currently available system has proven utility and is widely used, the efficiency of virus production by homologous recombination can be low and variable, and the system cannot always be used easily by those not skilled in the art. [0008] As demonstrated in (Anton, M. and Graham, F. L. "Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression," J. Virol. 69:4600-4606, 1995), and as described also in parent application Ser. No. 08/486,549 ("Adenoviruses for control of gene expression", hereby incorporated by reference), provision of Cre recombinase in Ad-infected cells can catalyse excision or rearrangement of viral DNA sequences that contain the target sites (lox P) for Cre-mediated site-specific recombination. Such techniques are applied in new ways in the present invention disclosure to provide a long-needed advancement in the art of adenoviral vector production. SUMMARY OF THE INVENTION [0009] In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox P sites positioned such that site-specific recombination between lox P sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Such cells (293Cre cells) have been described by Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. and Graham, F. L. "A new helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal," Proc. Natl. Acad. Sci. U.S. 93: 13565-13570, 1996, by Chen, L., Anton, M. and Graham, F. L., "Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre," Somat. Cell and Molec. Genet. 22: 477-488, 1996, in U.S. patent application Ser. No. 08/473,168, and in PCT publication WO96/40955, hereby incorporated by reference for this purpose. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing a contaminating parental adenovirus, with the attendant problems for removal thereof. [0010] In one embodiment, this invention provides a two plasmid system wherein homologous recombination via cellular enzymes is replaced by site specific recombination, via a recombinase such as Cre, to join (with high efficiency) two DNAs that separately are noninfectious to form an infectious DNA molecule. One application of the techniques disclosed herein is the isolation of "first generation" vectors with insertions of foreign DNA in E1. Such applications utilize a series of plasmids such as pBHG10lox (see FIG. 1, and variations and equivalents thereof), and various shuttle plasmids containing the left ITR, a packaging signal, an expression cassette, and a lox or other recombinase recognition site. Another application is in a sense the mirror image. Using a plasmid such as pFG173lox, sequences are rescued into the right end of the viral DNA, into E3 or into sites rightward of E3. The most important applications of this technology will likely be rescue of mutations into the fibre gene located immediately rightward of E3 (FIG. 9) (fibre is important because it is a major ligand for binding to cellular receptors) but one can also rescue mutations, deletions, insertions and other modifications in E4 genes (located between fibre and the right ITR) or use the method to rescue inserts of foreign DNA into E3 (cotransfection of a plasmid such as that depicted in FIG. 11 with pFG173lox). Note that the plasmid pFG173lox has a deletion of fibre, but E4 sequences could just as well be deleted as well as or instead of fibre. Note also that lox sites could be inserted at other locations in the Ad genome to enable the rescue of mutations engineered in other viral genes besides those of fibre or E4, or rescue of DNA inserts into other sites. [0011] In a further embodiment of this invention, DNA-TP complexes are utilized to combine the high efficiency of Cre-lox recombination with the high infectivity of DNA-TP. While the rescue of infectious virus via Cre mediated recombination is surprisingly efficient compared to homologous recombination, and is more than adequate to produce viral vectors and to introduce mutations into the viral genome for most applications, there may be certain applications for which even higher efficiencies are desirable or necessary. It is known by those skilled in the art that the infectivity of adenovirus DNA is up to 100 fold higher if the virion DNA is extracted and purified by methods that leave intact the terminal protein (TP) that is normally linked to the 5' end of each strand of the duplex Ad DNA molecule (Sharp P A, Moore C, Haverty J L, "The infectivity of adenovirus 5 DNA-protein complex," Virology 1976 December; 75(2):442-456, Chinnadurai G, Chinnadurai S, Green M, "Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex." J Virol 1978 April; 26(1):195-199). For rescue of cassettes, the two plasmid system is more than sufficiently efficient, especially with the approximately 30-fold enhancement demonstrated herein for Cre-lox mediated recombination over homologous recombination, and consequently would be preferred for most purposes. However, there may be times when even higher efficiencies are required, as when, for example, one wishes to develop a library of fibre mutations (a large number of different viruses--the more the better). Then the chore of preparing DNA-TP might be worthwhile and could be accomplished by those skilled in the art. Thus, an aspect of the present invention includes the combination of the Cre-lox recombination with the high specific infectivity of adenoviral DNA-TP complexes. [0012] Therefore, it is an object of the present invention to provide a highly efficient, reliable, and simple method for isolation of viral vectors based on site-specific recombination catalysed by a site-specific recombinase, such as but not limited to the Cre recombinase, rather than relying on homologous recombination, which depends on normal cellular recombinase pathways. [0013] It is a further object of this invention to use Cre-lox-mediated recombination and known two plasmid vector production systems to provide a simple method for introducing mutations or other modifications of viral genes into any desired location in the viral genome. [0014] It is a further object of this invention to provide a simple and useful system by which adenovirus cloning vectors may be developed. [0015] It is a further object of this invention to provide a kit for production of adenoviral vectors for vaccine and gene-therapeutic applications which relies on site-specific recombination, rather than homologous recombination, and which does not require a cold-chain for distribution. [0016] A further object of this invention is to provide a system whereby the high-efficiency of Cre-lox recombination is combined with enhanced infectivity achieved when adenovirus-TP complexes are utilized. [0017] Further objects of this invention will become apparent from a review of the complete disclosure and the claims appended hereto. BRIEF DESCRIPTION OF DRAWINGS [0018] FIG. 1 is a diagrammatic representation showing a method for isolation of an Ad vector containing an expression cassette in E1 using the Cre/lox recombination system. pBHG10lox comprises a circularized form of the Ad genome with a deletion of the E1 region including the packaging signal and a bacterial plasmid origin of replication and an ampicillin resistance gene. The plasmid has a loxP site near the 5' end of the pIX gene of the Ad genome and a deletion of E3 sequences. The "shuttle plasmid" contains an ITR of the virus genome and a packaging signal, a polycloning site for insertion of a foreign DNA (eg bacterial .beta.-galactosidase (LacZ)) and a loxP site inserted in the same relative orientation as the loxP site in pBHG10lox. Cotransfection of these two plasmids into 293Cre cells that express Cre results in Cre-mediated recombination and formation of joint molecules that generate infectious viruses containing the foreign DNA insert. [0019] FIG. 2 illustrates a cotransfection experiment similar to that depicted in FIG. 1 except that the shuttle plasmid contains Ad sequences 3' of the lox site that overlap (are homologous) with viral sequences in pBHG10lox to the right of the lox site. Therefore an Ad vector containing an expression cassette in E1 can be generated by Cre/lox recombination when the two plasmids are cotransfected into 293Cre cells or alternatively by homologous recombination between overlapping sequences. The shuttle plasmid in the illustration permits a comparison of the efficiency obtainable from the two recombination modes. Continue reading about Recombinase-based system for expression of foreign proteins using adenovirus vectors... 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