Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1999-03-05
2002-04-30
Schwartzmann, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S320100, C435S325000, C435S366000, C435S369000
Reexamination Certificate
active
06379943
ABSTRACT:
FIELD OF THE INVENTION
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 copending patent application Ser. No.08/250,885, a foreign equivalent of which published as WO95/00655, hereby incorporated by reference.
BACKGROUND OF THE INVENTION
As taught in WO95/00655, 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.
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.
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.
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 (&PSgr;) 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.
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 patent application S/N Ser. No.08/250,885, and in PCT publication number WO95/00655, 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.
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 (loxP) 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
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 loxP 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.
In one embodiment, this invention provides a two plasmid system wherein homologous recombination via cellular enzymes is replaced by site specific recomb
Graham Frank L.
Ng Philip
Parks Robin J.
Merck & Co. , Inc.
Schwartzmann Robert A.
Van Dyke & Associates, P.A.
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