Adenovirus gene expression system

Details Adenovirus gene expression system Adenovirus gene expression system

1996-05-24

2004-11-30

Nguyen, Dave T. (Department: 1635)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C435S069100, C435S456000, C435S320100, C435S325000, C424S093100, C536S023100, C536S024100, C514S04400A

Reexamination Certificate

active

06824770

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to a recombinant viral expression system. More particularly, the present invention relates to a highly efficient, recombinant adenovirus expression system capable of expressing a heterologous gene(s) in a host mammalian cell.
BACKGROUND OF THE INVENTION
The human adenovirus-5 (Ad5) genome consists of a double-stranded linear DNA molecule of 36 kilo-basepair (bp) (Ginsberg, 1984). The virus replication cycle has two phases: an early phase, during which 4 transcription units E1, E2, E3, and E4 are expressed, and a late phase occurring after the onset of viral DNA synthesis when late transcripts are expressed from the major late promoter (MLP). These late messages encode most of the viral structural proteins. E1, E2 and E4 gene products of human adenoviruses are involved in transcriptional activation, cell transformation, and viral DNA replication as well as other viral functions, and are essential for viral growth (Grand, 1987
, Biochem. J
., vol. 241, pp. 25-38; and Nevins, 1987
, Microbiol. Rev
., vol. 51, pp. 419-430). In contrast, E3 gene products are not required for viral replication in cultured cells (Ginsberg et al., 1989,), but appear to be involved in evading immune surveillance in vivo (Anderson et al., 1985
, Cell
, vol. 43, pp. 215-222; Burgert et al., 1985
, Cell
, vol. 41, pp. 987-997; Burgert et al., 1987
, EMBO J
., vol. 6, pp. 2019-2026; Carlin et al., 1989
, Cell
, vol. 57, pp. 135-144; Gooding and Wold, 1990
, Crit. Rev. Immunol
., vol. 10, pp. 53-71; Gooding et al., 1988
, Cell
, vol. 53, pp. 341-346; Horton et al., 1990
, J. Virol
., vol. 64, pp. 1250-1255; Tollefson et al., 1991
, J. Virol
., vol. 65, pp. 3095-3105; Wold and Gooding, 1989
, Mol. Biol. Med
., vol. 6, pp. 433-452; and Wold and Gooding, 1991
, Virology
, vol. 184, pp. 1-8).
E1 and E3 and a site upstream of E4 have been utilized as sites for insertion of foreign DNA sequences in the generation of recombinant adenoviruses (Berkner et al., 1984
, Nuc. Acids. Res
., vol. 12, pp. 1925-1941; Chanda et al., 1990
, Virology
, vol. 175, pp. 535-547; Haj-Ahmad et al., 1986
, J. Virol
., vol. 57, pp. 267-274; and Saito et al., 1985
, J. Virol
., vol. 54, pp. 711-719). Since the upper size limit for DNA molecules that can be packaged into adenovirus particles is approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987
, EMBO J
., vol. 6, pp. 1733-1739), only about 2 kb of extra DNA can be inserted without compensating deletions of viral DNA. Although E1 is essential for virus replication in cell culture, foreign DNA can be substituted for E1 sequences when the virus is grown in 293 cells which are transformed by adenovirus-5 DNA and constitutively express E1 (Graham et al., 1977
, J. Gen. Virol
., vol. 36, pp. 59-72). Several vectors having 1.9 kb deleted from E3 of adenovirus-5 have been constructed without interfering with virus replication in cell culture (Graham et al., 1992, Vaccines; New Approaches to Immunological Problems, R. W. Ellis (Ed.), Butterworth-Heinemann, Boston, Mass., pp. 364-390). Such vectors allow for insertion of up to 4 kb of foreign DNA. Recombinant adenoviruses containing inserts in E3 replicate in all adenovirus-permissive cell lines and may be suitable as live recombinant viral vaccines since a number of adenovirus vectors containing E3 inserts have been shown to express foreign genes efficiently both in vitro and in vivo (Berkner, 1988: Chanda et al., 1990; Dewar et al., 1989
, J. Virol
., vol. 63, pp. 129-136; Graham, 1990
, Trends Biotechnol
., vol. 8, pp. 85-87; Graham et al., 1992; Johnson et al., 1988
, Virology
, vol. 164, pp. 1-14; Lubeck et al., 1989
, Proc. Natl. Acad. Sci. USA
, vol. 86, pp. 6763-6767; McDermott et al. 1989
, Virology
, vol. 169, pp. 244-247; Morin et al., 1987
, Proc. Natl. Acad. Sci. USA
, vol. 84, pp. 4626-4630; Prevec et al., 1989
, J. Gen. Virol
., vol. 70, pp. 429-434; Prevec et al., 1990
, J. Inf. Dis
., vol. 161, pp. 27-30; Schneider et al., 1989
, J. Gen. Virol
. , vol. 70, pp. 417-427; Vernon et al., 1991
, J. Gen. Virol
., vol. 72, pp. 1243-1251; and Yuasa et al., 1991
, J. Gen. Virol
., vol. 72, pp. 1927-1934).
Adenoviruses are good mammalian cell expression vectors with potential utility as live recombinant vaccines, in gene therapy, or for high level protein production in mammalian cells.
Adenovirus expression vectors have been in use for the past decade (Thummel et al., 1981
, Cell
, vol. 23, pp. 825-836; Berkner et al., 1984, Nucleic Acids Res., vol. 12, pp. 1925-1941; and for a review see Grunhaus et al., 1992, Seminars in
Virology
3, pp. 237-252), and more recently exploited for the purpose of gene therapy (Herz et al., 1993
, Proc. Natl. Acad. Sci. U.S.A
., vol. 90, pp. 2812-2816; Rosenfeld et al., 1991
, Science
, vol. 252, pp. 431-434; and Rosenfeld et al., 1992
, Cell
, vol. 68, pp. 143-155). Features of adenovirus based expression vectors which make them attractive to gene therapy applications include very efficient uptake into cells which contain the appropriate adenovirus receptor and uptake pathway, and the ability to carry up to 7.5 kb of foreign DNA. Adenovirus vectors allow a reporter gene to be under the control of tissue specific promoter elements (Friedman et al., 1986
, Mol. Cell. Biol
., vol. 6, pp. 3791-3797; and Babiss et al., 1986
, Mol. Cell. Biol
., vol. 6, pp. 3798-3806) as well as a variety of viral and mammalian constitutive promoter elements (Mittal et al., 1993
, Virus Research
, vol. 28, pp. 67-90).
One such example of an adenovirus-based vector system is described in Mittal et al., 1993
, Virus Research
, vol. 28, pp. 67-90. The authors here describe a helper-independent adenovirus type 5-luciferase recombinant containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted into the early region 3 (E3) of the adenovirus-5 genome. A plasmid containing the luciferase gene and SV40 regulatory sequences in the E3 region was co-transfected with a plasmid containing the adenovirus-5 d1309 genome in circular form. Upon transfection of 293 cells, virus progeny produced by in vivo recombination between the two plasmids resulted in rescue of the adenovirus type 5-luciferase recombinant (i.e., E3 insert in Adenovirus-5 genome).
Gomez-Foix et al., 1992
, J. Biol. Chemistry
, vol. 267, no. 35, pp. 25129-25134, discloses adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes in culture. The preparation of a recombinant adenovirus containing the cDNA encoding rabbit muscle glycogen phosphorylase is described whereby the cytomegalovirus (CMV) early gene promoter/enhancer, pUC 18 polylinker, fragment of the SV40 genome that includes the small T-antigen intron and the polyadenylation signal, and cDNA that includes all of the protein coding region of the rabbit muscle glycogen phosphorylase, was inserted into vector pAC. The resulting plasmid was co-transfected into 293 cells with plasmid pJM17, which encodes a full-length adenovirus-5 genome. Homologous recombination between the recombinant plasmids in 293 cells generated a genome of packageable size in which the adenovirus early region 1 was replaced by the cloned chimeric gene encoding rabbit muscle glycogen phosphorylase.
Roessler et al., 1993
, J. Clin. Invest
., discloses using a recombinant adenoviral vector for the expression of the gene for
Escherichia coli
beta-galactosidase within synovium tissue. Replication defective adenoviral vectors are deleted of sequences spanning E1A, E1B and a portion of the E3 region, impairing the ability of this virus to replicate or transform nonpermissive cells. The early enhancer/promoter of the cytomegalovirus (CMV) was inserted into this vector to drive transcription of lacZ with a SV40 polyadenylation sequence cloned downstream from this reporter.
Yang et al.,
Proc. Natl. Acad. Sci. USA
, vol. 90, pp. 9480-9484, discloses the expression of cystic fibrosis transmembrane conductance regulator (CFTR) by adenovirus-mediated gene transfer. The recombinant adenoviruses were produ

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