Cells for the production of recombinant adenoviruses

Details Cells for the production of recombinant adenoviruses Cells for the production of recombinant adenoviruses

1997-07-17

2000-10-03

Mosher, Mary E.

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

435 691, 435 911, 435 9141, 435366, 4353201, 4352351, 4241991, 514 44, 536 2372, 536 241, C12P 2106, C12N 1500, C12N 510, C12N 701

Patent

active

061271756

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to new cell lines which can be used for the production of defective recombinant adenoviruses. It also relates to the purified viral preparations produced in these lines, as well as the plasmids allowing their construction. More particularly, the new cell lines according to the invention allow the transcomplementation of the E4 region and a clonal production with high titers of defective recombinant adenoviruses especially for all or part of the E4 region.
Adenoviruses exhibit certain properties which are particularly advantageous for use as vector for the transfer of genes in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated, up until now, with major pathologies in man. Adenoviruses have thus been used to transfer genes of interest into the muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), and the like.
Adenoviruses are viruses with a linear double-stranded DNA having a size of about 36 kb. Their genome comprises especially an inverted repeat sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes (Cf. FIG. 1). The principal early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the E1 region in particular are necessary for viral propagation. The principal late genes are contained in the L1 to L5 regions. The genome of the Ad5 adenovirus has been completely sequenced and is accessible on a database (see especially Genebank M73260). Likewise, parts or even the whole of other adenoviral genomes (Ad2, Ad7, Ad12 and the like) have also been sequenced.
For their use in gene therapy, various vectors derived from adenoviruses have been prepared, incorporating various genes (.beta.-gal, OTC, .alpha.-1AT, cytokines and the like). In each of these constructs, the adenovirus was modified so as to render it incapable of replicating in the infected cell. Thus, the constructs described in the prior art are adenoviruses from which the E1 region has been deleted, which region is essential for the viral replication and at the level of which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). These adenoviruses are produced in a complementation line (line 293) into which part of the adenovirus genome has been integrated. More precisely, line 293 contains the left end (about 11-12%) of the serotype 5 adenovirus (Ad5) genome comprising the left ITR, the encapsidation region, the E1 region, including E1a, E1b, the region encoding the pIX protein and part of the region encoding the pIVa2 protein. This line is capable of trans-complementing recombinant adenoviruses defective for the E1 region, that is to say lacking all or part of the E1 region, and of producing viral stocks having high titers. However, vectors deficient for the E1 region (E1.sup.- vectors, termed first generation vectors) exhibit certain disadvantages for a therapeutic use. In particular, they might not be completely defective for in vivo replication especially because of the existence of certain transcomplementing cellular functions. Thus, an E1 transcomplementation activity has been detected in F9 embryonic carcinoma cells (Imperiale et al., Mol. Cell. Biol. 4, 1984, 867-874). An activity of the same type, regulated by interleukin-6, has also been detected (Spergel et al., J. Virol. 66, 1992, 1021-1030). Other disadvantages linked to these vectors are the presence of numerous viral genes, capable of being expressed in vivo after gene transfer, and of inducing an immune and/or inflammatory response.
In order to overcome these disadvantages, it has been proposed to create other deletions or modifications in the adenovirus genome. Thus, a heat-sensitive point mutation has been introduced into the ts125 mutant, making it possible to

REFERENCES:
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