Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Patent
1996-07-25
1998-08-04
Achutamurthy, Ponnathapura
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
4242331, 4241991, 4352351, 435 691, 4351723, 536 235, A61K 39235, C12N 701, C12P 2100, C07H 2104
Patent
active
057893900
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel method for preparing vectors derived from adeno-associated viruses (AAVs) and to the cells used for this purpose. More specifically, the present invention relates to a process for preparing recombinant AAVs using an animal helper virus. The invention also relates to the use of the recombinant AAVs obtained, particularly for gene and/or cellular therapy.
Gene therapy consists of correcting a deficiency or an abnormality (mutation, aberrant expression, etc.), or of ensuring the expression of a protein of therapeutic interest, by introducing genetic information into the cell or organ concerned. This genetic information can be introduced either in vitro into a cell extracted from the organ, with the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue. Various techniques have been described for transferring this genetic information, including diverse transfection techniques involving complexes of DNA and DEAE dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), and of DNA and polylysine, the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. More recently, the use of viruses as vectors for transferring genes has appeared as a promising alternative to these physicochemical transfection techniques. In this respect, a variety of viruses have been tested for their ability to infect certain cellular populations; in particular, retroviruses (RSV, HMS, MMS, etc.), HSV virus, the adenoviruses and the adeno-associated viruses.
Among these viruses, the adeno-associated viruses (AAV denoting "adeno-associated virus") offer certain properties which are attractive as regards utilization for transferring genes. The AAVs are DNA viruses of relatively small size which integrate, in a stable and site-specific manner, into the genome of the cells they infect. They are able to infect a wide spectrum of cells without having any effects on cell growth, on cell morphology or on cellular differentiation. Moreover, they do not seem to be involved in human disease.
The genome of the AAVs has been cloned, sequenced and characterized. It comprises about 4,700 bases and contains, at each end, an inverted repeat region (ITR) of approximately 145 bases, serving as the origin of replication of the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: the left-hand part of the genome, containing the rep gene involved in replication of the virus and expression of the viral genes and; the right-hand part of the genome, containing the cap gene encoding the capsid proteins of the virus. The use of vectors derived from AAVs for transferring genes in vitro and in vivo has been described in the literature (see, particularly, WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). These applications describe different constructs derived from the AAVs, in which constructs the rep and/or cap genes are deleted and replaced by a gene of interest, and their use for transferring the said gene of interest in vitro (to cells in culture) or in vivo (directly into an organism).
Nevertheless, the therapeutic use of the AAVs as vectors for transferring genes is currently limited, particularly because of the risks of contaminating the recombinant AAVs with a wild-type helper virus, and of using potentially tumorigenic human cell lines for producing the recombinant viruses. In order to replicate, the AAVs require the presence of a helper virus. The term helper virus denotes any virus which is able to supply in trans the functions which are required for replicating the AAVs. This can, in particular, be an adenovirus, a herpes virus or a vaccinia virus. In the absence of a helper virus of this nature, the AAVs remain in latent form in the genome of the infected cells but are not able to replicate and thus to produce viral particles. In general, th
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Descamps Vincent
Perricaudet Michel
Achutamurthy Ponnathapura
Bui Phuong T.
Rhone-Poulenc Rorer S.A.
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