Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-03-09
2000-05-23
Guzo, David
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 914, 435 9142, 4353201, 435455, 435456, 435457, 435325, 435366, 435369, 536 232, 536 234, 536 235, 536 237, C12N 1564, C12N 15861, C12N 510, C07H 2104
Patent
active
060664789
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to novel helper vectors allowing defective recombinant viral vectors, which have the characteristic of being provided with recombination sequences recognized by a recombinase, to be complemented. It likewise relates to a complementation cell expressing the recombinase as well as a method of preparation of recombinant viral vectors in the form of infectious viral particles allowing the transfer and the expression of genes of interest in a cell or a host organism. The invention is of very particular interest for gene therapy prospects, especially in man.
The possibility of treating human diseases by gene therapy has passed in the course of a few years from the stage of theoretical considerations to that of clinical applications. The first protocol applied to man was initiated in the United States in September 1990 on a patient who was genetically immunodeficient because of a mutation affecting the gene coding for adenine deaminase (ADA). The relative success of this first experiment encouraged the development of new gene therapy protocols for various genetic or acquired diseases (infectious diseases and especially viral diseases such as AIDS or cancers). The great majority of the protocols described until now employ viral vectors to transfer and express the therapeutic gene in the cells to be treated.
The interest in adenoviruses as gene therapy vectors has already been touched on in numerous documents of the prior art. In fact, the adenoviruses have a wide spectrum of hosts, are not very pathogenic and do not have the disadvantages connected with the retroviruses since they are nonintegrative and replicate equally in quiescent cells. By way of information, their genome is formed of a linear and double-stranded DNA molecule of approximately 36 kb carrying regions acting in cis (ITR 5' and 5' encapsidation region of the viral genome and ITR 3') and additionally about thirty genes, at the same time early genes necessary for viral replication and late structure genes (see FIG. 1).
The early genes are divided into 4 regions dispersed in the adenoviral genome (E1 to E4; E for early). They comprise 6 transcriptional units which have their own promoters. The late genes (L1 to L5; L for late) partly cover the early transcription units and are, for the majority, transcribed starting from the major late promoter MLP.
At the present time, all the adenoviral vectors used in gene therapy protocols are devoid of the major part of the E1 region essential for replication, in order to avoid their distribution in the environment and the host organism (first generation vectors). This deletion makes the viral genome deficient for replication. However, the E1.sup.- viruses can be propagated in a cell line which complements the E1 function to generate an infectious viral particle. The 293 line, established starting from human embryonic kidney cells, is currently used, in the genome of which is integrated the left 5' end of the type 5 adenovirus (Graham et al., 1977, J. Gen. Virol. 36, 59-72).
The majority of adenoviral vectors of the prior art comprise supplementary deletions. Certain of these have been introduced in the E3 region with the aim of increasing the cloning capacities but do not need to be complemented to the extent where the E3 region is nonessential for replication. More recently, second generation vectors have been proposed in the literature. They conserve the in cis regions (ITRs and encapsidation sequences) and comprise important internal deletions aimed at suppressing the main part of the viral genes whose expression can be responsible for inflammatory responses in the host. In this respect, a minimal vector which is deficient for all of the coding viral regions represents a choice alternative.
The techniques of preparation of adenoviral vectors are widely described in the literature. Firstly, the complete genome is formed by homologous recombination in the 293 line (see especially Graham et Prevect, 1991, Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols;
REFERENCES:
Gage et al., J. Virol., vol. 66, No. 9, pp. 5509-5515, Sep. 1992.
W. French Anderson, Nature, vol. 392, pp. 25-30, Apr. 30,1998.
Ross et al., Human Gene Therapy, vol.7, pp.1781-1790, Sep. 1996.
Verma et al., Nature, vol. 389, pp. 239-242, Sep. 18, 1997.
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Proc. Natl. Acad. Sci. U.S.A. (1987), 84 (24), 9108-12 CODEN: PNASA6;ISSN: 0027-8424, XP002002088, Sauer, Brian et al, "Site-specific insertion of DNA into a pseudorabies virus vector".
Nucleic Acids Res. (1989), 17(1), 147-61 CODEN: NARHAD; ISSN: 0305-1048, XP002002089, Sauer, Brian et al: "Cre-stimulated recombination at loxP -- containing DNA sequences placed into the mammalian genome".
Lusky Monika
Mehtali Majid
Guzo David
Transgene S.A.
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