Chemistry: molecular biology and microbiology – Vector – per se
Patent
1996-10-04
1999-09-07
Campell, Bruce R.
Chemistry: molecular biology and microbiology
Vector, per se
514 44, 424 9321, 435 691, 435325, 435455, 536 231, C12N 1500
Patent
active
059486759
DESCRIPTION:
BRIEF SUMMARY
This application represents the U.S. national phase of PCT/FR95/00208 which claims priority to French application 94/01994, filed Feb. 22, 1994, and French application 94/15135, filed Dec. 15, 1994.
The present invention relates to a new approach for gene therapy applied especially to the elimination of certain categories of cells, such as tumor cells or certain cells infected by viruses, by the production of recombinant retroviruses from new genetic constructs.
The concept of gene therapy by killer genes or suicide genes has been developed since 1986 by numerous approaches. It involves the expression of a gene, this expression allowing the conversion of a nontoxic substance or a toxic substance by the cell. This concept of gene therapy is applicable and transposable to any gene whose expression in a target cell makes it possible to convert an inactive substance to an active substance, or vice versa, leading either to a destruction (example of the suicide gene), or to a restoration of certain functions.
In all that follows, the application of these concepts to suicide genes is more particularly developed given the accessibility of its experimental use. The transposition of the means of the invention to any type of transgene whose expression leads to the effect of a substance being modified in the direction of an activation or inactivation is nevertheless within the capability of persons skilled in the art.
Herpes Simplex virus type I thymidine kinase (HSV1-TK) is the enzyme which has been the subject of the highest number of investigations relating to suicide genes.
This enzyme, which is atoxic for eukaryotic cells, has the characteristic of being able to convert certain nucleoside analogs such as aciclovir (ACV) or ganciclovir (GCV) to monophosphate-containing molecules, of which cellular kinases are normally incapable (G. B. Elion, J. Antimicrob. Chemother. 12:9-17 (1983)). These nucleoside monophosphates are then converted by cellular enzymes to nucleotide triphosphates which can be used during the synthesis of DNA and block the elongation process thereby causing the death of the cell. The nucleoside triphosphate analog is therefore only toxic for dividing cells.
All the advantages which can be gained from the use of this type of conditional toxins can therefore be understood when applied to gene, especially anticancer, therapy.
Firstly, only a conditional toxicity makes it possible to generate stable cell clones producing the pseudo-viral particles capable of producing such a transgene and transferring the suicide gene into the target cells. Indeed, these cells simply have to be cultured in the absence of ACV or GCV since HSV1 thymidine kinase is not toxic for the cell in the absence of these drugs.
Next, in the event of a side effect of the treatment, stopping the administration of ACV or GCV causes the toxicity due to the transgene to cease immediately; in addition, adjustment of the doses of the nucleoside analog makes it possible to selectively destroy the cells strongly expressing the transgene while preserving the cells in which the gene is weakly expressed; this toxicity which is restricted to dividing cells is a great advantage especially for the treatment of cancer cells.
Finally, experimental data in vitro and in vivo have shown that cells not expressing HSV1-TK, but which are in contact with the latter, were also destroyed by the treatment with ACV ("Metabolic Cooperation" or "bystander effect") (Moolten F. L., 1986, Cancer Research, 46:5276-5281 and Culver K. W. et al., 1992, Science 256:1550-1552).
The mechanism of this effect is still not understood, but it is possible that the nucleoside triphosphate analogs can pass from one cell to another via "gap junctions".
Retroviruses appear to be the best vectors for transferring exogenous genes into eukaryotic cells, especially human cells.
However, an essential precondition for the use of retroviruses for therapeutic purposes and especially in gene therapy is to verify the safety of their use.
The principle danger of the use of retroviruses in
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Klatzmann David
Salzmann Jean-Loup
Campell Bruce R.
Nguyen Dave Trong
Universite Pierre Et Marie Curie (Paris VI)
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