Chemistry: molecular biology and microbiology – Vector – per se
Patent
1997-04-11
1999-12-21
Crouch, Deborah
Chemistry: molecular biology and microbiology
Vector, per se
435325, 435456, 435457, 536 232, C12N 1500, C12N 1586, C12N 500, C07H 2104
Patent
active
060048034
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an improvement made in the medicaments used in suicide gene-based gene therapy, and in particular in all the medicaments using the system for expression of a thymidine kinase gene in cells infected with a virus or in cancer cells, making said cells specifically sensitive to nucleoside analogs (N.A.) especially ganciclovir (GCV) or aciclovir (ACV).
The Herpes simplex virus type I thymidine kinase (HSV1-TK) gene has acquired a considerable importance in the developments of antiviral or anticancer therapy using a conditional toxicity and HSV1-TK is the enzyme which has been the subject of the largest number of investigations relating to suicide genes.
This enzyme, which is atoxic for eukaryotic cells, exhibits the characteristic of being able to convert certain nucleoside analogs such as ACV or GCV to monophosphate-containing molecules, of which cellular kinases are normally incapable (1,2). These monophosphate-containing nucleosides are then converted by cellular enzymes to nucleoside triphosphates which are used during the synthesis of DNA and block the extension process, thus causing the death of the cell.
Other viral thymidine kinases having the same properties can also be used, especially that of the varicella-zoster virus (VZV) as described in patent application EP 415 731. In the text which follows, where reference is made to HSV1-TK, persons skilled in the art will know how to generalize it to equivalent enzymes.
This conditional toxicity of HSV1-TK was first demonstrated in vitro and is now used in vivo for several purposes including: infected by viruses and especially retroviruses (4,7,8), secreting cells, or dendritic cells (D.C.) (10) in transgenic animal lines having the HSV1-TK gene under the control of specific cellular promoters; this system is a very powerful research means to know the physiological rose of either of these cellular types.
In antiviral therapy, it is the transactivation, under the effect of a viral infection, of a promoter regulating the synthesis of HSV1-TK which increases the synthesis of thymidine kinase, whose toxic effect manifests itself in the presence of an N.A.
All the advantages which may be gained from the use of this type of conditional toxins can therefore be understood when applied to gene, especially anticancer or antiviral, therapy.
Firstly, only a conditional toxicity makes it possible to generate stable cellular 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 in the patient, stopping the administration of ACV or GCV causes the toxicity due to the transgene to cease immediately; in addition, an adjustment of the doses of the nucleoside analog makes it possible to selectively destroy the cells strongly expressing the transgene while preserving as much as possible the cells in which the gene is weakly expressed. Finally, the 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 those expressing it, were also destroyed by the treatment with ACV ("metabolic cooperation" or "bystander effect") (3,4).
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" or intercellular communications.
In a transfer of exogenous genes in eukaryotic cells, especially human cells, the retroviruses appear to be the preferred vectors. However, an essential prerequisite for their use for therapeutic purposes is to ensure the safety of their use: the present invention offers a means of increasing the safety of the system without decreasing the therapeutic index of
REFERENCES:
Kappel et al (1992) Current Opin. Biotech. 3, 548-553.
Mullen (1994) Pharm. Ther. 63, 199-207.
Zemskova et al (1991) Gene 106, 249-253.
Burns et al (1993) Proc. Natl. Acad. 90, 8033-8037.
Culver et al (1992) Science 256, 1550-1552.
Salomon et al., Molecular and Cellular Biology, vol. 15, No. 10, pp. 5322-5328 (Oct. 1995).
Klatzmann David
Salomon Benoit
Crouch Deborah
Universite Pierre Et Marie Curie (Paris VI)
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