Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-09-10
2003-02-11
Priebe, Scott D. (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S069700, C435S194000, C435S325000, C435S455000, C536S023200, C536S023500, C536S023400, C424S093200, C424S093600, C514S04400A
Reexamination Certificate
active
06518062
ABSTRACT:
The present invention relates to the field of gene and cell therapy. It relates, in particular, to combinations of enzymes which can be used for the destruction of cells, especially proliferative cells. It also relates to vectors allowing the transfer and intracellular expression of these combinations of enzymes, as well as their therapeutic use, in particular in anticancer gene therapy.
Gene therapy, which consists in introducing genetic information into an organism or a cell, has undergone an extraordinary development over the past few years. The identification of genes involved in pathologies, the development of vectors for the administration of genes, the development of control or tissue-specific expression systems, in particular, have contributed to the development of these new therapeutic approaches. Thus, during the past 5 years, numerous clinical trials of gene or cell therapy have been undertaken in Europe and in the United States, in fields such as monogenic diseases (haemophilia, cystic fibrosis), cancer, cardiovascular diseases or disorders of the nervous system.
In the field of pathologies linked to a cellular hyperproliferation (cancer, restenosis, and the like), various approaches have been developed. Some are based on the use of tumour suppressor genes (p53, Rb), others on the use of antisenses directed against oncogenes (myc, Ras), still others on immunotherapy (administration of tumour antigens or of specific immune cells, and the like). Another approach consists in introducing into the affected cells a toxic or suicide gene capable of inducing the destruction of the said cells. Such genes are, for example, genes capable of sensitizing the cells to a pharmaceutical agent. They are generally genes encoding nonmammalian and nontoxic enzymes which, when they are expressed in mammalian cells, convert a prodrug, which is initially little or nontoxic, to a highly toxic agent. Such a mechanism of activation of prodrugs is advantageous in several respects: it makes it possible to optimize the therapeutic index by adjusting the prodrug concentration or the expression of the enzyme, to interrupt the toxicity by no longer administering the prodrug and to evaluate the mortality rate. In addition, the use of these suicide genes offers the advantage of not being specific to a particular type of tumour, but of general application. Thus, strategies based on the use of tumour suppressor genes or of anti-oncogene antisenses are applicable only to tumours exhibiting a deficiency in the said suppressor gene or an overexpression of the said oncogene. Likewise, approaches based on immunotherapy should be developed on a patient by patient basis to take into account immunorestrictions and immunocompetences. On the other hand, a strategy based on the use of a suicide gene is applicable to any type of tumour, and, more generally, to practically any type of cell.
Numerous suicide genes are described in the literature, such as, for example, the genes encoding cytosine deaminase, purine nucleoside phosphorylase or thymidine kinase, such as for example the thymidine kinases of the varicella virus or herpes simplex virus type 1.
The cytosine deaminase of
Escherichia coli
is capable of catalysing the deamination of cytosine to uracil. The cells which express the
E. coli
gene are therefore capable of converting 5-fluorocytosine to 5-fluorouracil, which is a toxic metabolite (Mullen et al. 1992 Proc. Natl. Acad. USA 89 p33).
The purine nucleoside phosphorylase of
Escherichia coli
allows the conversion of nontoxic analogues of deoxyadenosine to very toxic adenine analogues. As the eukaryotic enzyme does not exhibit this activity, if mammalian cells express the bacterial gene, the analogues of deoxyadenine such as 6-methylpurine-2′-deoxyribonucleoside will be converted to a product which is toxic for these cells (Sorscher et al. 1994 Gene Therapy 1 p233).
The thymidine kinase of the varicella virus allows the monophosphorylation of 6-methoxypurine arabinoside. If mammalian cells express this viral gene, this monophosphate is produced and then metabolized by the cellular enzymes to a toxic compound (Huber et al. 1991 Proc. Natl. Acad. USA 88 p8039).
Among these genes, the gene encoding thymidine kinase (TK) is most particularly advantageous from the therapeutic point of view because, unlike other suicide genes, it generates an enzyme capable of specifically eliminating dividing cells, since the prodrug is converted to a nondiffusible product which inhibits the synthesis of DNA. The viral thymidine kinase, and especially the thymidine kinases of the varicella virus or of the herpes simplex virus type 1, have a substrate specificity different from the cellular enzyme, and it has been shown that they are the target of guanosine analogues such as acyclovir or ganciclovir (Moolten 1986 Cancer Res. 46 p5276). Thus, ganciclovir is phosphorylated to ganciclovir monophosphate only when the mammalian cells produce the HSV1-TK enzyme, then cellular kinases allow the ganciclovir monophosphate to be metabolized to the diphosphate and then to the triphosphate which causes the synthesis of DNA to stop and leads to the death of the cell (Moolten 1986 Cancer Res. 46 p5276; Mullen 1994 Pharmac. Ther. 63 p199). The same mechanism is produced with other thymidine kinases and other guanosine analogues.
Moreover, a propagated toxicity effect (“by-stander” effect) was observed during the use of TK. This effect manifests itself by the destruction not only of the cells which have incorporated the TK gene, but also neighbouring cells. The mechanism of this process may be explained in three ways: i) the formation of apoptotic vesicles which contain phosphorylated ganciclovir or thymidine kinase, obtained from the dead cells, and then phagocytosis of these vesicles by the neighbouring cells; ii) the passage of the prodrug metabolized by thymidine kinase by a process of metabolic cooperation of the cells containing the suicide gene towards the cells not containing it and/or iii) an immune response linked to the regression of the tumour (Marini et al. 1995 Gene Therapy 2 p655).
For persons skilled in the art, the use of the suicide gene encoding the thymidine kinase of the herpes virus is very widely documented. In particular, the first studies in vivo on rats having a glioma show regressions of tumour when the HSV1-TK gene is expressed and when 150 mg/kg doses of ganciclovir are injected (K. Culver et al. 1992 Science 256 p1550). However, these doses are highly toxic in mice (T. Osaki et al. 1994 Cancer Research 54 p5258) and therefore completely proscribed in gene therapy in man.
A number of therapeutic trials are also underway in man, in which the TK gene is delivered to the cells by means of various vectors such as especially retroviral or adenoviral vectors. In clinical trials of gene therapy in man, much smaller doses, of the order of 5 mg/kg, have to be administered and for a short duration of treatment (14 days) (E. Oldfield et al. 1995 Human Gene Therapy 6 p55). For higher doses or for more prolonged treatments, undesirable toxic side effects are indeed observed.
To overcome these disadvantages, it has been proposed to synthesize more specific or more active thymidine kinase derivatives to phosphorylate the guanosine analogues. Thus, derivatives obtained by site-directed mutagenesis have been described. However, no precise biochemical characterization on the pure enzymes has been carried out, no cellular test using these mutants has been published and no functional improvement has been reported (WO 95/30007; Black et al., 1993 Biochemistry 32 p11618). In addition, the inducible expression of an HSV1-TK gene, deleted of its first 45 codons, has been performed in eukaryotic cells, but the prodrug doses used remain comparable to those described in all the trials in the literature (B. Salomon et al. 1995 Mol. Cell. Biol. 15 p5322). Consequently, none of the variants described up until now exhibits an improved activity in relation to thymidine or towards ganciclovir.
The present invention provides an improve
Blanche Francis
Cameron Beatrice
Couder Michel
Crouzet Joël
Aventis Pharma S.A.
Coppola William C.
Kaushal Sumesh
Krupen Karen I.
Priebe Scott D.
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