Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1998-08-06
2001-03-27
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C435S194000, C435S325000, C435S252300, C435S320100, C435S006120, C536S023200
Reexamination Certificate
active
06207150
ABSTRACT:
The present invention relates to nucleic acid sequences coding for enzymes derived from the wild-type thymidine kinase, TK, enzyme, and possessing improved functions for the purpose of therapeutic use. It relates more especially to new enzymes possessing an improved substrate specificity and/or efficacy relative to the wild-type thymidine kinase enzyme. It also relates to vectors containing these nucleic acid sequences and to their therapeutic uses, in particular in gene therapy.
The present invention relates more especially to the field of gene therapy which employs suicide genes for the purpose of inducing the cell death of specific cells such as cells infected with a virus such as the HIV (human immunodeficiency virus), CMV (cytomegalovirus) or RSV (respiratory syncytial virus) type virus. This type of therapeutic treatment, consisting in causing a suicide gene to be expressed within a cell, is also applied for the treatment of cancers and of some cardiovascular diseases.
As suicide gene, it is preferable to use, in gene therapy, genes whose expression product endows the cell with a sensitivity to a therapeutic agent. More generally, the genes in question are ones that code for non-mammalian and non-toxic enzymes which, when they are expressed in mammalian cells, transform a prodrug which initially has little or no toxicity to a highly toxic agent. Such a mechanism of action of prodrugs is advantageous on several counts: 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.
Numerous suicide genes are described in the literature, such as, for example, the genes coding for cytosine deaminase, purine nucleoside phosphorylase or a thymidine kinase such as, for example, the chickenpox virus or the herpes simplex virus type 1 thymidine kinases. Among these genes, the gene coding for herpes simplex virus type 1 thymidine kinase is most especially advantageous from a therapeutic standpoint since, in contrast to the other suicide genes, it generates an enzyme, thymidine kinase, capable of specifically eliminating dividing cells. This enzyme has a different substrate specificity from the cellular enzyme, and it has been shown to be the target of guanosine analogues such as acyclovir or ganciclovir (Moolten 1986 Cancer Res. 46, p. 5276).
In the particular case of the HSV1-TK/ganciclovir system, the mechanism of action may be outlined as follows: mammalian cells modified to express the HSV1-TK enzyme implement the first step of phosphorylation of ganciclovir to yield ganciclovir monophosphate. This step appears to be limiting. Subsequently, cellular kinases enable this ganciclovir monophosphate to be metabolized successively to diphosphate and then triphosphate. The ganciclovir triphosphate thus generated then produces toxic effects by becoming incorporated in the DNA, and partially inhibits the cellular DNA polymerase alpha, thereby causing DNA synthesis to be stopped and hence leading to cell death (Moolten 1986 Cancer Res. 46, p. 5276; Mullen 1994 Pharmac. Ther. 63, p. 199).
Moreover, a propagated toxicity effect (“bystander” effect) has been observed when TK is used. This effect manifests itself in the destruction not only of the cells which have incorporated the TK gene, but also the neighbouring cells. The mechanism of this process may be explained in three ways: i) the formation of apoptotic vesicles which contain thymidine kinase or phosphorylated ganciclovir, originating from dead cells, followed by phagocytosis of these vesicles by the neighbouring cells, ii) transfer of the prodrug metabolized by thymidine kinase, by a process of metabolic cooperation, from the cells containing the suicide gene to the cells not containing it, and/or iii) an immune response linked to regression of the tumour (Marini et al., 1995 Gene Therapy 2, p. 655).
For a person skilled in the art, the use of the suicide gene coding for herpesvirus thymidine kinase is very amply documented. In particular, the initial in vivo studies on rats having a glioma show regression of tumours when the HSV1-TK gene is expressed and when doses of 150 mg/kg of ganciclovir are injected (K. Culver et al., 1992 Science 256, p. 1550). However, these doses are highly toxic in mice (T. Osaki et al., 1994 Cancer Research 54, p. 5258) and hence totally banned in gene therapy in man.
A number of therapeutic trials are also in progress in man, in which the TK gene is delivered to the cells by means of different vectors such as, in particular, retroviral or adenoviral vectors. In clinical trials of gene therapy in man, the doses which have to be administered are much smaller, of the order of 5 mg/kg, and for a short treatment period (14 days) (E. Oldfield et al., 1995 Human Gene Therapy 6, p. 55). With higher doses or treatments over a longer period of time, adverse side effects are, in effect, observed.
It will hence be especially advantageous to have at one's disposal a suicide gene related to the gene coding for wild-type thymidine kinase, capable of generating a variant of the wild-type TK enzyme which would be more specific and/or more active in phosphorylating ganciclovir. Advantageously, such a variant may also be employed at a significantly reduced dose compared to the dose of wild-type suicide gene and, in addition, enable the dose of substrate which is traditionally combined with it to be reduced.
The objective of the present invention is, specifically, to provide a nucleic acid sequence coding for an enzyme of the thymidine kinase type having more potent activating behaviour in relation to ganciclovir or a nucleoside analogue.
The sequence of the gene coding for the herpes simplex virus type 1 thymidine kinase enzyme has been described in the literature (see, in particular, McKnight 1980 Nucl. Acids Res. 8, p. 5949). Natural variants of it exist, leading to proteins having a comparable enzyme activity with respect to thymidine, or ganciclovir (M. Michael et al., 1995 Biochem. Biophys. Res. Commun 209, p. 966). Similarly, derivatives have been described which were obtained by directed mutagenesis at the binding site of the enzyme with the substrate. However, no precise biochemical characterization has been carried out on the pure enzymes, and no cellular test using these mutants has been published (Black et al., 1993 Biochemistry 32, p. 11618). In addition, the inducible expression of an HSV1-TK gene from which its first 45 codons have been deleted has been carried out in eukaryotic cells, but the doses of prodrug used remain comparable to those described in all the trials in the literature (B. Salomon et al., 1995 Mol. Cell. Biol. 15, p. 5322). Consequently, none of the variants described hitherto displays improved activity in relation or with respect to ganciclovir.
The present invention describes the construction of new thymidine kinase variants possessing improved enzymatic properties. The present application also describes the construction of nucleic acid sequences coding for these variants, as well as vectors containing the said sequences and permitting their administration in vivo and the in vivo production of mutants.
Unexpectedly, the Applicant has, in effect, prepared, isolated and characterized a series of particular nucleic acid sequences coding for thymidine kinase variants possessing the requisite activating behaviour, that is to say significantly improved compared to that of wild-type thymidine kinase. The Applicant has, in particular, demonstrated that new thymidine kinase variants having improved enzyme properties could be obtained, in particular, by modification of the region of the protein responsible for the binding with ATP.
Thus, a first subject of the invention lies in a nucleic acid sequence coding for a thymidine kinase, characterized in that it possesses, in relation to the wild-type sequence, at least one mutation in the region corresponding to the ATP-binding site combined with at least one mutation in the N-termi
Blanche Francis
Cameron Beatrice
Couder Michel
Crouzet Joel
Aventis Pharma S.A.
Brobeck Phleger & Harrison LLP
Monshipouri M S
Prouty Rebecca E.
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