Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-07-06
2002-11-05
Nguyen, Dave T. (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093200, C435S320100, C435S455000, C435S456000, C435S325000
Reexamination Certificate
active
06475996
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the use of a vector which expresses DNA polymerase &bgr; as a DNA medicinal product in the context of the molecular treatment of cancer and viral diseases such as AIDS.
Essentially two types of genetic treatment exist for these diseases, namely immunotherapy and the introduction of “suicide” genes by viral vectors into the target cells.
Genetic immunotherapy is the use of tumor-infiltrating lymphocytes (TILs), which are highly tumoricidal. When they are transformed, they convey interleukin (IL2, IL4, IL6, &ggr;-interferon) genes to the tumor and these activate the immune response locally, allowing both a limitation of the side effects on the body and an amplification of their antitumor effect.
The approach directed toward transferring a “medicinal DNA” or therapeutic gene into a tumor cell has already been proposed (Culver et al., 1994) in the context of the use of genes such as the HSV tk gene of thymidine kinase from the human herpesvirus HSV-1 (Moolten et al., 1990; Culver et al., 1992) or VZV tk if it is from the chickenpox virus (Huber et al., 1991), the bacterial genes gpt coding for xanthine/guanine phosphoribosyl-transferase (Mroz et al., 1993) or codA coding for cytosine deaminase (Mullen et al., 1992). The products of these “suicide” genes convert initially nontoxic agents, such as ganciclovir (GCV) in the case of HSV-TK, 6-methoxypurine (ara-M) with VZV-TK, 5-fluorocytosine (5-FC) with CodA and 6-thioxanthine (6-TX) with GPT, into products which are highly toxic to the cell.
Viral thymidine kinase (TK) genes have been used in particular to destroy several types of cancer cells (Moolten et al., 1990; Huber et al., 1991; Vile et al., 1993), making these cells sensitive to purine or pyrimidine analogs such as acyclovir (ACV), ganciclovir (GCV) or bromovinyldeoxyuridine (BVDU). These nucleoside analogs are converted by the viral TKs into diphosphorylated forms which are then triphosphorylated with endogenous cell enzymes before incorporation into the tumoral DNA by DNA polymerases. The incorporation of these chain terminators (absence of 3′ OH) blocks the replication of the DNA and leads to cell death.
DETAILED DESCRIPTION OF INVENTION
The present invention proposes to use a novel type of suicide gene whose function consists in facilitating the incorporation of a nucleotide analog into the DNA of the target cell after phosphorylation of the nucleoside prodrug optionally by a “standard” suicide gene.
The incorporation of nucleotides into DNA is naturally carried out in eukaryotic cells by DNA polymerases. Among the mammalian DNA polymerases, DNA polymerase &bgr; has a number of specific features.
DNA polymerase &bgr; is a polypeptide of 39 kD and is an enzyme which is highly conserved in higher eukaryotes (Kornberg et al., 1992). Its primary function is believed to be the repair of damaged DNA (Sobol et al., 1996), but it also has a role in the replication of native DNA (Jenkins et al., 1992; Sweasy et al., 1992). DNA polymerase &bgr; is expressed at a constant level during the cell cycle (Zmudzka et al., 1988) and exposure of the cell to xenobiotic agents such as radiations induces its expression (Srivastava et al., 1995; Fornace et al., 1989). It differs from the other polymerases in its small size and its unfaithful nature during DNA replication, this infidelity being linked to the absence of associated corrective exonuclease activities (Kunkel et al., 1986).
in vitro, it has been shown that DNA polymerase &bgr; incorporates ddCMP (triphosphorylated dideoxycytidine), an inhibitor of DNA synthesis, with an efficacy which is comparable to that observed for the incorporation of the natural antagonist dCMP or deoxycytidine monophosphate (Copeland et al., 1992). Very similar results have been published in relation to AZT (Copeland et al., 1992; Parker et al., 1991). in vivo, AZT-MP (azidothymidine monophosphate) is in fact incorporated into cellular DNA (Sommadossi et al., 1989) and it has been suggested that DNA polymerase &bgr; plays a role in this process (Parker et al., 1991).
Thus, a subject of the present invention is, in particular, the use of a vector which expresses DNA polymerase &bgr; or an analog of DNA polymerase &bgr; in order to incorporate nucleotide analogs with antiviral or antitumor activity into a cell's DNA, for the manufacture of a medicinal product intended for the treatment of cancer or a viral disease such as AIDS.
The expression “DNA polymerase &bgr; analog” means any nucleotide sequence which has at least 80% homology with the nucleotide sequence of mammalian DNA polymerase &bgr; and which fulfils the same functions.
The present invention thus consists in inducing an intracellular overproduction of an enzyme which is normally present in low amount in the cell, namely DNA polymerase &bgr;, in order to amplify its unfaithful and mutagenic nature and thus force the incorporation of nucleotide analogs into the DNA.
A subject of the present invention is also an expression vector comprising a gene coding for DNA polymerase &bgr; or an analog of DNA polymerase &bgr;. The vector is intended to express DNA polymerase &bgr; in tumor cells or cells infected with a virus such as the AIDS virus. It is thus advantageous to place said gene under the control of an expression system which is effective in the target cells.
According to one advantageous embodiment of the present invention, the expression vector in accordance with the present invention comprises a target sequence and/or expression sequence which is specific for tissues in which it is desired to express the DNA polymerase &bgr; (or an analog), such as tumors or tissues infected with viruses; by way of example, mention may be made of hematopoietic strain cells for the eradication of CD4
+
lymphocytes infected with the HIV virus.
The expression vector according to the present invention can be any vector commonly used in gene therapy, and particularly a viral vector derived from a virus chosen from adenoviruses, adeno-associated viruses, retroviruses (including HIV), herpesviruses, poxviruses, parvoviruses, plasmoviruses, Semliki Forest viruses and Sindbis viruses.
As indicated above, DNA polymerase &bgr; allows the incorporation of nucleotide analogs into DNA. Now, certain nucleoside analogs are known to have antiviral activity when they are phosphorylated, i.e. in the form of nucleotides. This is the case in particular for AZT and ddC. These nucleoside analogs are normally atoxic to cells. However, after phosphorylation and in the presence of DNA polymerase &bgr; (or analog), they are incorporated into DNA and block its replication. The phosphorylation in question can be carried out in particular by thymidine kinase and/or thymidilate kinase.
Thus, according to one particularly advantageous embodiment, the use of a vector which expresses DNA polymerase &bgr; (or analog) in accordance with the present invention can be potentiated by the expression, in these same target cells or tissues, of the thymidine kinase and/or thymidilate kinase gene.
The cells in which the DNA polymerase &bgr; and the thymidine kinase and/or thymidilate kinase are expressed are thus made more sensitive to nucleoside analogs such as AZT or ddC, which, in situ, are phosphorylated before being incorporated into the DNA.
The thymidine kinase and/or thymidilate kinase gene can be inserted on the same vector as the one which expresses DNA polymerase &bgr; or on another vector. In the latter case, the target cell will undergo a co-transfection in order to allow the expression of each of the genes concerned. In any case, the gene coding for thymidine kinase and/or thymidilate kinase will be placed under the control of an expression system which is effective in the target cells to be reached.
The subject of the present, invention is also cells transformed with an expression vector in accordance with the invention comprising a gene coding for DNA polymerase &bgr; (or analog) and optionally also comprising a gene coding for thymidine kinase and/or thymidilate k
Cazaux Christophe
Fons Pascal
Hoffmann Jean-Sébastien
Tiraby Gérard Jean
Centre National De La Recherche Scientifique (CNRS)
Nguyen Dave T.
Nguyen Quang
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