Gene therapy for restenosis using an adenoviral vector

Details Gene therapy for restenosis using an adenoviral vector Gene therapy for restenosis using an adenoviral vector

1996-06-20

2002-06-25

Priebe, Scott D. (Department: 1632)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C514S04400A, C435S320100, C435S455000, C435S456000

Reexamination Certificate

active

06410011

ABSTRACT:

The present invention relates to a method for treatment of restenosis by gene therapy, comprising the administration of a recombinant adenovirus containing a suicide gene. It also relates to particular pharmaceutical compositions permitting the local and effective administration of recombinant viruses.
Atherosclerosis is a complex, polygenic disease which is defined in histological terms by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in the wall of the large arteries (aorta, coronary arteries, carotid). These plaques, which are more or less calcified according to the degree of progression of the process, may be coupled with lesions and are associated with the accumulation in the arteries of fatty deposits consisting essentially of cholesterol esters. These plaques are accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism, which occur in those patients who are most affected. These lesions can hence lead to very serious cardiovascular pathologies such as infarction, sudden death, cardiac insufficiency, stroke, and the like.
Since 1977, the technique of angioplasty has been developed to permit a non-surgical intervention in respect of the atherosclerosis plaque. However, the treatment of an atherosclerotic lesion by angioplasty results very frequently (up to 50% of cases in some studies) in a restenosis following mechanical injury of the arterial wall. A key event in this mechanism is the proliferation and migration of vascular smooth muscle cells (VSMC) from the media to the intima, in particular as a result of the absence of protection and/or feedback control exercised by the endothelial cells of the intima.
The treatment of restenosis by administration of chemical or proteinaceous substances capable of killing vascular smooth muscle cells has been proposed in the prior art. Thus, psolaren derivatives, incorporated by proliferative cells and then sensitizing these cells to the action of light, have been used (March et al., 1993, circulation, 87:184-191). Similarly, some cytotoxins consisting of a fusion protein between a plant or bacterial toxin fragment and a growth factor have also been used (Pickering et al., J. Clin. Invest., 1993, 91:724-729; Biro et al., 1992, Circ. Res., 71:640-645; Casscells et al., Proc. Natl. Acad. Sci. USA, 1992, 89:7159-7163). However, these treatments have many drawbacks, such as their low specificity, their indifferent efficacy, a considerable delay in acting and a potential toxicity.
The present invention offers an advantageous approach to this problem. The present invention provides, in effect, an especially effective and selective method for the treatment of postangioplasty restenosis by gene therapy. The method of the present invention consists mainly in administering a recombinant adenovirus containing a suicide gene, capable of specifically sensitizing proliferating vascular smooth muscle cells to a therapeutic agent. Simultaneous or subsequent administration of this therapeutic agent then brings about the selective death of the sensitized cells.
The advantages of the present invention lie, in particular, in the high capacity of the adenoviruses of the invention to infect proliferating vascular smooth muscle cells. This enables relatively small amounts of active principle (recombinant adenovirus) to be used, and also permits an effective and very rapid action on the sites to be treated. The adenoviruses of the invention are also capable of expressing at very high levels the suicide genes introduced, thereby endowing them with a very effective therapeutic action. Furthermore, on account of their episomal character, the adenoviruses of the invention have a limited persistence in proliferative cells, and hence a transient effect entirely suited to the desired therapeutic effect. Lastly, the Applicant has also developed an especially advantageous method of administration, which enables certain target cells essential to the desired therapeutic effect to be infected with great efficacy.
A first object of the invention hence relates to the use of a defective recombinant adenovirus containing a suicide gene for the preparation of a pharmaceutical composition intended for the treatment of restenosis.
As mentioned above, for the purposes of the present invention, suicide gene is understood to mean any gene whose expression product endows the infected cell with a sensitivity to a therapeutic agent. As an example, there may be mentioned the thymidine kinase gene, whose expression product endows mammalian cells with a sensitivity to certain therapeutic agents such as ganciclovir or acyclovir, or the cytosine deaminase gene, whose expression product endows mammalian cells with a sensitivity to 5-fluorocytosine (5-FC).
Herpes simplex virus thymidine kinase is capable of phosphorylating nucleoside analogues such as acyclovir and ganciclovir. These modified molecules may be incorporated in a DNA chain undergoing elongation, which results in the cessation of DNA synthesis and brings about the death of the cell (F. L. Moolten, Cancer Res. 46 (1986) 5276). This strategy thus enables cells expressing the TK gene to be specifically eliminated. Furthermore, since the DNA synthesis is the target of the toxicity, only cells undergoing division are affected.
More preferably, the human herpesvirus thymidine kinase (hHSV-1 TK) gene is used in the context of the present invention. The sequence of this gene has been described in the literature (see, in particular, McKnight et al., Nucleic Acid. Res. 8 (1980) 5931). It is also possible to use derivatives of this sequence displaying greater substrate specificity or better kinase activity. Such derivatives may, in particular, be obtained by mutagenesis at the binding site, as described previously (Balasubramaniam et al., J. Gen. Virol. 71 (1990) 2979; Munir et al., JBC 267 (1992) 6584).
It is also possible to use the cytosine deaminase gene, whose expression product endows mammalian cells with a sensitivity to 5-fluorocytosine (5-FC). Cytosine deaminase is capable of catalyzing the deamination of cytosine to uracil. Cells which express this gene are hence capable of converting 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which is a toxic metabolite. The sequence of this gene has been described in the literature (Anderson et al., Arch. Microbiol. 152 (1989) 115).
More generally, any gene capable of endowing infected cells with a sensitivity to a therapeutic agent may be used in the context of the present invention. The thymidine kinase gene constitutes an especially advantageous embodiment.
For the construction of the adenoviruses according to the invention, different serotypes may be used. There are, in effect, many serotypes of adenovirus, whose structure and properties vary somewhat. Among these serotypes, it is preferable however to use, in the context of the present invention, human adenoviruses type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see Application FR 93/05954). Among adenoviruses of animal origin which are usable in the context of the present invention, adenoviruses of canine, bovine, murine (for example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or alternatively simian (for example: SAV) origin may be mentioned. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan or A26/61 (ATCC VR-800) strain, for example]. It is preferable to use adenoviruses of human or canine or mixed origin in the context of the invention.
As stated above, the adenoviruses according to the invention are defective, that is to say they are incapable of replicating autonomously in the target cell. Generally, the genome of the defective adenoviruses used the context of the present invention hence lacks at least the sequences needed f

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