AU691008B2 - Adenovirus comprising a gene coding for an no synthase - Google Patents

Description

WO 96/01902 1 ICT/FR95/00913 ADENOVIRUS CONTAINING A GENE CODING FOR AN NO

SYNTHASE

The present invention relates to a recombinant adenovirus containing a gene coding for an NO synthase.

It also relates to pharmaceutical compositions permitting the local and effective administration of these recombinant viruses, and more especially to their use for the treatment of restenosis.

Dyslipoproteinaemias are disorders of the metabolism of the lipoproteins responsible for the transport of lipids such as cholesterol and triglycerides in the blood and the peripheral fluids.

They lead to major pathologies associated, respectively, with hypercholesterolaemia or hypertriglyceridaemia, such as, in particular, atherosclerosis.

Atherosclerosis is the main cause of myocardial infarction, and is responsible for nearly half of the mortality rate in developed countries.

Physiologically, it manifests itself chiefly in the formation, in the wall of the large arteries, of plaques consisting of cholesterol, lipoproteins, foam cells and/or fibrous tissues. Probably, these atherosclerotic plaques are the outcome of an excessive inflammatory response to a chronic lesion in the endothelium of the arterial walls. This type of atheromatous plaque protrudes very markedly from the REPLACEMENT SHEET (RULE 26) I I 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.

The technique of angioplasty is the therapy most commonly employed to reestablish blood circulation through the artery obstructed in this way. However, in approximately 40 of cases, this technique fails as a result of r,.stenosis following the mechanical injury of the arterial wall. This latter pathology is mainly linked to the migration and proliferation of vascular smooth muscle cells (VSMC), which take place in the absence of the protection and/or the feedback control exercised by the endothelial cells of the intima.

A treatment proposed for countering this proliferation of VSMC cells consists in eliminating them by administering appropriate chemical or proteinaceous substances. To this end, psolaren derivatives are incorporated in proliferative cells so as to sensitize them to the action of light [March et al., Circulation, 87, 184-191, (1993)]. Similarly, some cytotoxins, consisting of a fusior protein between a fragment of plant or bacterial toxin and a growth factor, have also been used for these purposes [Pickering et al., J. Clin. Invest., 91, 724-729 (1993); Biro et al., Circ. Res., 71, 640-645, (1992); Casscells et al., Proc. Natl. Acad. Sci. USA, 89,,7159- REPLACEMENT SHEET (RULE 26) 7163, (1992)].

However, these treatments are not completely satisfactory on account, in particular, of their low specificity, their unexceptional efficacy and a considerable delay in acting.

The subject of the present invention is specifically to propose a w method, by gene therapy, which is especially effective and selective for the treatment of postangioplasty restenosis.

The approach adopted in the context of the present invention is completely different from the one mentioned above, and consists in intervening at the level of the expression of one of the factors involved the proliferation of VSMC. Preferentially, it is iA:; ted towards increasing the content of a factor which plays the part of a blocking agent with respect to this proliferation.

More specifically, the present invention is directed towards increasing the concentration of nitrogen monoxide, NO, in the vascular wall subjected to angioplasty, via the expression of NO synthase, an enzyme catalysing the synthesis of NO from arginine.

It may be accepted that the amounts of NO produced in the physiological state by the endothmlial cells correspond to the concentrations needed for the integrity, i.e. proper functioning, of the vascular wall. The mechanical destruction of the endothelium following angioplasty hence leads to a fall in NO production.

Conversely, it is established that pharmacological modulation of the NO levels is capable of interfering with the proliferation of VSMC observed during restenosis; McNamara et al. [Biochem. Biphys.

Res. Com., 193(1), 291-296, (1993)] have shown, in particular, that the administration of L-arginine, a precursor of NO, reduces intimal hyperplasia in a model of postangioplasty stenosis in rabbits.

The expression of NO synthase, according to the present invention, directly in the damaged wall, advantageously makes it possible to reestablish rapidly sufficient concentrations of NO, to improve the vascular remodelling in this way via EDRF-dependent vasodilatation and to benefit, in addition, from advantageous associated effects such as an inhibition of VSMC proliferation and a reduction in platelet aggregation. Moreover, NO is also involved in the mechanisms of angiogenesis (Ziche et al., J.Clin.Invest. 94, 2036-2044 (1994). This angiogenic activity is reflected in vitro by the capacity of NO donors such as SNP (sodium nitroprusside) to stimulate the growth and migration of endothelial cells (Ziche et al., J.Chin.Invest. 94, 2036-2044 (1994). NO might hence act as an especially important autocrine factor of neovascularization in situations of ischaemia, in particular myocardial ischaemia.

The present invention relates mainly to a II i recombinant adenovirus containing at least one gene coding, in its entirety or otherwise, for the whole or an active portion of an enzyme catalysing the synthesis of nitrogen oxide.

The advantages of the present invention lie, in particular, in the great ability 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 a very rapid effective action on the sites to be treated. The adenoviruses of the invention are also capable of expressing the genes introduced at very high levels, endowing them with a very effective therapeutic action. As a result of their episomal character, the adenoviruses of the invention have a limited persistence in the proliferative cells and hence a transient effect entirely suited to the desired therapeutic effect.

Nitrogen monoxide is known to be involved in the biological activity of EDRF (endothelium-derived relaxing factor). EDRF plays an important part in the regulation, in particular, of blood flow by inhibiting contractions of slack muscles and platelet aggregation [Radomski et al., Lancet 2, 1057-1058 (1987)]. It should be noted that NO is also present in plasma in the form of S-nitrosothiol groups bound to proteins such as albumin. The S-nitroso proteins thus generated, which are more stable than NO, are at least partially 6 responsible for EDRF activity. Beside their vasodilatory properties, the S-nitroso proteins are also potent platelet aggregation-inhibitory agents [Simon et al., Arteriosclerosis and Thrombosis, 13, 791-799 (1993)]. In the case of cardiovascular diseases, an impaired production of EDRF is associated with the pathogenicity of atherosclerosis and of pulmonary and systemic hypertension. Nitrogen monoxide is also recognized as functioning in the nervous system as a neurotransmitter, and as participating, in the immune system, in the cytotoxic activity of macrophages. More recently, nitrogen monoxide has been described as an agent capable of acting directly on VSMC proliferation since, in vitro, different NO-donor compounds inhibit the mitogenesis of VSMC in culture [Garg et al., J. Clin. Invest., 83, 1774-1777, (1989)].

The enzymes responsible for the production of nitrogen monoxide in each of these systems, hereinafter designated NO synthases, are traditionally divided into three categories termed isoform isoform II and isoform III.

S The NO synthase termed isoform I is expressed continuously and in a calcium-dependent manner in brain cells. This neuronal NO synthase is constitutive. It has recently been cloned in man [Nakane M., Schmidt H. Pollock F8rstmann U. and Murad F.; Lett. 316 175-180 (1993)].

Isoform II is expressed in mouse macrophages and induced by TNF or IL-1. The distinctive feature of inducible type NOSs is mainly their capacity to produce high concentrations of NO in response to certain cytokines. In the context of the present invention, their expression can be readily envisaged under the control of an exogenous promoter, viral or otherwise, not containing a cytokine response element. The control of nitrogen monoxide production is, in this case, effected by a promoter present in the adenovirus. Two inducible human NO synthases have, in particular, been cloned [Geller et al., Proc. Natl. Acad. Sci. USA, 3491-3495 (1993) and Charles et al., Proc. Natl. Acad.

Sci. USA, 90, 11419-11423 (1993)].

Isoform III is, for its part, expressed in endothelial cells and is calcium/calmudolin-dependent.

This so-called constitutive or endothelial isoform is normally expressed in the vascular wall and associated with EDRF activity. Human endothelial NO synthase has been cloned [Janssens et al., J. Bio. Chem. 267, 14519-14522 (1992) and Marsden et al., FEBS Lett. 307, 287-293 (1992)]. As a result of its EDRF properties, the nitrogen monoxide produced by this NO synthase plays an essential part in the relaxation of VSMC.

According to the present invention, any gene coding for the whole or only an active portion of an NO synthase or one of its derivatives, and preferably coding for one of these three isoforms of NO synthases, may be incorporated in an adenovirus for the purpose of s RA -T a 7- o.

8 its expression in vivo. NO synthase derivative is understood to denote any polypeptide obtained by modification and which retains a biological activity.

Modification is understood to mean any mutation, substitution, deletion, addition or modification of a genetic and/or chemical nature.

The NO synthase, or its derivative, produced in the context of the present invention can be a human NO synthase or an animal NO synthase. It can, in particular, be a bovine NO synthase.

The DNA sequence coding for the NO synthase, or one of its derivatives, used in the context of the present invention can be a cDNA, a genomic DNA (gDNA) or a hybrid construction consisting, for example, of a cDNA into which one or more introns might be inserted.

Synthetic or semi-synthetic sequences are a further option in this context.

It is especially advantageous to use a cDNA or a gDNA. According to a preferred embodiment of the invention, the sequence in question is a cDNA sequence coding for a human NO synthase.

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 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: MavI, Beard et al., Virology 75, 81, (1990)], 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 for replication of the said virus in the infected cell.

These regions may be either removed (in their entirety or partially), or rendered non-functional, or replaced by other sequences, and in particular by the suicide gene. Preferably, the defective adenovirus nevertheless retains the sequences of its genome which are needed for encapsidation of the viral particles.

Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence permitting encapsidation and the gene coding for an NO synthase enzyme. Still more preferably, in the genome of the adenoviruses of the invention, the El gene and at least one of the genes E2, E4, L1-L5 are non-functional. The viral gene of interest may be rendered non-functjinal by any technique known to a person skillad in the art, and in particular by total elimination, substitution, partial deletion or addition of one or more bases in the gene or genes of interest. Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example by means of genetic engineering techniques, or alternatively by treatment using mutagenic agents.

The defective recombinant adenoviruses according to the invention may be prepared by any technique known to a person skilled in the art [Levrero et al., Gene, 101, 195, (1991), EP 185,573; Graham, EMBO J. 3, 2917, (1984)]. In particular, they may be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the gene coding for the NO synthase. Homologous recombination takes place after cotransfection of the said adenovirus and said plasmid into a suitable cell line. The cell line used should preferably be transformable by the said elements, and (ii) contain the sequences capable of complementing the portion of the genome of the defective adenovirus, preferably in integrated form in order to avoid risks of recombination. As an example of a line, there may be mentioned the human embryonic kidney line 293 [Graham et al., J. Gen. Virol. 36, 59, (1977)] which contains, in particular, integrated in its genome, the left-hand portion of the genome of an adenovirus (12 Strategies of construction of vectors derived from adenoviruses have also been described in Application Nos. FR 93/05954 and FR 93/08596. Thereafter, the adenoviruses which have multiplied are recovered and purified according to standard techniques of molecular biology, as illustrated in the examples.

Advantageously, in the adenoviruses of the invention, the gene coding for an NO synthase enzyme is placed under the control of a promoter permitting its expression in infected cells. This promoter can be the one belonging to the gene, a heterologous promoter or a synthetic promoter. In particular, promoters originating from eukaryotic or viral genes may be used.

For example, it is possible to use promoter sequences originating from the genome of the cell which it is desired to infect. Similarly, the promoter sequences may originate from the genome of a virus, including that of the virus used. In this connection, the E1A, MLP, CMV, RSV LTR, and the like, promoters may, for example be mentioned. In addition, these expression sequences may be modified by adding activation or regulatory sequences or sequences permitting a tissue-specific expression. It can, in effect, be especially advantageous to use expression signals which are active specifically or predominantly in vascular I- 12 smooth muscle cells which are preferably dividing, so that the therapeutic gene is expressed and produces its effect only when the virus has actually infected a vascular smooth muscle cell.

In a particular embodiment of the invention, a defective recombinant adenovirus is used which comprises a gene coding for an NO synthase under the control of a viral promoter, preferably chosen from the RSV LTR and the CMV early promoter.

The present invention is directed towards the use of the adenoviruses according to the invention for therapeutic purposes, and more especially their application for the treatment of restenosis. This application can be extended, in particular, to pathological situations of ischaemia where the properties of NO as an angiogenic intermediary may be favourably exploited. Their use may also be envisaged for the treatment of pathologies associated with the central nervous system.

Another subject of the present invention hence relates to a pharmaceutical composition comprising at least one defective recombinant adenovirus according to the invention, where appropriate combined with a suitable excipient.

The doses of defective recombinant adenovirus used may be adapted in accordance with different parameters, and in particular in accordance with the mode of administration used, the pathology in question

I

or alternatively the desired duration of the treatment.

Generally speaking, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu/ml, and preferably 106 to 1010 pfu/ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a solution of virus, and is determined by infecting a suitable cell culture and then measuring, generally after 48 hours, the number of plaques of infected cells. The techniques of determination of the pfu titre of a viral solution are well documented in the literature.

Another subject of the invention relates to an; mammalian cell infected with one or more defective recombinant adenoviruses as described above. More especially, the invention relates to any human cell population infected with these adenoviruses. Such cells can be, in particular, endothelial cells, smooth muscle cells, neuronal cells, tumour cells, and the like.

The cells according to the invention can originate from primary cultures. They can be removed by any technique known to a person skilled in the art and then cultured under conditions permitting their proliferation. In the case of fibroblasts more especially, the latter can be readily obtained from biopsies, for example according to the technique described by Ham [Methods Cell.Biol. 21a (1980) 255].

These cells may be used directly for infection with the 14 adenoviruses, or preserved, for example by freezing, for the establishment of autologous libraries for the purpose of subsequent use. The cells according to the invention can also be secondary cultures, obtained, for example, from pre-established libraries.

The cells in culture are then infected with recombinant adenoviruses in order to endow them with the capacity to produce NO synthase. Infection is carried out in vitro according to techniques known to a person skilled in the art. In particular, depending on the cell type used and the desired number of copies of virus per cell, a person skilled in the art can adapt the multiplicity of infection. It is obvious that these steps must be performed under suitable sterility conditions when the cells are intended for administration in vivo. The doses of recombinant adenovirus used for infecting the cells may be adapted by a person skilled in the art according to the desired objective. The conditions described above for in vivo administration may be applied to in vitro infection.

Another subject of the invention relates to an implant comprising mammalian cells infected with one or more defective recombinant adenoviruses as described above, and an extracellular matrix. Preferably, the implants according to the invention comprise 105 to 1010 cells. More preferably, they comprise 106 to 108 cells.

More especially, in the implants of the invention, the extracellular matrix comprises a gelling

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compound and, optionally, a support permitting anchoring of the cells.

For the preparation of the implants according to the invention, different types of gelling agents may be employed. The gelling agents are used for inclusion of the cells in a matrix having the constitution of a gel, and in order to promote anchoring of the cells to the support, where appropriate. Different cellular adhesion agents may hence be used as gelling agents, such as, in particular, collagen, gelatin, glycosaminoglycans, fibronectin, lectins, agarose, and the like.

As stated above, the compositions according to the invention advantageously comprise a support permitting anchoring of the cells. The term anchoring denotes any form of biological and/or chemical and/or physical interaction bringing about adhesion and/or binding of the cells to the support. Moreover, the cells can either cover the support used, or penetrate inside this support, or both. It is preferable to use, in the context of the invention, a solid, non-toxic and/or biocompatible support. In particular, polytetrafluoroethylene (PTFE) fibres or a support of biological origin such as, for example, a venous graft, may be used.

The implants according to the invention may be implanted at different sites of the body. In particular, implantation may be performed in the I 16 peritoneal cavity, in the subcutaneous tissue (suprapubic region, iliac or inguinal fossae, and the like), in an organ, a muscle, a tumour or the central nervous system or alternatively under a mucosa. The implants according to the invention are especially advantageous in the sense that they enable the release of the therapeutic product in the body to be controlled: This release is determined, in the first place, by the multiplicity of infection and by the number of cells implanted. Thereafter, the release may be controlled either by withdrawal of the implant, thereby stopping the treatment permanently, or by the use of regulable expression systems, enabling the expression of the therapeutic genes to be induced or repressed.

Moreover, it has been noted that the biological properties of NO are closely controlled by oxidation reactions. In particular, the modification of NO- by the superoxide ion leads to the formation of peroxynitrite ion ONOO-, and consequently to the loss of EDRF activity. According to White et al., [Proc.

Natl. Acad. Sci. USA, 91, 1044-1048, (1 this oxidation process is involved in the development of atherosclerosis. Two complementary mechanisms are, in fact, brought into play: a reduction in the guanylate cyclase activity of the VSMC which is inherent in the inactivation of NO, and an increase in the oxidation of lipoproteins resulting from the appearance of peroxynitrite.

The combination of an antioxidant treatment with an increase in NO synthase activity may consequently be desirable in order to preserve the therapeutic benefit associated with the local production of NO. With this in mind, the administration of superoxide dismutase SOD [White et al., Proc. Natl.

Acad. Sci. USA, 91, 1044-1048, (1994)] enables the vascular environment to be kept free from the production of peroxynitrite ion. It is perfectly possible to envisage carrying out a joint administration of an adenovirus according to the invention with at least one second adenovirus containing a gene coding for a superoxide dismutase, or alternatively employing an adenovirus according to the invention containing, besides the gene coding for an NO synthase, a gene coding for a superoxide dismutase.

The adenovirus and the pharmaceutical composition according to the present invention constitute especially advantageous means for the treatment of postangioplasty restenosis.

The present invention will be described more completely by means of the examples which follow, which are to be considered to be illustrative and nonlimiting.

Legend to the figures Figure 1: Diagram of the vector pXLCMV-humNOS Figure 2: Diagrai of the vector pXLRSV-humNOS 18 Figure 3: Immunodetection of human neuronal NOS on VSMC transfected with the vector pXLRSV-humNOSn Figure 4: Validation of the vector pXLRSV-humNOSn on VSMC (detection of NOS activity by measuring the conversion of arginine to citrulline).

General techniques of molecular biology The methods traditionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in a caesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenol/chloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in a saline medium, transformation in Escherichia coli, and the like, are well known to a person skilled in the art and are amply described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, (1982); Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley Sons, New York, (1987)].

Plasmids of the pBR322 and pUC type and phages of the M13 series are of commercial origin (Bethesda Research Laboratories).

To carry out ligation, the DNA fragments may be separated according to their size by agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol/chloroform mixture, precipitated with i- I _e 19 ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the supplier's recommendations.

The filling in of 5' protruding ends may be performed with the Klenow fragment of E.coli DNA polymerase I (Biolabs) according to the supplier's specifications. The destruction of 3' protruding ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of 5' protruding ends is performed by a controlled treatment with S1 nuclease.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides may be performed accordir- it* the method developed by Taylor et al. [Nucleic Acid* 13, 8749-8764, (1985)] using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by the so-called PCR [Polymerase-catalysed Chain Reaction, Saiki R.K. et al., Science 230, 1350-1354, (1985); Mullis K.B. and Faloona Meth. Enzym. 155, 335-350, (1987)] technique may be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.

The verification of the nucleotide sequences may be performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by 1 'mersham.

c Example 1: Construction of the vector PXLCMV-humNOS carrying the gene coding for NO synthase under the control of the cytomegalovirus early promoter (Figure 1) This example describes the construction of a vector containing the cDNA coding for human endothelial NO synthase, under the control of a promoter consisting of the cytomegalovirus (CMV) early promoter, as well as a region of the Ad5 adenovirus needed for homologous recombination.

The EcoRI fragment of human endothelial NO synthase cDNA, described by Janssens et al., Biol.

Chem. 267, 14519-14522, (1992)], was recloned into pBluescript II SK (STRATAGENE) in the orientation placing the 5' bases of the cDNA on the same side as the Not I site of pBluescript. A Sal I site is created by introducing two paired oligonucleotides into the Not I site.

The Cla I-Sal I fragment of the resulting plasmid contains the human endothelial NO synthase cDNA. This fragment was inserted between the Cla I and Sal I sites into plasmid PXL2375 (PCT/FR94/00422), which comprises the sequences of the cytomegalovirus (CMV) early promoter and the Ad5 regions permitting homologous recombination. The plasmid obtained was designated PXLCMV-humNOS.

Example 2: Construction of the vector pXLRSV-humNOSn carrying human neuronal NO synthase (type I) under the

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21 control of the RSV LTR promoter (Figure 2) This example describes the construction of a vector containing the cDNA coding for human neuronal NO synthase under the control of a promoter consisting of the LTR of Rous sarcoma virus (RSV LTR), as well as a region of the Ad5 adenovirus needed for homologous recombination.

The EcoRI fragment of human neuronal NO synthase cDNA, cloned into the vector pcDNAI (Nakane et al., FEBS 316: 175-180, 1993), was recloned into the vector pIC-20H in the orientation placing the 5' end of the neuronal NOS cDNA on the same side as the Cla I site (Marsh et al., Gene 32: 481-485, 1984). The Cla I-Sal I fragment of the resulting plasmid was inserted between Cla I and Sal I sites of plasmid pXL-RSV-LPL (FR 94 06758), which comprises the sequences of the RSV LTR promoter and the Ad5 regions permitting homologous recombination. The plasmid obtained was designated pXLRSV-humNOSn.

Example 3: Check on the activity of the vectors carrying the gene coding for an NO synthase on in vitro cell culture models The activity of vectors containing the NO synthase cDNA is checked on in vitro models. Cells are transfected with the vectors containing the different isoforms of NO synthase (see Example 1, 2).

Determination of the NO synthase activity of the transfected cells makes it possible to compare the i activity of the NO synthases as well as the efficacy of the promoters used RSV LTR, CMV).

The amount of N0 2 oxidation products of NO, is determined in the culture supernatant by the Griess method or, if necessary, by more sensitive fluorometric methods [Misko et al., Analytical Biochemistry 214, 11-16, (1993)]. Moreover, intracellular NO synthase activity may be quantified directly by measuring the conversion of arginine to citrulline [Nakane et al., FEBS Letters, 316, 175-180, (1993)]. NO synthase activity is sensitive to the addition of N-monomethyl-L-arginine. Conversely, an increase in intracellular calcium is associated with a stimulation of neuronal and endothelial NO synthase activity.

Different cell models are developed. Stable clones expressing the NO synthases are selected from CHO cell lines. Transient transfections are also performed on other cell models, such as rat and rabbit vascular smooth muscle cells.

The construction pXLRSV-humNOSn (see Example 2) was validated in this way in vitro on rabbit smooth muscle primary cell cultures. Briefly, as shown in Figure 3, the presence of NOS was monitored by immunofluorescence using antibodies specific for neuronal NOS (anti-NOS B220-1, Interchim). In parallel, NO synthase activity was measured by the method of conversion of arginine to citrulline (see above). Thus, Ir 23 NOS activity reflected by the presence of tritiated citrulline is detected in rabbit smooth muscle cells transfected with plasmid pXLRSV-humNOSn. This NOS activity is not to be found in cells transfected with plasmid pXLRSV-humNOSn. This NOS activity is not to be found in cells transfected with a control plasmid expressing the b-galactosidase gene of E.Coli under the control of the RSV LTR promoter (see Figure 4).

Moreover, in order to verify the functionality of the vectors and to supplement the initial data of Garg and Hassid Clin. Invest., 83, 1774-1777, (1989)], the effect of the expression of NO synthase is also measured on the proliferation of vascular smooth muscle cells. We have demonstrated previously in our rabbit VSMC cell model that NO donors such as SNP or hydroxylamine reduce the proliferation of rabbit VSMC incubated in the presence of optimal concentrations of foetal calf serum. At high concentration, the effect of NO donors is also associated with a cell death which has similarities to apoptosis. Thus, we have detected a characteristic fragmentation of the DNA into oligomers of 180 base pairs.

Example 4: Construction of a recombinant adenovirus containing a sequence coding for an NO synthase The plasmid described in Example 1 is linearized and cotransfected for homologous recombination with the defective adenoviral vector into I I 24 helper cells (line 293) supplying in trans the functions encoded by the adenovirus El (E1A and E11B) regions.

More specifically, the adenovirus Ad-CMV-humNOS was obtained by homologous recombination in vivo between the mutant adenovirus Ad-d11324 [Thimmappaya et al., Cell 31, 543, (1982)] and plasmid pXL-CMV humNOS according to the following protocol: the linearized plasmid pXL-CMV humNOS and the adenovirus Ad-d11324, linearized with the enzyme Clal, were cotransfected into line 293 in the presence of calcium phosphate, to permit homologous recombination. The recombinant adenovirus Ad-RSV-NOSn containing a sequence coding for a human neuronal NO synthase was constructed in a similar manner, plasmid pXLRSV-humNOSn having been linearized with the enzyme Fsp I. The recombinant adenoviruses thus generated were selected by plaque purification. After isolation, the DNA of the recombinant adenovirus was amplified in the cell line 293, leading to a culture supernatant containing the unpurified defective recombinant adenovirus having a titre of approximately 1010 pfu/ml.

The viral particles are then purified by centrifugation on a caesium chloride gradient according to known techniques (see, in particular, Graham et al., Virology 52, 456, 1973). The adenoviruses Ad-CMV-humNOS and Ad-RSV-humNOSn may be stored at -80 0 C in 20 glycerol.

POPUnMULR\29306.95.SP 4-9/ Example 5: In vitro validation of the recomnb:,ant adenovirus Ad-CMV.NOS containing a sequence coding for an NO synthase Vascular smooth muscle cells are first infected with the adenovirus [Lee et al., Circulation Research 73, 797-807, (1993)]. The amount of NO generated is then measured, together with the effect of the production of NO on cell proliferation, by the teclmiques described in Example 3. Cell proliferation is determined by measuring incorporation of BrdU into the DNA (Cell Proliferation Assay RPN210, Amersham).

Throughout this specification and the claims which follow, unless the context requires otherwise, 10 the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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Claims (24)

1. Defective recombinant adenovirus containing at least one gene coding, in its entirety or partially, for the whole or an active portion of an NO synthase or one of its derivatives.

2. Adenovirus according to claim 1, characterized in that the NO synthase is an endothelial NO synthase.

3. Adenovirus according to claim 1, characterized in that the NO synthase is a neuronal NO synthase.

4. Adenovirus according to claim 1, characterized in that the NO synthase is an inducible NO synthase.

5. Adenovirus according to one of claims 1 to 4, characterized in that the gene is a cDNA sequence.

6. Adenovirus according to one of claims 1 to 4, characterized in that the gene is a gDNA sequence.

7. Adenovirus according to one of claims 1 to 6, characterized in that the DNA sequence codes for a bovine NO synthase.

8. Adenovirus according to one of claims 1 to 6, characterized in that the DNA sequence codes for a human NO synthase.

9. Adencvirus according to one of the. preceding claims, characterized in that the gene is placed under the control of a promoter permitting its expression in infected cells.

Adenovirus according to claim 9, characterized in that the promoter is chosen from viral promoters, preferably the RSV LTR and CMV promoters.

11. Adenovirus according to one of the preceding claims, characterized in that the adenovirus comprises, besides the gene, the ITRs and a sequence permitting encapsidation.

12. Adenovirus acc-rding to claim 11, characterized in that the adenovirus comprises, besides the gene, the ITRs and a sequence permitting encapsidation, and in which the El gene and at least one of the genes E2, E4, L1-L5 are non-functional.

13. Adenovirus according to one of the preceding claims, characterized in that the adenovirus is an adenovirus of human origin, preferably chosen from the serotypes Ad2 and

14. Adenovirus according to one of claims 1 to 12, characterized in that the adenovirus is an adenovirus of animal origin, preferably chosen from canine adenoviruses.

Use of an adenovirus according to one of the preceding claims, for the preparation of a pharmaceutical composition intended for the treatment of restenosis.

16. Pharmaceutical composition, characterized in that it comprises as active principle at least one adenovirus as defined in claims 1 to 14, combined with a suitable excipient.

17. Pharmaceutical composition according to claim 16, characterized in that it comprises between and 1014 pfu/ml, and preferably 106 to 1010 pfu/ml, of defective recombinant adenovituses.

18. Mammalian cell infected with one or more defective recombinant adenoviruses according to one of claims 1 to 14.

19. Cell according to claim 18, characterized in that it is a human cell.

Cell according to claim 19, characterized in that it is a hunan cell of the endothelial, neuronal, smooth muscle or tumour type.

21. Implant comprising infected cells according to claims 18 to 20 and an extracellular matrix.

22. Implant according to claim 21, characterized in that the extracellular matrix comprises a gelling compound preferably chosen from collagen, gelatin, glucosaminoglycans, fibronectin, agarose and lectins.

23. Implant according to claims 21 or 22, characterized in that the extracellular matrix also comprises a support permitting anchoring of the infected cells.

24. Implant according to claim 23, characterized in that the support preferably consists P'OPaRU LR 9306 95 24/2/98 -29- ofpolytetrafluoroethylene fibres. A defective recombinant adenovirus according to any one of claims 1-14, or the use of an adenovirus according to claim 15, or a pharmaceutical composition according to claim 16 or 17, or a mammalian cell according to any one of claims 18 to 20, or an implant according to any one of claims 21 to 24, substantially as hereinbefore described with reference to the figures and/or Examples. DATED this 24th day of February, 1998 Rhone-Poulenc Rorer S.A. by their Patent Attorneys DAVIES COLLISON CAVE 9. o 9 9o