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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/45—Transferases (2)
• • A—HUMAN NECESSITIES
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  • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

• the present invention relates to a method for the treatment of restenosis by gene therapy, comprising the administration of a recombinant adenovirus comprising a suicide gene. It also relates to particular pharmaceutical compositions allowing the local and effective administration of the recombinant viruses.
• Atherosclerosis is a complex, polygenic disease, which is defined histologically by deposits (lipid or fibro-lipid plaques) of lipids and other blood derivatives in the wall of the large arteries (aorta, coronary arteries, carotid) . These plaques, more or less calcified depending on the progress of the process, can be combined with lesions and are linked to 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 enlargement of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue.
• the atherosclerotic plaque is very clearly in relief on the wall, which gives it a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism which occur in the most affected patients. These lesions can therefore lead to very serious cardiovascular pathologies such as infarction, sudden death, heart failure, cerebrovascular accidents, etc.
• vascular smooth muscle cells LVMC
• the present invention provides an advantageous solution to this problem.
• the present invention indeed provides a particularly effective and selective method for the treatment of post-angioplasty restenosis by gene therapy.
• the method of the present invention consists mainly in administering a recombinant adenovirus comprising a suicide gene, capable of specifically sensitizing the proliferating smooth vascular muscle cells to a therapeutic agent.
• the simultaneous or subsequent administration of this therapeutic agent then leads to the selective death of the cells.
• the advantages of the present invention lie in particular in the strong capacity of the adenoviruses of the invention to infect proliferating vascular smooth muscle cells. This makes it possible to use relatively small amounts of active principle (recombinant adenovirus), and also allows 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, which gives them a very effective therapeutic action.
• the adenoviruses of the invention due to their episomal nature, have a limited persistence in proliferative cells and therefore a transient effect perfectly suited to the desired therapeutic effect.
• the applicant has also developed a method of particularly advantageous administration which makes it possible to infect with great efficiency certain target cells essential to the desired therapeutic effect
• a first object of the invention therefore relates to the use of a defective recombinant adenovirus comprising a suicide gene for the preparation of a pharmaceutical composition intended for the treatment of restenosis
• the term “suicide gene” is intended to mean any gene the expression product of which confers on the infected cell sensitivity to a therapeutic agent.
• the thymidine kinase gene the expression product of which confers on mammalian cells a sensitivity to certain therapeutic agents such as ganciclovir or acyclovir
• the cytosine deaminase gene the expression product of which confers on mammalian cells a sensitivity to 5-fluoro-cytosine (5-FC)
• the herpes simplex virus thymidine kinase is capable of phosphorylating nucleoside analogs such as acyclovir and ganciclovir
• nucleoside analogs such as acyclovir and ganciclovir
• the human herpes virus thymidine kinase gene (hTK HSV-1) is used.
• 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 exhibiting greater substrate specificity or better kinase activity. Such derivatives can 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)
• cytosine deaminase the expression product of which confers on mammalian cells a sensitivity to 5-fluorocytosine (5-FC).
• Cytosine deaminase is capable of catalyzing the deamination of cytosine to uracil
• the cells which express this gene are therefore capable of converting 5-fluoro-cytosine (5-FC) into 5-fluoro-uracil (5-FU), which is a toxic metabolite
• the sequence of this gene has been described in literature (Anderson et al Arch Microbiol 152 (1989) 115)
• thymidine kinase gene constitutes a particularly advantageous embodiment
• adenovirus serotypes For the construction of the adenoviruses according to the invention, different serotypes can be used. There are indeed many adenovirus serotypes, the structure and properties of which vary somewhat. Among these serotypes, it is however preferred to use in the context of the present invention.
• adenoviruses type 2 or 5 Ad 2 or Ad 5
• Ad 2 or Ad 5 adenoviruses of animal origin
• adenoviruses of animal origin mention may be made of adenoviruses of canine, bovine, urine origin (example Mavl, Beard et al, Virology 75 (1990) 81), ovine, porcine , avian or even simian (example.
• the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example]
• adenoviruses of human or canine or mixed origin Preferably, one uses in the context of the invention adenoviruses of human or canine or mixed origin.
• the adenoviruses according to the invention are defective, that is to say that they are unable to replicate autonomously in the target cell
• the genome of defective adenoviruses used in the context of The present invention therefore lacks at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the suicide gene
• the defective adenovirus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles
• the defective adenoviruses of the invention comprise the ITRs, a sequence allowing the packaging and the suicide gene.
• the El gene and at least one of the E2 genes, E4, L1-L5 are non-functional
• the viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the or the genes considered Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents
• a defective adenovirus is used, made non-functional by a total or partial deletion of the region E 1 and a deletion in the region E4
• the region E4 comprises 7 reading phases
• the deletion in the region E4 can be transcomplemented by the presence, in the cell line used for the multiplication of viruses, either simply from the reading phase ORF6 or from the reading phases ORF6 and ORF6 / 7
• the preferred adenoviruses according to the invention are chosen from the following - Defective recombinant adenovirus ⁇ E1, ⁇ E4 comprising a deletion of all or part of the E1 region and a deletion of all or part of the E4 region -Defective recombinant adenovirus ⁇ E1, ORF3 ", ORF6", comprising a deletion of all or part of the E1 region and nucleotides 34801-34329 and 341 15-33126 of the E4 region
• ORFl + comprising a deletion of all or part of the E1 region and a deletion of the E4 region with the exception of the ORF1 reading phase. More specifically, the deletion in the E4 region at its 5 ′ end included in the reading phase ORF7 and its 3 ′ end included in the reading phase ORF2 For example in the region covering nucleotides 33093- 35053.
• adenovirus ⁇ E1, ⁇ E4 comprising a deletion of all or part of the E1 region and a deletion covering the entire E4 region, for example among the following deletions nucleotides 32720-35835, or 33466- 35355, or 33093-35355
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573, Graham, EMBO J 3 (1984) 2917) In particular , they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, among other things, the suicide gene. Homologous recombination occurs after cotransfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used must preferably (i) be transformable by said elements, and (ii), contain sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al, J Gen Virol 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of an adenovirus Ad5 (12%).
• Strategies for the construction of vectors derived from adenoviruses have also been described in applications Nos. FR 93 05954 and FR 93 08596.
• the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• the suicide gene is placed under the control of a promoter allowing its expression in the infected cells.
• a promoter can be the promoter of the suicide gene, a heterologous promoter or a synthetic promoter.
• they may be promoters derived from eukaryotic or viral genes.
• they may be promoter sequences originating from the genome of the cell which it is desired to infect.
• they may be promoter sequences originating from the genome of a virus, including the virus used.
• these expression sequences can be modified by adding activation, regulation sequences or allowing tissue-specific expression. It may in fact be particularly advantageous to use expression signals which are active specifically or mainly in vascular smooth muscle cells, so that the suicide gene is not expressed and does not produce its effect until the virus has actually infected a vascular smooth muscle cell.
• expression signals which are active specifically or mainly in vascular smooth muscle cells, so that the suicide gene is not expressed and does not produce its effect until the virus has actually infected a vascular smooth muscle cell.
• the promoters active specifically or mainly in vascular smooth muscle cells mention may be made in particular of the promoter of ⁇ -actin of smooth muscle.
• a defective recombinant adenovirus comprising a suicide gene under the control of a viral promoter, preferably chosen from LTR-RSV or the early promoter of CMV.
• it is a promoter active specifically or mainly in vascular smooth muscle cells.
• the present invention thus provides a particularly effective method for the treatment of restenosis. Furthermore, to further increase the efficiency and specificity of the treatment, the Applicant has developed a method allowing local administration of the recombinant adenoviruses at the sites to be treated. More particularly, this method is based on the use of an angioplasty balloon coated with a hydrophilic film (for example a hydrogel) soaked with adenovirus, which can thus be applied in a precise manner on the site to be treated, and allow a local and effective release of the adenoviruses at the level of the cells to be treated.
• a hydrophilic film for example a hydrogel
• the applicant has also shown that the viruses and the method according to the invention allow an efficient and selective transfer of genes into an atheromatous artery. More particularly, the Applicant has demonstrated for the first time the ability of adenoviruses to transfer a therapeutically effective gene into an atheromatous artery. This is absolutely essential since the therapeutic efficacy of the treatment of restenosis requires demonstrating the capacity to transfer the therapeutic gene, in the right cells and with appropriate efficiency, under the pathophysiological conditions.
• the atheromatous arteries are characterized by the presence at the intima level (i) of deposits of extracellular matrix, (ii) of lipid deposits consisting essentially of foam cells of macrophagic type and (iii) of smooth muscle cells in proliferation.
• the results presented below show that, in these atheromatous arteries, the viruses according to the invention allow a lower percentage of infection but of greater specificity (taking into account the presence of cells of macrophagic type in this case , the macrophagic cells not being transduced) and being accompanied by a significant therapeutic efficacy.
• the results obtained show in particular a very selective transfer of the adenovirus into the target cells, that is to say the smooth muscle cells in proliferation. Over the entire cell population present in the atheromatous zone, more than 95% of the infected cells are vascular smooth muscle cells. Thus, the macrophagic cells present at the level of the intima are not infected at all (no infected macrophagic cell has been detected).
• the treatment according to the invention makes it possible to infect a percentage less than 1% (0.2% for example). This is much lower than the results described previously in healthy arteries or with lesions of the wall but which do not represent a pathophysiological situation of restenosis (endothelial abrasion of a healthy artery).
• the Applicant has also shown that the infection of this small percentage of cells nevertheless allows an important therapeutic effect, demonstrated in particular by measuring the diameter luminal This result is particularly surprising and implies the existence of an induced cytotoxic effect ("by-stander" effect) in vivo.
• the invention therefore describes for the first time a method allowing the selective transfer of genes into vascular smooth muscle cells.
• Another subject of the present invention relates to a pharmaceutical composition comprising a defective recombinant adenovirus and a hydrogel More specifically, the invention relates to a composition comprising a defective recombinant adenovirus comprising a suicide gene, and a hydrogel
• the hydrogel used in the context of The present invention can be prepared from any biocompatible and non-cytotoxic polymer (homo or hetero). Such polymers have, for example, been described in application WO93 / 08845. Some of them, such as in particular those obtained from ethylene oxide and / or propylene, are commercial
• the treatment method of the invention therefore advantageously consists in introducing, at the site to be treated, a composition comprising a hydrogel soaked in recombinant adenoviruses.
• the hydrogel can be deposited directly on the surface of the tissue to be treated, for example during a surgical procedure.
• the hydrogel can be introduced at the site to be treated by means of a catheter, for example a catheter. balloon, especially during angioplasty, which avoids any additional trauma due to a new intervention at the angioplasty site.
• the soaked hydrogel is introduced at the site to be treated by means of a balloon catheter, protected by a sleeve.
• the hydrogel has many advantages; it makes it possible to improve the sliding.
• the hydrogel can be used with any type of angioplasty balloon, which in particular allows the use of infusion balloons.
• the adenoviruses according to the invention are administered by means of infusion balloons, in particular of balloon catheters with channels ("channeled balloon angioplasty catheter", Mansfield Medical, Boston Scientific Corp., Watertown, MA) .
• the latter consists of a conventional balloon covered with a layer of 24 perforated channels which are perfused by an independent lumen through an additional infusion orifice.
• infusion balloons which maintain blood flow and thus reduce the risk of myocardial ischemia, when the balloon is inflated, also make it possible to locally deliver a drug at normal pressure, for a relatively long time, more than twenty minutes. , which is necessary for optimal infection.
• hydrogel-coated infusion balloon catheter It is particularly advantageous to use a hydrogel-coated infusion balloon catheter.
• Another object of the present invention relates to a pharmaceutical composition comprising a defective recombinant adenovirus and poloxamer. More specifically, the invention relates to a composition comprising a defective recombinant adenovirus comprising a suicide gene, and poloxamer.
• Poloxamer 407 is a non-toxic biocompatible polyol, it is commercially available (BASF, Parsippany, NJ).
• a treatment method of the invention therefore advantageously consists in introducing, at the site to be treated, a composition comprising poloxamer soaked in recombinant adenoviruses.
• the poloxamer can be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention.
• the poloxamer be introduced at the site to be treated by means of a catheter, for example a balloon catheter, in particular during angioplasty, which makes it possible to avoid any additional trauma due to a new intervention at the angioplasty site.
• the soaked poloxamer is introduced at the site to be treated by means of a balloon catheter, protected by a sleeve.
• the poloxamer has essentially the same advantages as the hydrogel while having a lower viscosity. It is particularly advantageous to use an infusion balloon catheter coated with poloxamer, in particular with channel balloon catheters. In this case, the advantages of both are combined, ie the possibility of keeping the balloon inflated during a period of longer time while keeping the sliding properties and site-specificity of the poloxamer easy In this case, we also obtain optimal infection efficiency
• the plasmids of the pBR322, pUC type and the phages of the Ml 3 series are of commercial origin (Bethesda Research Laboratories)
• the DNA fragments can be separated according to their size by electrophoresis in agarose gels or acrylamide, extracts with phenol or with a phenol / chloroform mixture, precipitated with ethanol then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the supplier's recommendations
• the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
• the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
• the destruction of the protruding 5 ′ ends is carried out by a gentle treatment with nuclease S 1.
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 1_3 (1985) 8749-8764] using the kit distributed by Amersham. Enzymatic amplification of DNA fragments by the so-called PCR technique
• Example 1 Construction of the vector Ad-LTR-TK carrying the TK gene under the control of the promoter of the LTR of the sorcoma virus (LTR-RSV) (FIG. 1).
• This example describes the construction of a recombinant adenovirus comprising the thymidine kinase gene of the herpes simplex virus (tk) under the control of a viral promoter (LTR-RSV promoter).
• This adenovirus was constructed by homologous recombination between the defective adenovirus Ad-dll324 and the plasmid pRSVtk carrying the " tk gene under the control of the RSV promoter (example 1.3).
• This plasmid was constructed from the plasmid pONTtk (example 1.1), by substitution of the promoter transactivable by EBNA1 by the promoter RSV (example 1.2.).
• the BglII-NcoI fragment containing the thymidine kinase (tk) gene of the he ⁇ ippo simplex virus type 1 was isolated from the plasmid pHSV-106 (marketed by Gibco BRL), repaired by the action of the klenovv fragment and then inserted into the site Smal of the plasmid pGEM7zf (+) (marketed by Promega) The Smal and BglII sites are destroyed during this step, the Ncol site is preserved
• the plasmid obtained was designated p7tkl
• This example describes the construction of a plasmid containing a chimeric promoter consisting of a sequence necessary for transactivation by the EBNA1 antigen and of the TPI promoter of the EBV virus.
• the EcoRI (7315) -SmaI (8191) fragment of the EBV virus was isolated from strain B95-8 The complete sequence of the EBV virus has been described by Baer et al (Nature 310 (1984) 207) This fragment contains the sequences necessary for transactivation by nuclear antigen 1 (EBNA1) (D Reisman & B Sugden, 1986, Molecular and Cellular Biology, vol 6 pp 3838-3846)
• This fragment was then fused to the fragment Nrul (166 241) -PstI (166 559) of EBV B95-8 (the PstI site a was digested with T4 polymerase), containing the TPI promoter
• the chimeric promoter thus obtained was then inserted into the cloning multisite of the plasmid pBlue
• the plasmid pONTtk comprises the gene for the thymidine kinase of the heres simplex virus (tk) cloned in the plasmid p7tkl, under the control of the chimeric promoter EBNA1-RE / TP1 cloned in the plasmid pONTl
• the BamHI-XhoI fragment from pONTl which contains the chimeric promoter transactivated by EBNA-1 and EBNA-2, and the Xhol-ClaI fragment from p7tkl which contains the open reading phase of tk were cloned at the BamHI sites (478) and ClaI (4550) of the plasmid pAd RSVbgal
• the plasmid pAd RSVbGal contains, in the orientation 5 ′ -> 3 ′,
• the PvuII fragment corresponding to the left end of the Ad5 adenovirus comprising the ITR sequence, the origin of replication, the packaging signals and the amplifier E 1 A, - the gene coding for b-galactosidase under the control of the RSV promoter (from Rous sarcoma virus),
• the plasmid obtained was designated pONTtk.
• plasmid pRSVtk This plasmid was constructed from the plasmid pONTtk (example 1.1.), By substitution of the promoter transactivable by EBNA1 by the promoter RSV. For this, the RSV promoter was isolated in the form of a BamHI-SalI fragment from the plasmid pAd.RSV. ⁇ gal (Stratford-Perricaudet et al., J. Clin. Invest 90 (1992) 626), then cloned with BamHI (478) and Sall (1700) sites of the plasmid pONTtk. The resulting plasmid was designated pRSVtk ( Figure 1).
• the vector pRSVtk was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trcms the functions coded by the El regions (El A and El 1B) of adenovirus.
• the adenovirus Ad-RSV-tk was obtained by homologous recombination in vivo between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pRSVtk, according to the following protocol: the plasmid pRSVtk, linearized by Xmnl, and the adenovirus Ad-dll324, linearized by the enzyme ClaI, were co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated were selected by plaque purification.
• the DNA of the recombinant adenovirus was amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer of approximately 10 ° pfu / ml.
• the viral particles are then purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
• the Ad-RSV-tk adenovirus can be stored at -80 ° C in 20% glycerol.
• Example 2 Activity of an adenovirus according to the invention in the presence of ganciclovir on smooth muscle cells in culture.
• the activity of the adenovirus containing the TK gene prepared in Example 1 was checked on in vitro models of smooth muscle cells.
• the smooth muscle cells isolated from aorta of rats and rabbits were infected. by the recombinant adenovirus Ad-RSV-tk, and incubated in the presence of ganciclovir.
• the effect of the Ad-RSV-tk / ganciclovir combination on cell viability is then confirmed by MTT colorimetric test, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, according to the technique. described by Mosman (J. Immunol.Meth 65 (1983) 55), or more precisely by cell counting
• vascular smooth muscle cells are cultured by enzymatic digestion of rabbit aorta NZW according to a method adapted from Chamley et al (Cell Tissue Res 177 • 503-522 1977) The cells are maintained in the presence of 20 % of fetal calf serum and used for all of the tests (see below) before the tenth passage
• smooth muscle cells are characterized by immunostaining using anti- ⁇ SM- antibodies actin (Sigma)
• the rabbit aorta CMLVs are incubated in the presence of the adenovirus diluted in culture medium (DMEM, 0.5% FCS) After approximately one hour at 37 ° C in a humid atmosphere, the medium containing the adenoviral solution is aspirated and replaced with culture medium (DMEM, 0.5% FCS) for a period of 18 to 24 hours
• DMEM adenovirus diluted in culture medium
• Different concentrations of ganciclovir are then added to a medium rich in SVF (10%)
• the cells are counted (100% of cell viability corresponding to cells not transduced by Ad-RSV-TK and not treated with ganciclovir)
• the Ad-RSV-TK ganciclovir combination induces a cytotoxic effect against rabbit CMLV (see Figure 3) This cytotoxicity varies depending on the concentration of ganciclovir and the multiplicity of Ad-RSV-TK infection .
• the combination Ad-RSV-TK (MOI 1000) / ganciclovir (25 ⁇ M) results in total cytolysis
• the IC50 is 0.3 ⁇ M
• the concentrations of ganciclovir active in vitro, on CML are compatible with therapeutic use.
• the immunodetection of the HSV-TK protein therefore illustrates the importance of the "bystander” effect observed on CMLVs treated with the Ad-RSV-TK / ganciclovir combination.
• This "bystander” effect can have its counterpart in vivo.
• these data strongly suggest that a limited transfer of Ad-RSV-TK adenovirus, especially in a pathological artery, can lead to a significant reduction in the neointimal mass, rich in CML and responsible for restenosis in the patient.
• This example describes the development of a particularly effective technique for percutaneous gene transfer.
• This technique is based on the use of a hydrogel balloon catheter.
• the results presented show that, quite advantageously, this technique makes it possible to effectively infect certain privileged cell populations, in particular for the treatment of restenosis
• This example was carried out using a defective recombinant adenovirus comprising the E coli ⁇ -galactosidase gene under the control of the RSV-RSV promoter and a nuclear localization signal
• the construction of this adenovirus has been described notably in Stratford-Perricaudet et al, (J Clin Invest 90 (1992) 626)
• Ad-RSV ⁇ -Gal adenovirus (1-2 10 10 pfu in 100 ⁇ l of phosphate buffer) was deposited on a balloon catheter previously coated with hydrogel (Hydroplus, Mansfield Médical, Boston Scientific Corp, Watertown, MA) (Riessen et al, Hum Gene Ther 4 (1993) 749)
• the catheter used is a balloon catheter of 2 cm in length, and with a diameter between 2.5 and 3 mm
• the catheter was then introduced, using a sleeve protector, in the right femoral artery A pressure of one atmosphere was then applied, then the catheter was directed to the external iliac artery where a pressure of 6 atmosphere was then applied to the balloon for 30 minutes
• This experiment was carried out on 27 rabbits 3 to 28 days after administration, the animals were sacrificed by overdose of pentobarbital
• the arteries of the sacrificed animals were isolated, and the expression of ⁇ -galactosidase was detected by staining in the presence of X-gal according to the technique of
• the transfer efficiency was determined on 7 rabbits treated. All these animals received 5.10 ⁇ pfu of adenovirus to infect an arterial segment 2 cm in length, so that the multiplicity of infection is similar for each animal.
• tissue samples from the liver, brain, testes, heart, lung, kidney, and skeletal muscle, as well that an arterial segment adjacent to the treated site were removed immediately after the sacrifice.
• the transfer and the expression of the gene were demonstrated by PCR (by means of probes directed against the gene coding for protein IX of the adenovirus and against the lacZ gene) and histochemistry.
• the results obtained show that none of the samples taken from the animals treated with a balloon catheter coated with hydrogel, present a coloration in the tissues tested. Similarly, no presence of virus could be detected by PCR in the samples tested, even using an optimized and very sensitive protocol of 45 amplification cycles
• Example 4 Arterial transfer of the adenovirus Ad-RSV-TK.
• the efficiency of arterial transfer was evaluated in a restenosis model in the New Zealand white rabbit.
• the rabbits were previously put on a high cholesterol diet (1%) for two weeks.
• the iliac artery was abraded using a latex balloon (4F) by five successive inflations.
• the animals were again placed on a cholesterol-lowering diet for six weeks.
• the arterial transfer was performed percutaneously, according to the procedure described above, at the level of the injured artery.
• the Ad-RSV-TK adenovirus (4.10 ⁇ pfi in 40 ⁇ l of phosphate buffer) was therefore deposited on a balloon catheter previously coated with hydrogel (Hydroplus, Mansfield Médical, Boston Scientific Co ⁇ ., Watertown, MA) (Riessen et al., Hum. Gene Ther. 4 (1993) 749).
• the catheter used is a balloon catheter 2 cm in length and 2.5 mm in diameter.
• the severity of the lesion therefore makes it possible to assess the effectiveness of a gene transfer treatment on a pathological artery.
• the lesions induced in this animal model are rich in macrophages but also rich in smooth muscle cells which constitute the therapeutic target of gene transfer.

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