EP0753067A1

EP0753067A1 - RECOMBINANT ADENOVIRUSES CODING FOR BASIC FIBROBLAST GROWTH FACTORS (bFGF)

Info

Publication number: EP0753067A1
Application number: EP95914419A
Authority: EP
Inventors: Marc Abitbol; Jacques Mallet; Michel Perricaudet; Frédéric Revah; Paul Roustan; Emmanuelle Vigne
Original assignee: Rhone Poulenc Rorer SA
Current assignee: Aventis Pharma SA
Priority date: 1994-03-29
Filing date: 1995-03-24
Publication date: 1997-01-15
Also published as: FR2718150B1; WO1995026409A1; FR2718150A1; AU2142595A; JPH09510621A; ZA952563B; CA2184755A1; US6685934B1; IL113152A0

Abstract

Recombinant adenoviruses comprising a heterologous DNA sequence coding for basic fibroblast growth factors (bFGF), preparation and uses thereof for the treatment and/or prevention of neurodegenerative diseases.

Description

RECOMBINANT ADENOVIRUSES ENCODING FOR GROWTH FACTORS

BASIC FIBROBLASTS (bFGF)

The present invention relates to recombinant adenoviruses comprising a DNA sequence encoding the growth factors of basic fibroblasts. The invention also relates to the preparation of these vectors, the pharmaceutical compositions containing them and their therapeutic use, in particular in gene therapy for the treatment and / or prevention of neurodegenerative diseases.

The increase in lifespan in Western countries is accompanied by a regular growth of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, etc. Parkinson's disease, for example, affects 4% of people over the age of 65, and Alzheimer's disease affects 10% of those over 70 and 30% of those over 80. In general, all these diseases result from a progressive loss of neuronal cells in the central nervous system, or even within very localized structures as in the case of Parkinson's disease.

In recent years, many researches have been developed with a view to understanding the mechanisms of these degenerations linked to aging in order to develop means of treatment but also means of prevention, through gene therapy.

Trophic factors are a class of molecules with properties that stimulate neuritic growth or the survival of nerve cells. The first factor with neurotrophic properties, NGF ("Nerve Growth Factor"), was characterized some forty years ago (for review, see Levi- Montalcini and Angelleti, Physiol. Rev. 48 (1968) 534) . Other neurotrophic factors have been identified, including fibroblast growth factors (FGF). The growth factors of fibrobasts (FGF) whose acid and basic forms have been identified, undergo retrograde transport in several neuronal populations including the nigro-striated system (Fergusson and Johnson, 1991J. Comp. Neurol. 313: 693) and allow in vitro the survival of dopaminergic neurons of the midbrain (Knûsel et al, 1990, J. Neurosci. 10, 558). The majority forms of human bFGF have molecular weights of 22.5 kd, 21 kd and 18 kd, respectively (Prats H., Kagahd M., Parts A.C., Klagsbrun M., Lelias J.M., Liauzun P.,

ACEMENT RULE 26) Chalon P., Tauber JP, Amalric F., Smith JA and Caput D .. Proc. Natl. Acad. Sci. USA 86, 1836-1840 (1989)).

The growth factors of human fibroblasts, in their basic form, (bFGF) are in particular recommended for preventing and / or treating hereditary or acquired retinopathies, of degenerative character. Likewise, they can restore a normal morphology of photoreceptors.

To do this, bFGFs are conventionally injected directly by the intravitreal or subretinal route at the site to be treated. However, this form of administration is not entirely satisfactory. In fact, given the tumorigenic nature of bFGFs, it is important to control their localization essentially at the level of the desired region of the organism. General injection cannot safely prevent the spread of bFGFs into the bloodstream.

The object of the present invention is in particular to propose a particularly advantageous solution to this problem.

The present invention resides in the development of vectors which are particularly effective for delivering in vivo and in a localized manner therapeutically active amounts of bFGF and therefore making it possible to overcome undesirable side effects.

The present invention is particularly advantageous for the application of bFGF as a therapeutic agent.

More specifically, the present invention aims to develop vectors which are particularly effective for delivering in vivo and in a localized manner, therapeutically active amounts of the specific gene coding for bFGF in the nervous system. In copending application No. PCT / EP93 / 02519, it has been shown that adenoviruses can be used as a vector for the transfer of a foreign gene in vivo into the nervous system and the expression of the corresponding protein.

The present invention relates more particularly to new constructions, which are particularly suitable and effective for the transfer of basic fibroblast growth factors (bFGF).

More specifically, it relates to a recombinant adenovirus comprising a DNA sequence coding for bFGF or one of its derivatives, its preparation, and its use for the treatment and / or prevention of neurodegenerative diseases. The Applicant has thus demonstrated that it is possible to construct recombinant adenoviruses containing a sequence coding for bFGF, to administer these recombinant adenoviruses in vivo, and that this administration allows stable and localized expression of therapeutically active amounts of bFGF in vivo, in the nervous system and in particular in the treatment of retinopathies and without cytopathological effect.

A first object of the invention therefore resides in a defective recombinant adenovirus comprising at least one DNA sequence coding for all or an active part of the basic fibroblast growth factor (bFGF) or one of its derivatives.

The basic fibroblast growth factor (bFGF) produced in the context of the present invention can be human bFGF or animal bFGF. It may in particular be that of the rat.

The DNA sequence coding for bFGF, used in the context of the present invention may be a cDNA, a genomic DNA (gDNA), or a hybrid construct consisting, for example, of a cDNA into which one or more introns would be inserted. They can also be synthetic or semi-synthetic sequences. Particularly advantageously, a cDNA or a gDNA is used.

According to a preferred embodiment of the invention, it is a gDNA sequence coding for bFGF. Its use can allow better expression in human cells.

Of course, prior to its incorporation into an adenovirus vector according to the invention, the DNA sequence is advantageously modified, for example by site-directed mutagenesis, in particular for the insertion of appropriate restriction sites. The sequences described in the prior art are in fact not constructed for use according to the invention, and prior adaptations may prove to be necessary, in order to obtain important expressions.

Within the meaning of the present invention, the term “bFGF derivative” means any sequence obtained by modification and coding for a product retaining at least one of the biological properties of bFGF (trophic and / or differentiating effect). By modification, we must understand any mutation, substitution, deletion, addition or modification of genetic and / or chemical nature. These modifications can be carried out by techniques known to a person skilled in the art (see general molecular biology techniques below). The derivatives within the meaning of the invention can also be obtained by hybridization from nucleic acid libraries, using as probe the native sequence or a fragment thereof.

These derivatives are in particular molecules having a greater affinity for their binding sites, sequences allowing improved expression in vivo, molecules exhibiting greater resistance to proteases, molecules having greater therapeutic efficacy or lesser side effects, or possibly new biological properties.

Among the preferred derivatives, mention may be made more particularly of natural variants, molecules in which one or more residues have been substituted, derivatives obtained by deletion of regions having little or no effect on the interaction with the binding sites considered or expressing an undesirable activity, and the derivatives comprising, relative to the native sequence, additional residues, such as for example a secretion signal and / or a junction peptide.

According to a preferred embodiment of the invention, the DNA sequence, coding for bFGF or one of its derivatives, also integrates a secretion signal making it possible to direct the bFGF synthesized in the secretory pathways of infected cells. In this way, the synthesized bFGF is advantageously released in the extracellular compartments and can thus activate its receptors. However, it can also be a heterologous or even artificial secretion signal. As far as possible, the secretion signal is advantageously the bFGF's own signal. This will be the case in particular for rat bFGF which has its own signal peptide. In contrast, human bFGF does not have it.

The DNA sequence, coding for all or part of bFGF or one of its derivatives, can also be an antisense sequence, the expression of which in the target cell makes it possible to control the expression of bFGF. Preferably, the heterologous DNA sequence comprises a gene coding for an antisense RNA capable of controlling the translation of the mRNA of bFGF. The antisense sequence may be all or only part of the DNA sequence, coding for bFGF, inserted in the reverse orientation into the vector according to the invention.

Advantageously, the sequence coding for bFGF is placed under the control of signals allowing its expression in nerve cells, including especially retinal cells. Preferably, these are heterologous expression signals, that is to say signals different from those naturally responsible for the expression of bFGF. They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences. In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus, including the adenovirus used. In this regard, mention may be made, for example, of promoters El A, MLP, CMV, LTR-RSV, etc. In addition, 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 nerve cells, so that the DNA sequence is only expressed and produces its effect when the virus has actually infected a nerve cell. In this respect, mention may be made, for example, of the promoters of neuron-specific enolase, of GFAP, and more particularly those of rodopsin and tyrosinase.

In a first particular embodiment, the invention relates to a defective recombinant adenovirus comprising a cDNA sequence coding for the human basic fibroblast growth factor (hbFGF) under the control of the LTR-RSV promoter.

The invention also relates to a defective recombinant adenovirus comprising a cDNA sequence coding for human basic fibroblast growth factor (hbFGF) under the control of the rhodopsin or tyrosinase promoter as well as to a defective recombinant adenovirus comprising a cDNA sequence encoding the growth factexir of rat basic fibroblasts (bFGF) under the control of the rhodopsin or tyrosinase promoter.

In another particular embodiment, the invention relates to a defective recombinant adenovirus comprising a gDNA sequence coding for the basic fibroblast growth factor (bFGF) under the control of the LTR-RSV promoter.

The Applicant has in fact shown that the promoter LTR of the sore coma virus (RS V) allows a lasting and significant expression of bFGF in the cells of the nervous system, in particular central. Still in a preferred embodiment, the invention relates to a defective recombinant adenovirus comprising a DNA sequence coding for the neurotrophic factor bFGF under the control of a promoter allowing predominant expression in the nervous system. A particularly preferred embodiment of the present invention resides in a defective recombinant adenovirus comprising the ITR sequences, a sequence allowing the packaging, a DNA sequence coding for the basic fibroblast growth factor (bFGF) or a derivative. of the latter under the control of a promoter allowing majority expression in the nervous system, and in which the El gene and at least one of the E2, E4, L1-L5 genes is non-functional.

The defective adenoviruses according to the invention are adenoviruses incapable of replicating autonomously in the target cell. Generally, the genome of the defective adenoviruses used in the context of the present invention is therefore devoid of 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 DNA sequence coding for bFGF.

Preferably, the defective virus of the invention conserves the sequences of its genome which are necessary for the packaging of the viral particles. Even more preferably, as indicated above, the genome of the defective recombinant virus according to the invention comprises the ITR sequences, a sequence allowing the packaging, the non-functional E1 gene and at least one of the E2, E4, L1-L5 genes. nonfunctional.

There are different serotypes of adenovirus, the structure and properties of which vary somewhat. Among these serotypes, it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application FR 93 05954). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-

800) for example]. Preferably, in the context of the invention, adenoviruses of human or canine or mixed origin are used. 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 inter alia the DNA sequence coding for bFGF. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ϋ), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of 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 an Ad5 adenovirus (12%). Strategies for constructing vectors derived from adenoviruses have also been described in applications No. FR 93 05954 and FR 93 08596 which are incorporated herein by reference.

Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.

The particularly advantageous properties of the vectors of the invention derive in particular from the construction used (defective adenovirus, deleted from certain viral regions), from the promoter used for the expression of the sequence coding for bFGF (viral promoter or tissue-specific preferably ), and methods of administration of said vector, allowing efficient expression and in appropriate tissues of bFGF. The present invention thus provides viral vectors usable directly in gene therapy, particularly suitable and effective for directing the expression of bFGF in vivo. The present invention thus offers a particularly advantageous new approach for the treatment and / or prevention of neurodegenerative diseases.

The present invention also relates to any use of an adenovirus as described above for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of neurodegenerative diseases. More particularly, it relates to any use of these adenoviruses for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, epilepsy, vascular dementia and also retinopathies.

With regard more particularly to retinopathies, it may be any retinal degeneration, central, peripheral or mixed, as well as any retinopathy, acquired or not, and in particular diabetic retinopathies.

The present invention also relates to a pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above. These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, especially for direct injection into the nervous system of the patient. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. Direct injection into the patient's nervous system is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues. The direct injection into the central nervous system of the patient is advantageously carried out by means of a stereotaxic injection device. The use of such a device makes it possible to target with great precision the injection site.

In this regard, the invention also relates to a method of treatment of neurodegenerative diseases comprising the administration to a patient of a recombinant adenovirus as defined above. More particularly, the invention relates to a method of treatment of neurodegenerative diseases comprising the stereotaxic administration of a recombinant adenovirus as defined above.

The doses of defective recombinant adenovirus used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned or even the duration of the treatment sought. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ^ and 10 ^ pfu / ml, and preferably 10 ^ to 10 ^ 0 pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, then measurement, generally after 48 hours, of the number of plaques infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

Another subject of the invention relates to any mammalian cell infected with one or more defective recombinant adenoviruses as described above. More particularly, the invention relates to any population of human cells infected with these adenoviruses. It can in particular be fibroblasts, myoblasts, hepatocytes, keratinocytes, endothelial cells, Glial cells, etc.

The cells according to the invention can come from primary cultures. These can be removed by any technique known to those skilled in the art, then cultured under conditions allowing their proliferation. As they are more particularly fibroblasts, these can be easily obtained from biopsies, for example according to the technique described by Ham [Methods Cell.Biol. 21a (1980) 255]. These cells can be used directly for infection by adenoviruses, or stored, for example by freezing, for the establishment of autologous libraries, for later use. The cells according to the invention can also be secondary cultures, obtained for example from pre-established banks.

The cultured cells are then infected with recombinant adenoviruses, to give them the capacity to produce bFGF. The infection is carried out in vitro according to techniques known to those skilled in the art. In particular, according to the type of cells used and the number of copies of virus per cell desired, a person skilled in the art can adapt the multiplicity of infection and possibly the number of infection cycles carried out. It is understood that these steps must be carried out under conditions of appropriate sterility when the cells are intended for administration in vivo. The doses of recombinant adenovirus used for the infection of the cells can be adapted by a person skilled in the art according to the aim sought. The conditions described above for administration in vivo can be applied to infection in vitro.

The present invention also relates to a pharmaceutical composition comprising a sufficient quantity of defective recombinant virus as described above.

According to a preferred embodiment of the invention, these compositions are very particularly suitable for the treatment of retinopathies. In particular, the defective recombinant virus may be in the form of an injectable solution, eye drops, ophthalmic ointment, etc. The pharmaceutically acceptable vehicles for such formulations suitable for ocular use are in particular saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), petrolatum, l petroleum jelly, etc.

In the case of eye drops or ophthalmic ointments, it is understood that the therapeutic applications may be more limited due to a weaker diffusion of the defective recombinant virus.

In their use for the treatment of ocular pathologies, the defective recombinant viruses according to the invention can be administered according to different modes, and in particular by subretinal injection, possibly preceded by a vitrectomy, or by intravitreal injection, single or multiple. The sub-retinal injection can be carried out selectively in different compartments of the eye, and in particular, the injection can be carried out at the level of the vitreous, the anterior chamber or the retrobulbar space. These different modes of injection allow targeted infection of the various tissues of the eye, and in particular, the corneal endothelium, photoreceptor cells, bipolar cells, ganglion cells or even the cells of the oculomotor muscles.

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 10 ^ to 10 ^ cells. More preferably, they include 10 ^ to 10 ^.

More particularly, in the implants of the invention, the extracellular matrix comprises a gelling compound and optionally a support allowing the anchoring of the cells.

For the preparation of the implants according to the invention, different types of gelling agents can be used. The gelling agents are used for the inclusion of cells in a matrix having the constitution of a gel, and to promote the anchoring of the cells on the support, if necessary. Different cell adhesion agents can therefore be used as gelling agents, such as in particular collagen, gelatin, glycosaminoglycans, fibronectin, lectins, etc. Preferably, one uses in the part of the present invention of collagen. It can be collagen of human, bovine or murine origin. More preferably, type I collagen is used.

As indicated above, the compositions according to the invention advantageously comprise a support allowing the anchoring of the cells. The term anchoring designates any form of biological and / or chemical and / or physical interaction resulting in the adhesion and / or fixing of the cells on the support. Furthermore, the cells can either cover the support used, or penetrate inside this support, or both. It is preferred to use within the framework of the invention a solid, non-toxic and / or biocompatible support. In particular, polytetrafluoroethylene (PTFE) fibers or a support of biological origin can be used.

The implants according to the invention can be implanted at different sites in the body. In particular, the implantation can be carried out in the peritoneal cavity, in the subcutaneous tissue (suprapubic region, iliac or inguinal fossa, etc.), in an organ, a muscle, a tumor, the central nervous system , or under a mucous membrane. The implants according to the invention are particularly advantageous in that they make it possible to control the release of the therapeutic product in the organism: This is first of all determined by the multiplicity of infection and by the number of cells implanted . Then, the release can be controlled either by the withdrawal of the implant, which definitively stops the treatment, or by the use of regulable expression systems, making it possible to induce or repress the expression of the therapeutic genes.

The present invention thus provides a very effective means for the treatment or prevention of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and ALS diseases, but also retinopathies. The adenoviral vectors according to the invention also have significant advantages, linked in particular to their very high efficiency of infection of nerve cells, making it possible to carry out infections from small volumes of viral suspension. In addition, infection with the adenoviruses of the invention is very localized at the injection site, which avoids the risks of dissemination to neighboring brain structures.

In addition, this treatment can concern both humans and any animal such as sheep, cattle, mice, pets (dogs, cats, etc.), horses, fish, etc.

The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and not limiting. JL-égenfle fles figures

Figure 1: Representation of the vector pLTR IX-hbFGF

General molecular biology techniques

Methods conventionally used in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extraction of proteins with phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Escherichia coli, etc. are well known in the art. skilled in the art and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].

The pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).

For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (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. At (1985) 8749-8764] using the kit distributed by Amersham.

Enzymatic amplification of DNA fragments by the technique called PCR [Polymerase-catalyzed hate Reaction, Saiki RK et al., Science 22û (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin El er Cetus) according to the manufacturer's specifications Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

Examples

Example 1: Construction of the vector pLTR IX-rbFGF.

This example describes the construction of a vector comprising a DNA sequence coding for rat bFGF under the control of a promoter constituted by the LTR of RSV.

1.1. Starting vector (pLTR IX): The vector pLTR IX contains in particular the left region of the Ad5 adenovirus comprising ÎTTR and the packaging site, the RSV LTR promoter, and a region of the Ad5 adenovirus ranging from the pIX gene to the EagI restriction site, allowing homologous recombination in vivo. This vector has been described by Stratford-Perricaudet et al. (J. Clin. Invest. 90 (1992) 626).

1.2. Construction of a cDNA sequence coding for rbFGF.

To allow the production of vectors according to the invention, a cDNA sequence coding for rat bFGF is constructed as follows:

An 820 base pair fragment containing the coding sequence for rat bFGF and its own signal peptide was isolated from the plasmid pUC13-bFGF by digestion with the enzyme EcoRI. This fragment was then subcloned at the corresponding site of the Bluescript vector (Pharmacia).

1.3. Construction of the vector pLTR IX -rbFGF

This example describes the construction of the vector pLTR IX-bFGF containing the sequence coding for rat bFGF (Shimasaki S., Emoto N ;, Koba A., Mercado M., Shibata F., Cooksey K., Baird A. and lin N. Complementary DNA cloning and sequencing of the rate ovarian basic fibroblasts growth f actor and tissue distribution study of its mRNA, BBRC 152, 256-263, 1988) under the control of the RSV virus LTR, as well as Ad5 adenovirus sequences allowing in vivo recombination.

The DraTI-SacII fragment was isolated by enzymatic digestion from the construct prepared in Example 1.2. This fragment contains the coding sequence for bFGF. This fragment was isolated and purified by electrophoresis on an LMP agarose gel. ("Low Melting Point"), then treated with T4 DNA polymerase to obtain blunt ends. This fragment was then inserted at the EcoRV site of the vector pLTR IX (Example 1.1.) To generate the vector pLTR IX-rbFGF. The entire nucleotide sequence of the rbFGF insert was then verified by dideoxynucleotide sequencing.

Example 2: Construction of the vector pLTR IX-hbFGF.

This example describes the construction of a vector comprising a DNA sequence coding for human bFGF under the control of a promoter constituted by the LTR of RSV.

A 1.63 kb fragment, coding for human bFGF, was isolated from the plasmid pSCT40 by enzymatic digestion at the Xhol and EcoRV sites. This fragment was then inserted at the SalI (1028) and EcoRV (1119) sites of the pLTR IX vector (example 1.1.) To generate the pLTR IX-hbFGF vector (FIG. 1). The entire nucleotide sequence of the bFGF insert was then verified by dideoxynucleotide sequencing.

Example 3. Construction of the recombinant adenoviruses containing a sequence coding for human BFGF.

The vector pLTR IX-bFGF was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions encoded by the El (El A and E1B) regions of adenovirus.

More specifically, the adenovirus Ad-bFGF was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pLTR IX-bFGF, according to the following protocol : the plasmid pLTR IX-bFGF and the adenovirus Ad-dll324, linearized by the enzyme Clal, were cotransfected into line 293 in the presence of calcium phosphate, to allow homologous recombination. 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, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer of approximately 10 × 0 pfu / ml.

The viral particles are then purified by centrifugation on a gradient.

Claims

1. Defective recombinant adenovirus comprising at least one DNA sequence coding for all or an active part of bFGF or one of its derivatives.

2. Adenovirus according to claim 1 characterized in that the DNA sequence is a cDNA sequence.

3. Adenovirus according to claim 1 characterized in that the DNA sequence is a gDNA sequence

4. Adenovirus according to claim 1 or 2 characterized in that the DNA sequence codes for human bFGF.

5. .Adenovirus according to claim 1 or 2 characterized in that the DNA sequence codes for the rat bFGF.

6. Adenovirus according to claim 1 characterized in that the DNA sequence is an antisense sequence whose expression makes it possible to control the expression of the bFGF gene.

7. Adenovirus according to claim 6 characterized in that it is a gene coding for an antisense RNA capable of controlling the translation of the mRNA of bFGF.

8. Adenovirus according to one of claims 1 to 7 characterized in that the DNA sequence is placed under the control of signals allowing its expression in nerve cells.

9. Adenovirus according to claim 8 characterized in that the expression signals are chosen from viral promoters, preferably from El A, MLP, CMV and LTR-RSV promoters.

10. Adenovirus according to claim 1 comprising a gDNA sequence coding for human bFGF under the control of an LTR-RSV promoter.

11. Adenovirus according to claim 1 comprising a DNA sequence coding for all or an active part of the basic fibroblast growth factor (bFGF) or a derivative thereof under the control of a promoter allowing a majority expression in nerve cells.

12. Adenovirus according to claim 11 characterized in that the promoter is chosen from the promoter of the specific neuron enolase and the promoter of GFAP and preferably from the promoters of rhodopsin and of tyrosinase.

13. An adenovirus according to claim 1 comprising a cDNA sequence coding for human bFGF under the control of a rhodopsin or tyrosinase promoter.

14. The adenovirus according to claim 1, comprising a cDNA sequence coding for rat bFGF under the control of a rhodopsin or tyrosinase promoter.

15. Adenovirus according to one of claims 1 to 14 characterized in that it is devoid of the regions of its genome which are necessary for its replication in the target cell.

16. Adenovirus according to claim 15 characterized in that it comprises the ITRs and a sequence allowing the encapsidation, and in which the El gene and at least one of the E2, E4, L1-L5 genes are non-functional.

17. Adenovirus according to claim 15 or 16 characterized in that it is a human adenovirus type Ad 2 or Ad 5 or canine type CAV-2.

18. Use of an adenovirus according to one of claims 1 to 17 for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of neurodegenerative diseases.

19. Use according to claim 18 for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of Parkinson's, Alzheimer's, Huntington's or ALS disease.

20. Use according to claim 19 for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of retinopathies.

21. Pharmaceutical composition comprising one or more defective recombinant adenoviruses according to one of claims 1 to 17.

22. Pharmaceutical composition according to claim 21 characterized in that it is in injectable form.

23. Pharmaceutical composition according to claim 21 characterized in that it is in the form of a form suitable for ocular use.

24. Pharmaceutical composition according to claim 23 characterized in that it comprises a sufficient amount of defective recombinant adenovirus in the form of eye drops or ophthalmic ointment suitable for ocular use.

25. Pharmaceutical composition according to one of claims 21 to 24 characterized in that it comprises between 10 ^ and 10 ^ pfu / ml, and preferably 10 ^ to 10 0 pfu / ml defective recombinant adenoviruses.

26. A mammalian cell infected with one or more defective recombinant adenoviruses according to one of claims 1 to 17.

27. Cell according to claim 26 characterized in that it is a human cell.

28. Cell according to claim 27 characterized in that it is a human cell of the retinal type, fibroblast, myoblast, hepatocyte, endothelial cell, Glial cell or keratynocyte.

29. Implant comprising infected cells according to claims 26 to 28 and an extracellular matrix.

30. Implant according to claim 29 characterized in that the extracellular matrix comprises a gelling compound preferably chosen from collagen, gelatin, glucosaminoglycans, fibronectin and lectins.

31. Implant according to claims 29 or 30 characterized in that the extracellular matrix also comprises a support allowing the anchoring of the infected cells.

32. Implant according to claim 31 characterized in that the support preferably consists of fibers of polyethylene rafluoroethylene.