EP0750675A1 - REKOMBINANTE ADENOVIREN,DIE FÜR aFGF KODIEREN - Google Patents

REKOMBINANTE ADENOVIREN,DIE FÜR aFGF KODIEREN

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Publication number
EP0750675A1
EP0750675A1 EP95911370A EP95911370A EP0750675A1 EP 0750675 A1 EP0750675 A1 EP 0750675A1 EP 95911370 A EP95911370 A EP 95911370A EP 95911370 A EP95911370 A EP 95911370A EP 0750675 A1 EP0750675 A1 EP 0750675A1
Authority
EP
European Patent Office
Prior art keywords
afgf
adenovirus
sequence
cells
defective recombinant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95911370A
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English (en)
French (fr)
Inventor
Pascal Barneoud
Pia Delaere
Michel Perricaudet
Laurent Pradier
Emmanuelle Vigne
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
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Publication date
Application filed by Rhone Poulenc Rorer SA filed Critical Rhone Poulenc Rorer SA
Publication of EP0750675A1 publication Critical patent/EP0750675A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]

Definitions

  • the present invention relates to recombinant vectors of viral origin and their use for the treatment and / or prevention of neurodegenerative diseases. More particularly, it relates to recombinant adenoviruses comprising a DNA sequence coding for the acidic fibroblast growth factor (aFGF, "acidic fibroblast growth factor”).
  • aFGF acidic fibroblast growth factor
  • the invention also relates to the preparation of these vectors, the pharmaceutical compositions containing them and their therapeutic use, in particular in gene therapy.
  • Neurodegenerative diseases account for a large share of health spending in Western countries, a share that continues to increase following the aging of the population. These diseases include Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, etc.
  • the pathological signs and etiology of these diseases are very varied, but all these diseases result from a progressive loss of neuronal cells in the central nervous system, sometimes within very localized structures like the substantia nigra in Parkinson's disease.
  • the present invention describes a novel therapeutic approach, which is particularly advantageous for the treatment of these diseases. More particularly, the present invention describes vectors making it possible to directly promote the survival of the neuronal cells involved in these pathologies, by efficient and localized expression of certain trophic factors.
  • 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) . It is only recently that other neurotrophic factors have been identified, and in particular the brain-derived neurotrophic factor (BDNF) (Thoenen, Trends.in NeuroSci. 14 (1991) 165), CNTF, etc.
  • BDNF brain-derived neurotrophic factor
  • the Applicant is more particularly interested in the acid fibroblast growth factor (aFGF).
  • AFGF is a protein of approximately 134 to 155 amino acids and of molecular weight between 15 and 17.
  • AFGF fibroblast growth properties
  • aFGF undergoes retrograde transport in several neuronal populations, including the nigro-striated system (Fergusson and Johnson, J. Comp. Neurol. 313 (1991) 693), and that it could, in in vitro, the survival of midbrain dopaminergic neurons (Knhalesel et al., J. Neurosci. 10 (1990) 558).
  • the therapeutic application of aFGF encounters various obstacles.
  • the lack of bioavailability of aFGF limits any therapeutic use.
  • the aFGF delivered is active and can exert a therapeutic activity in vivo.
  • the present invention provides a particularly advantageous solution to these problems.
  • the present invention resides in fact in the development of vectors which are particularly effective for delivering in vivo and in a localized manner, therapeutically active amounts of aFGF.
  • adenoviruses can be used for the transfer of genes in vivo into the nervous system.
  • the present invention relates to new constructions, which are particularly suitable and effective for the transfer of a specific gene into the nervous system. More particularly, the present invention relates to a recombinant adenovirus comprising a DNA sequence coding for the acid fibroblast growth factor (aFGF), its preparation, and its use for the treatment and / or prevention of neurodegenerative diseases.
  • aFGF acid fibroblast growth factor
  • the Applicant has now shown that it is possible to construct recombinant adenoviruses containing a sequence coding for aFGF, to administer these recombinant adenoviruses in vivo, and that this administration allows stable and localized expression of therapeutically active amounts of aFGF in vivo. , and in particular in the nervous system, and without cytopathological effect.
  • 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 aFGF (viral promoter or tissue-specific preferably ), and methods of administration of said vector, allowing efficient expression and in appropriate tissues of aFGF.
  • the present invention thus provides viral vectors usable directly in gene therapy, particularly suitable and effective for directing the expression of aFGF in vivo.
  • the present invention thus offers a particularly advantageous new approach for the treatment and / or prevention of neurodegenerative diseases.
  • a first object of the invention therefore resides in a defective recombinant adenovirus comprising a DNA sequence coding for the acid fibroblast growth factor (aFGF) or a derivative thereof.
  • aFGF acid fibroblast growth factor
  • the invention also relates to the use of such a defective recombinant adenovirus for the preparation of a pharmaceutical composition intended for the treatment or prevention of neurodegenerative diseases.
  • the acid fibroblast growth factor (aFGF) produced in the context of the present invention may be human aFGF or an animal aFGF.
  • aFGF acid fibroblast growth factor
  • the DNA sequence encoding human aFGF has been cloned and sequenced (Jaye et al., Science 273 (1986) 541).
  • these sequences Prior to their incorporation into an adenovirus vector according to the invention, these sequences are 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 (see example 1.2.).
  • a construct coding for a derivative of aFGF in particular a derivative of human aFGF.
  • a derivative of aFGF includes for example any sequence obtained by mutation, deletion and / or addition with respect to the native sequence, and coding for a product retaining at least one of the biological properties of aFGF (trophic and / or differentiating effect).
  • 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.
  • aFGF natural variants of aFGF.
  • different forms of aFGF exist, and in particular a form comprising 154 amino acids, a form comprising 140 amino acids, and a form comprising 134 amino acids. It is understood that the term aFGF includes these different forms.
  • Other preferred derivatives are in particular the molecules in which one or more residues have been substituted, the derivatives obtained by deletion of regions having little or no involvement in 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.
  • the sequence used within the framework of the present invention also contains a secretion signal making it possible to direct the aFGF synthesized in the secretory pathways of the infected cells, so that the aFGF synthesized is released more efficiently in the extracellular compartments and can activate its receptors.
  • the secretion signal used can be a heterologous or even artificial secretion signal.
  • a functional secretion signal is used in nerve cells, such as the secretion signal from a cytokine.
  • the DNA sequence coding for the acid fibroblast growth factor 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 would be inserted one or more several introns. They can also be synthetic or semi-synthetic sequences. Particularly advantageously, a cDNA or a gDNA is used. In particular, the use of a gDNA can allow better expression in human cells.
  • the adenovirus therefore comprises a cDNA sequence coding for the acid fibroblast growth factor (aFGF).
  • the adenovirus includes a gDNA sequence encoding the acid fibroblast growth factor (aFGF).
  • the DNA sequence codes for aFGF precedes a heterologous secretory signal functional in nerve cells.
  • the sequence coding for aFGF is placed under the control of signals allowing its expression in nerve cells.
  • these are heterologous expression signals, that is to say signals different from those naturally responsible for the expression of aFGF.
  • They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences.
  • they may be promoter sequences of 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 adenovirus 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 nerve cells, so that the DNA sequence is only expressed and produces its effect when the virus has actually infected a nerve cell.
  • 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.
  • the invention relates to a defective recombinant adenovirus comprising a cDNA sequence coding for the human acid fibroblast growth factor (haFGF) under the control of the LTR-RSV promoter.
  • haFGF human acid fibroblast growth factor
  • the invention relates to a defective recombinant adenovirus comprising a gDNA sequence coding for the human acid fibroblast growth factor (haFGF) under the control of the LTR-RSV promoter.
  • LTR-RSV sorcoma virus
  • the invention relates to a defective recombinant adenovirus comprising a DNA sequence coding for the factor growth of human acid fibroblasts (haFGF) under the control of a promoter allowing majority expression in the nervous system.
  • haFGF human acid fibroblasts
  • a particularly preferred embodiment of the present invention resides in a defective recombinant adenovirus comprising the ITR sequences, a sequence permitting packaging, a DNA sequence coding for the human acid fibroblast growth factor (haFGF) or a derivative thereof 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.
  • 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 aFGF.
  • the defective virus of the invention conserves the sequences of its genome which are necessary for the packaging of the viral particles.
  • 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. They are preferably the El and E4 genes.
  • adenoviruses of 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, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service).
  • 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 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).
  • they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence coding for aFGF. 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 (ii), 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.
  • a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol.
  • the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
  • 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 and vascular dementia.
  • ALS amyotrophic lateral sclerosis
  • the present invention also relates to a pharmaceutical composition
  • 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.
  • 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 according to the case 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.
  • 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 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.
  • the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu / ml, and preferably 10 6 to 10 10 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 of 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. They 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 regards 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 aFGF.
  • 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 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.
  • 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.
  • the implants according to the invention comprise 10 ⁇ > to 10 * 0 cells. More preferably, they include 10 "at 10 °.
  • the extracellular matrix comprises a gelling compound and optionally a support allowing the anchoring of the cells.
  • 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.
  • collagen is used. It can be collagen of human, bovine or murine origin. More preferably, type I collagen is used.
  • the compositions according to the invention advantageously comprise a support allowing the anchoring of the cells.
  • 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.
  • 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.
  • PTFE polytetrafluoroethylene
  • the implants according to the invention can be implanted at different sites in the body.
  • 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. It is particularly suitable for the treatment of Alzheimer's, Parkinson's, Huntington's, and ALS diseases.
  • 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.
  • infection with the adenoviruses of the invention is very localized at the injection site, which avoids the risks of dissemination to neighboring brain structures.
  • this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.
  • Figure 1 Representation of the vector pXL2244
  • Figure 2 Representation of the vector pSh-Ad-aFGF General molecular biology techniques
  • the pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).
  • 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 gentle treatment with nuclease SI.
  • Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 1. (1985) 8749-8764] using the kit distributed by Amersham.
  • 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 pSh-Ad-aFGF.
  • This example describes the construction of a vector comprising a DNA sequence coding for aFGF preceded by a heterologous secretion sequence, under the control of a promoter constituted by the LTR of the russet sarcoma virus (LTR-RSV). .
  • the plasmid pXL2244 contains the ApoAI cDNA under the control of the LTR promoter of the RSV virus, as well as sequences of the Ad5 adenovirus (FIG. 1). It was constructed by insertion of a Clal-EcoRV fragment containing the cDNA coding for preproApoAI into the vector pLTR RSV- ⁇ gal (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626) by the same enzymes.
  • a cDNA sequence coding for aFGF was constructed as follows:
  • aFGF ⁇ A cDNA sequence coding for human aFGF comprising 134 amino acids (aFGF ⁇ ) was isolated from the plasmid pMJ26 (Jaye et al., J. Biol. Chem.
  • Plasmid pMJ26 contains a cDNA sequence encoding human aFGF preceded by the EcoRI restriction site sequence.
  • the sequence of the plasmid in this region is as follows (EcoRI site underlined and codon start in bold):
  • This sequence was then modified by insertion of a secretory sequence.
  • the introduction of this sequence is particularly advantageous since it allows the more efficient extracellular release of the aFGF synthesized by the cells infected with the vectors of the invention. More particularly, the sequence obtained above was modified by insertion, at the 5 ′ end and in the phase of the coding sequence of aFGF, of a synthetic double-stranded oligonucleotide corresponding to the secretion sequence of the interferon of human fibroblasts (Taniguchi et al., Gene 10 (1980) 11). Each strand of this oligonucleotide was synthesized chemically using a nucleic acid synthesizer, then hybridized to reconstitute the double stranded DNA.
  • the sequence used contains the sequence of the following 70 bp (SEQ ID No. 1):
  • This oligonucleotide comprises, at the 5 ′ end of the secretion sequence, an Xhol site (underlined in the sequence above).
  • This double-stranded oligonucleotide was ligated with the sequence coding for the aFGF digested with EcoRI and the nuclease mung bean as described above. A clone with the insert in the correct orientation was then isolated and verified by sequencing. The complete fragment present in this clone, containing the sequence coding for aFGF preceded by the secretion sequence, was then isolated by digestion with the enzymes Xhol and BglII (BglII site located after the stop codon), then ligated with the vector p267. (Jaye et al., EMBO J. 7 (1988) 963) previously opened with the same enzymes. The resulting vector was designated pMJ35.
  • PCR amplification steps on the plasmid pMJ35 make it possible to introduce on either side of the chimeric sequence "secretion sequence / aFGF" appropriate restriction sites. More precisely, these steps allow the incorporation of a ClaI site at the 5 ′ end (sequence underlined in single in the 5 ′ oligonucleotide, which indicates its position relative to the start codon underlined in double) and of a site Kpnl at the 3 'end (sequence underlined in single in the 3' oligonucleotide). The sites created allow the insertion of the sequence under conditions of expression in the vectors of the invention.
  • the PCR fragment prepared in Example 1.2. has been digested by enzymes
  • the capacity of the vector pSh-Ad-aFGF to express on cell culture a biologically active form of aFGF has been demonstrated by transient transfection of COS1 cells.
  • the cells (2.10 ⁇ cells per 10 cm diameter dish) were transfected (8 ⁇ g of vector) in the presence of Transfectam. After 48 hours, the cell culture supernatant was harvested. Serial dilutions (1/200 and 1/50) of this supernatant were then added to cultures of NIH 3T3 cells. The trophic effect on these cultures was observed by incorporation of radiolabelled thymidine.
  • the production of aFGF by the transfected cells has also been demonstrated by immunodetection.
  • the vector pSh-Ad-aFGF was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus.
  • the adenovirus Ad-aFGF was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pSh- Ad-aFGF, according to the protocol. following: the plasmid pSh- Ad-aFGF 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 titre of approximately 10 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-aFGF adenovirus can be stored at -80 ° C in 20% glycerol.
  • Ad-aFGF adenovirus The capacity of the Ad-aFGF adenovirus to infect cells in culture and to express in the culture medium a biologically active form of aFGF has been demonstrated by infection of human 293 and rat PCI 2 lines. The presence of active aFGF in the culture supernatant was then determined under the same conditions as in Example 2.
  • This example describes the transfer of the aFGF gene in vivo using an adenoviral vector according to the invention. It shows on an animal model of the fimbria- fornix lesion, that the vectors of the invention make it possible to induce the expression in vivo of therapeutic amounts of aFGF.
  • the septo-hippocampal pathway (fimbria- fornix) was cut in the left hemisphere. This mechanical injury was performed using a retractable surgical knife.
  • the stereotaxic coordinates used for this purpose are, relative to the bregma: AP: -1.7; ML: +1.5; V: -5.5 to -0.5.
  • the recombinant adenovirus aFGF was injected immediately after the lesion, in the median nucleus of the septum and in the dorsal part of the degenerated hippocampus
  • the adenovirus injected is the adenovirus Ad-aFGF prepared in Example 3.1., Used in purified form (3.5 10 ⁇ pfu / ⁇ l), in a phosphate saline solution (PBS).
  • PBS phosphate saline solution
  • the injections are carried out using a cannula (outside diameter 280 ⁇ m) connected to a pump.
  • the injection speed is fixed at 0.5 ⁇ l / min, after which the cannula remains in place for an additional 4 minutes before being reassembled.
  • the injection volumes into the hippocampus and the septum are 3 ⁇ l and 2 ⁇ l respectively.
  • the concentration of adenovirus injected is 3.5. 10 "pfu / ⁇ l.
  • the cannula has an angle of 9 degrees relative to vertical (in the medio-lateral direction) in order to avoid the median venous sinus.
  • the therapeutic effects of the administration of the adenovirus according to the invention have been demonstrated by three types of analysis: a histological and immunohistochemical analysis, a quantitative analysis and a behavioral analysis.
  • the mechanical lesion of the fimbria-fornix induces a loss of cholinergic neurons (revealed in immunohistology by an anti-choline acetyl transferase antibody, ChAT) in the median septum, as well as cholinergic denervation in the hippocampus (detected in histochemistry by activity acetyl choline esterase, AChE).
  • the histological analysis of the injected brains is carried out 3 weeks after the intracerebral injection of the adenovirus Ad-aFGF.
  • the animals are sacrificed, under anesthesia, by intracardiac perfusion of 4% paraformaldehyde.
  • the brain is cut in cryomat according to the coronal plane: serial coronal sections 30 ⁇ m thick are made over the entire length of the median septum and at the anterior, median and posterior levels of the hippocampus.
  • sections spaced 180 ⁇ m apart (1 section out of 6) are stained with cresyl violet (to assess neuronal density) and immunolabeled with an anti-ChAT antibody (Biochem) (to identify cholinergic neurons).
  • the immunohistochemical method is that of streptavidin-biotin peroxidase revealed by DAB.
  • sections spaced 180 ⁇ m apart are stained using the histochemical method for AChE (acetyl choline esterase) in order to detect cholinergic innervation. The sections are mounted on glass slides.
  • the number of cholinergic neurons (ChAT positive) in the middle septum is the parameter used to assess the effects of the Ad-aFGF adenovirus.
  • the counting is carried out on a sample (1 cut out of 6 over the entire length of the median septum). For each section, the positive CHAT neurons are counted separately from the 2 sides of the septum. The cumulative results for all sections are expressed by the ratio of the number of positive ChAT neurons on the injured side to the number of positive ChAT neurons on the uninjured side.
  • This example describes the transfer of the aFGF gene in vivo using an adenoviral vector according to the invention. It shows on an animal model of the nigro-striated pathway lesion, that the vectors of the invention make it possible to induce the expression in vivo of therapeutic amounts of aFGF.
  • the nigro-striated pathway was injured at the level of the median mesencephalic bundle (MFB) by injection of the toxin 6-hydroxy dopamine (6OH-DA).
  • This chemical injury by injection was unilateral, according to the following stereotaxic coordinates: AP: 0 and -1; ML: +1.6; V: -8.6 and -9 (the AP and ML coordinates are determined relative to the bregma, the V coordinate relative to the dura mater).
  • the incisor bar is fixed at the +5 mm level.
  • the recombinant adenovirus aFGF was injected immediately after the lesion into the substantia nigra and the striatum on the side of the lesion. More particularly, the adenovirus injected is the adenovirus Ad-aFGF prepared in Example 3.1., Used in purified form (3.5 10 ⁇ pfu / ⁇ l), in a phosphate saline solution (PBS).
  • PBS phosphate saline solution
  • the injections were carried out using a cannula (outside diameter 280 ⁇ m) connected to a pump.
  • the injection speed is fixed at 0.5 ⁇ l / min, after which the cannula remains in place for an additional 4 minutes before being reassembled.
  • the injection volumes into the striatum and the substantia nigra are respectively 2x3 ⁇ l and
  • the concentration of adenovirus injected is 3.5. 10 ⁇ pfu / ⁇ l.
  • the therapeutic effects of the administration of the adenovirus according to the invention have been demonstrated by three types of analysis: a histological and immunohistochemical analysis, a quantitative analysis and a behavioral analysis.
  • the chemical lesion of the nigro-striated pathway induces neuronal loss in the substantia nigra as well as dopaminergic denervation in the striatum (revealed in immunohistology by an anti-tyrosine hydroxylase, TH antibody).
  • the histological analysis of the injected brains is carried out 3 weeks after the intracerebral injection of the adenovirus Ad-aFGF under the conditions described in Example 6.
  • the serial coronal sections 30 ⁇ m thick are made in the substantia nigra and the striatum.
  • the number of dopaminergic neurons (TH positive) in the substantia nigra is the parameter for evaluating the effects of the adenovirus Ad-aFGF.
  • the counting is carried out on a sample (1 section out of 6 over the entire length of the black substance). For each section, TH positive neurons are counted separately from the 2 sides of the substantia nigra. The cumulative results for all sections are expressed in proportion: number of positive TH neurons on the injured side compared to the number of positive TH neurons on the uninjured side.
  • Ad-aFGF adenovirus The protective functional effects of an injection of Ad-aFGF adenovirus on the lesion of the nigro-striated pathway are demonstrated by analysis of the sensory-motor performance of animals during 2 behavioral tests: the rotation test induced by dopamine agonists (apomorphine, amphetamine and levodopa), and the grip test ("paw-reaching").
  • This example describes the transfer of the aFGF gene in vivo using an adenoviral vector according to the invention. It shows on an animal model of the perforating pathway lesion, that the vectors of the invention make it possible to induce the expression in vivo of therapeutic amounts of aFGF.
  • the recombinant adenovirus aFGF is injected immediately after the lesion, either at the level of the lesion, or at the level of the hippocampus and the entorhinal cortex. More particularly, the adenovirus injected is the adenovirus Ad-aFGF prepared in the example
  • the injections were carried out using a cannula (outside diameter 280 ⁇ m) connected to a pump.
  • the injection speed is fixed at 0.5 ⁇ l / min, after which the cannula remains in place for an additional 4 minutes before being reassembled.
  • the injection volumes into the hippocampus, the entorhinal cortex and the site of lesion of the perforating tract are 3 ⁇ l, 2 ⁇ l and 2 ⁇ l respectively.
  • the concentration of adenovirus injected is 3.5. 10 ⁇ pfu / ⁇ l.
  • the therapeutic effects of the administration of the adenovirus according to the invention can be demonstrated by a behavioral analysis under the conditions of Example 5.
  • NAME RHONE-POULENC RORER S.A.

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EP95911370A 1994-03-18 1995-03-02 REKOMBINANTE ADENOVIREN,DIE FÜR aFGF KODIEREN Withdrawn EP0750675A1 (de)

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FR9403190A FR2717495B1 (fr) 1994-03-18 1994-03-18 Virus recombinants, préparation et utilisation en thérapie génique.
FR9403190 1994-03-18
PCT/FR1995/000249 WO1995025803A1 (fr) 1994-03-18 1995-03-02 ADENOVIRUS RECOMBINANTS CODANT POUR LE FACTEUR DE CROISSANCE DES FIBROBLASTES ACIDES (aFGF)

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FR2792531B1 (fr) * 1999-04-26 2003-01-31 Aventis Pharma Sa Utilisation d'adenovirus recombinant defectif comprenant un acide nucleique codant pour un facteur angiogenique pour le traitement de l'hypertension arterielle pulmonaire
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US20070134204A1 (en) * 2005-12-09 2007-06-14 Henrich Cheng Method for treating nerve injury and vector construct for the same
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FR2717495A1 (fr) 1995-09-22
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IL112993A0 (en) 1995-06-29

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