EP0774008A1 - Adenovirus comprenant un gene codant pour une superoxyde dismutase - Google Patents

Adenovirus comprenant un gene codant pour une superoxyde dismutase

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Publication number
EP0774008A1
EP0774008A1 EP95924371A EP95924371A EP0774008A1 EP 0774008 A1 EP0774008 A1 EP 0774008A1 EP 95924371 A EP95924371 A EP 95924371A EP 95924371 A EP95924371 A EP 95924371A EP 0774008 A1 EP0774008 A1 EP 0774008A1
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EP
European Patent Office
Prior art keywords
adenovirus
superoxide dismutase
dna sequence
human
cell
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.)
Ceased
Application number
EP95924371A
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German (de)
English (en)
French (fr)
Inventor
Martine Barkats
Jacques Mallet
Michel Perricaudet
Frédéric Revah
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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Filing date
Publication date
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Publication of EP0774008A1 publication Critical patent/EP0774008A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • 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
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • 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
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • 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
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to recombinant adenoviruses comprising a DNA sequence encoding a superoxide dismutase and its uses in gene therapy.
  • the latter formed from superoxide and hydrogen peroxide according to the Haberiss reaction, is the most reactive free radical. Due to the presence of a free electron in their outer layer, these radicals are highly reactive. This reactivity can be detrimental to important biological molecules such as DNA, essential cellular proteins and membrane lipids. In addition, these free radicals can initiate chain reactions such as lipid peroxidation which can alter the integrity of cells and cause their destruction.
  • the object of the present invention is precisely to propose a means to compensate for this type of deficiency in the natural regulatory mechanisms, and this by intervening more particularly in the activity of superoxide dismutase.
  • the main function of this enzyme in mammals, is to destroy the superoxide radicals which are generated during the various biological redox reactions. This enzyme is therefore particularly important since it provides a defense against oxygen toxicities and any damage that can be caused to cells by carcinogenic hydrocarbons.
  • Superoxide dismutase is actually a variety of different enzymes found in most living things.
  • SOD single-chain diotes
  • MnSOD dependent on manganese produced at the level of mitrochondria in eukaryotes and prokaryotes (Creagan R. and al. Humangenetic 20 203-209 1973)
  • FeSOD iron-dependent, cytosolic and present mainly in prokaryotes (Hendrickson D et al. Genomics 8, 736-738 1990).
  • the intracellular CuZn superoxide dismutase constitutes approximately 85 to 90% of the total cellular SOD activity. It is a dimeric protein, apparently composed of two identical subunits, linked non-covalently, each of them having a molecular weight of the order of 16,000 to 19,000 (Lieman-Hurwitz J. and al; Biochem Int. 3: 107-115, 1981).
  • the locus for human cytoplasmic superoxide dismutase is found on chromosome 21. (Tan Y.H. et al. J. Exp. Med. 137: 317-330, 1973).
  • the endogenous CuZn superoxide dismutase is present in tissues in limited quantities and when large quantities of superoxide anions are produced, its concentration is clearly insufficient.
  • SOD is an enzyme of protection normally ensuring a minimum level of superoxide radicals within the cell. To do this, it catalyzes the interaction of free radicals in order to oxidize one and reduce the other, a disproportionation reaction, which leads to the formation of hydrogen peroxide.
  • the superoxide radical is not particularly toxic. The danger comes from its ability to interact with hydrogen peroxide to generate singlet oxygen and hydroxyl radicals, two highly reactive and extremely toxic forms of oxygen. An increased amount of superoxide dismutase can therefore lead to increased production of hydrogen peroxide with the consequences explained above. This phenomenon is expressed physiologically in particular by an increase in lipoperoxidation with a decrease in the content of unsaturated fatty acid in cell membranes and the main consequence is a disturbance of membrane functions.
  • the present invention resides in the development of vectors which are particularly effective for delivering in vivo and in a localized manner, therapeutically active quantities of the specific gene coding for a superoxide dismutase or one of its derivatives.
  • 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 controlling the expression of superoxide dismutase.
  • adenovirus comprising a DNA sequence suitable for controlling the expression of superoxide dismutase, its preparation and its use for therapeutic treatments and / or the prevention of various pathologies.
  • the Applicant has thus demonstrated that it is possible to construct recombinant adenoviruses containing a sequence coding for a superoxide dismutase, to administer these recombinant adenoviruses in vivo, and that this administration allows stable and localized expression of therapeutically active amounts of superoxide dismutase in vivo.
  • 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 a superoxide dismutase or one of its derivatives.
  • the superoxide dismutase produced in the context of the present invention may be a human or animal superoxide dismutase. According to a preferred embodiment of the invention, it is one of the three forms of the human superoxide dismutase previously described, CuZnSOD (SOD ⁇ ), MnSOD (SOD2) and extracellular SOD (SOD3). More preferably, the DNA sequence integrated into the adenovirus according to the invention codes for all or an active part of the human intracellular CuZn superoxide dismutase, hSODl, or one of its derivatives.
  • the DNA sequence coding for superoxide dismutase 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. H can also be synthetic or semi-synthetic sequences. Particularly advantageously, a cDNA or a gDNA is used.
  • gDNA genomic DNA sequence
  • the DNA sequence prior to its incorporation into an adenovirus vector according to the invention, can be advantageously modified, for example by site-directed mutagenesis, in particular for the insertion of appropriate restriction sites.
  • 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.
  • the term “superoxide dismutase derivative” means any sequence obtained by modification and coding for a product retaining at least one of the biological properties of superoxide dismutase.
  • modification one 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.
  • superoxide dismutase derivative is also understood to cover, in the context of the present invention, the so-called dominant negative mutants of superoxide dismutase. More specifically, in this case, the cloned gene is altered so that it codes for a mutant product capable of inhibiting the cellular activity of wild superoxide dismutase. This type of derivative is particularly advantageous when it is sought, for example, to suppress a natural overexpression of superoxide dismutase.
  • the DNA sequence, coding for all or part of superoxide dismutase 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 superoxide dismutase.
  • the heterologous DNA sequence comprises a gene coding for an antisense RNA capable of controlling the translation of the corresponding mRNA.
  • the antisense sequence may be all or only part of the DNA sequence, coding for superoxide dismutase, inserted in the reverse orientation into the vector according to the invention
  • the DNA sequence, coding for superoxide dismutase or one of its derivatives also includes a secretion signal for directing the synthesized superoxide dismutase into the secretion pathways of infected cells.
  • a secretion signal for directing the synthesized superoxide dismutase into the secretion pathways of infected cells.
  • the superoxide dismutase synthesized is advantageously released in the extracellular compartments.
  • it can also be a heterologous or even artificial secretion signal.
  • the secretion signal can advantageously be the own SOD3 signal.
  • the sequence coding for superoxide dismutase is placed under the control of signals allowing its expression in the target cells.
  • these are heterologous expression signals, that is to say signals different from those naturally responsible for the expression of superoxide dismutase.
  • 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 predominantly in the target cells, so that the DNA sequence is not expressed and does not produce its effect until the virus has actually infected a target cell.
  • the invention relates to a defective recombinant adenovirus comprising a cDNA sequence coding for human intracellular CuZn superoxide dismutase under the control of the LTR-RSV promoter.
  • the invention relates to a defective recombinant adenovirus comprising a gDNA sequence coding for human intracellular CuZn superoxide dismutase under the control of the LTR-RSV promoter.
  • a particularly preferred embodiment of the present invention resides in a defective recombinant adenovirus comprising the ITR sequences, a sequence allowing encapsidation, a DNA sequence coding for the intracellular human CuZn superoxide dismutase or a derivative thereof. under control a promoter allowing a majority expression in the target tissues and in which the E1 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 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 superoxide dismutase.
  • 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.
  • serotypes of adenoviruses there are different serotypes of adenoviruses, 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).
  • 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).
  • 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 a person 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 superoxide dismutase. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
  • the cell line used should preferably (i) be transformable by said elements, and (ii), include 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. 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 No. FR 93 05954 and FR 93 08596 which are incorporated into the present application by reference.
  • the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology.
  • 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 superoxide dismutase (viral promoter or tissue-specific of preferably), and methods of administration of said vector, allowing efficient expression and in appropriate tissues of superoxide dismutase.
  • 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 the pathologies mentioned above. 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 neurodegenerative diseases such as for example Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) ), and trisomy 21. They can also be advantageously used in the treatment of atherosclerosis, cardiovascular diseases, cirrhosis of the liver, diabetes, cataract formation and the aging process.
  • ALS amyotrophic lateral sclerosis
  • an adenovirus according to the invention with at least one second adenovirus comprising a gene coding for catalase (P. Amstad et al. Biochemistry 1991, 30, 9305-9313) , another important enzyme in the regulation of free radical production.
  • catalase P. Amstad et al. Biochemistry 1991, 30, 9305-9313
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one or more defective recombinant adenoviruses such as described above, associated, where appropriate, with a recombinant adenovirus comprising a gene coding for catalase.
  • compositions of the invention 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 patient.
  • a pharmaceutically acceptable vehicle for an injectable formulation especially for direct injection into the patient.
  • injectable formulation especially for direct injection into 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.
  • 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.
  • the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ⁇ and 10 14 pfu / ml, and preferably 10 ⁇ to 10 * 0 pfu ml.
  • the term pfu (“plaque fc ming unit”) corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and then measures, generally after 48 hours, 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.
  • ⁇ 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 CelLBiol. 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 superoxide dismutase.
  • 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. 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 in vivo administration 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 ⁇ to 10 °.
  • the extracellular matrix comprises a gelling compound and optionally a support allowing the anchoring of the cells.
  • gelling agents can be used for the preparation of the implants according to the invention.
  • 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, agarose, etc.
  • 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.
  • cells can 11
  • 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 viral vectors usable directly in gene therapy, particularly suitable and effective for directing the expression of superoxide dismutase in vivo.
  • the present invention thus offers a particularly advantageous new approach for the treatment and / or prevention of numerous pathologies such as those mentioned above.
  • the adenoviral vectors according to the invention also have significant advantages, linked in particular to their very high infection efficiency of the target 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 diffusion to neighboring brain structures. This treatment can concern man as well as any animal such as sheep, cattle, mice, pets (dogs, cats, etc.), horses, fish, etc.
  • Rgure 1 Enzymatic activity of human CuZnSOD (hSOD-1) on NS2OY cells infected with a recombinant adenovirus encoding hSOD-1 (0 to 500 pfu / cell).
  • 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. 12. (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 Protocol for constructing the vectors pLTRIX-hSODl, pLTRIX-hSODl Gly37, and pLTRIX-hSODlAsnl39
  • vectors contain the sequences coding for human SOD1 wild type or mutated under the control of the LTR of the RSV virus, as well as sequences of the adenovirus allowing recombination in vivo.
  • cDNAs encoding the different types of SOD used are described in Rosen et al., Nature, vol. 362. 52-62, and Deng et al., Science, vol. 261. 1047-1051.
  • Each cDNA is inserted into a Bluescript plasmid (Stratagene) between the PstI and HindIII sites.
  • a polyadenylation sequence originating from SV40 has previously been introduced into the Xhol site of the same plasmid.
  • These plasmids are SK-hSOD-PolyA, SK-hSODgly-PolyA and SK-hSODasn-PolyA.
  • the vectors pLTRIX-hSODl, pLTRIX-hSODlgly and pLTRIX-hSODl are obtained by introducing into the EcoRV site of the plasmid pLTRIX an insert obtained by cleavage of SK-hSOD-PolyA, SK-hSODgly-PolyA and SK-hSODasn-PolyA by Kpn Sacl (Kpnl and Sacl ends made blunt).
  • Example 2 Construction of recombinant adenoviruses containing a sequence coding for human intracellular CuZn superoxide dismutase.
  • the pLTR IX-hSOD1 vector is linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the El (El A and E1B) regions of adenovirus.
  • the adenovirus Ad-hSOD1 was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Eye 31 (1982) 543) and the vector pLTR IX-hSODl, according to the following protocol : the plasmid pLTR IX-hSODl and the adenovirus Ad-dll324, linearized by the enzyme Clal, 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 ⁇ 0 pfu / ml.
  • Example 3 Control of the in vitro expression of hSOD-1.
  • NP-40 extract is produced from 500,000 NS2OY cells (mouse neuroblastomas), loaded onto non-denaturing acrylamide gel, and electrophoresis is carried out for 3 hours at 100 V.
  • the superoxide dismutase is localized by soaking the gel in a solution of tetrazolium nitroblue (NBT) and riboflavin and then in a solution of tetramethylethylenediamine (TEMED).
  • NBT tetrazolium nitroblue
  • TEMED tetramethylethylenediamine
  • the gel is then illuminated, and thus becomes uniformly blue except at the positions containing the superoxide dismutase, (the riboflavin reduced in the presence of TEMED generates superoxide radicals after reoxidation in air.
  • the superoxide radicals produced will reduce the colorless NBT to a compound blue (formazan).
  • SOD by neutralizing the superoxide radicals produced, will inhibit the color reaction, and will appear as a colorless spot).

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EP95924371A 1994-06-29 1995-06-27 Adenovirus comprenant un gene codant pour une superoxyde dismutase Ceased EP0774008A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9408029A FR2721943B1 (fr) 1994-06-29 1994-06-29 Adenovirus comprenant un gene codant pour une superoxyde dismutase
FR9408029 1994-06-29
PCT/FR1995/000854 WO1996000790A1 (fr) 1994-06-29 1995-06-27 Adenovirus comprenant un gene codant pour une superoxyde dismutase

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EP (1) EP0774008A1 (fi)
JP (1) JPH10505485A (fi)
AU (1) AU2890595A (fi)
CA (1) CA2190883A1 (fi)
FI (1) FI965231A0 (fi)
FR (1) FR2721943B1 (fi)
IL (1) IL114273A0 (fi)
MX (1) MX9606327A (fi)
NO (1) NO965406L (fi)
WO (1) WO1996000790A1 (fi)
ZA (1) ZA955289B (fi)

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US6465253B1 (en) 1994-09-08 2002-10-15 Genvec, Inc. Vectors and methods for gene transfer to cells
US5846782A (en) 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
DK0833934T4 (da) 1995-06-15 2012-11-19 Crucell Holland Bv Pakningssystemer til human rekombinant adenovirus til anvendelse ved genterapi
AU8671798A (en) * 1997-07-30 1999-02-22 Betagene, Inc. Methods and compositions relating to no-mediated cytotoxicity
US6171856B1 (en) 1997-07-30 2001-01-09 Board Of Regents, The University Of Texas System Methods and compositions relating to no-mediated cytotoxicity
US20030017138A1 (en) 1998-07-08 2003-01-23 Menzo Havenga Chimeric adenoviruses
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6492169B1 (en) 1999-05-18 2002-12-10 Crucell Holland, B.V. Complementing cell lines
US20050232900A1 (en) * 1999-05-18 2005-10-20 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US6913922B1 (en) * 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
FI20010898A0 (fi) * 2001-04-30 2001-04-30 Ylae Herttuala Seppo Ekstrasellulaarinen superoksididismutaasi (EC-SOD) geeniterapia restenoosoin ehkäisemiseksi
ATE447037T1 (de) * 2002-04-25 2009-11-15 Crucell Holland Bv Mittel und verfahren zur herstellung von adenovirusvektoren
EP1499344A1 (en) * 2002-04-30 2005-01-26 FIT Biotech Oyj Plc Medical device
US20050036988A1 (en) * 2003-05-28 2005-02-17 Ruian Xu Compositions and methods for preventing and treating liver cirrhosis
CN102639700A (zh) 2009-09-30 2012-08-15 哈佛大学校长及研究员协会 通过调节自噬增强基因产物调节自噬的方法
EP2953640B1 (en) * 2013-02-07 2020-04-08 The Cleveland Clinic Foundation Nanoencapsulated superoxide dismutase and catalase for treating spinal cord injury
CN109833468A (zh) * 2019-03-21 2019-06-04 吉林大学 一种金属有机框架-超氧化物歧化酶组装体、制备方法及其在制备治疗帕金森药物中的应用

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EP0213628A3 (en) * 1985-09-03 1988-09-21 Yeda Research And Development Company, Ltd. Expression of superoxide dismutase in eukaryotic cells
US5082670A (en) * 1988-12-15 1992-01-21 The Regents Of The University Of California Method of grafting genetically modified cells to treat defects, disease or damage or the central nervous system

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FI965231A (fi) 1996-12-27
US20050244381A1 (en) 2005-11-03
ZA955289B (en) 1996-01-31
CA2190883A1 (fr) 1996-01-11
JPH10505485A (ja) 1998-06-02
NO965406D0 (no) 1996-12-16
FR2721943A1 (fr) 1996-01-05
US20030059455A1 (en) 2003-03-27
NO965406L (no) 1996-12-16
FI965231A0 (fi) 1996-12-27
FR2721943B1 (fr) 1996-08-02
IL114273A0 (en) 1995-10-31
AU2890595A (en) 1996-01-25
MX9606327A (es) 1997-03-29
WO1996000790A1 (fr) 1996-01-11

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