Classifications

• • 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
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
• • 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
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/11—Surgical instruments, devices or methods, e.g. tourniquets for performing anastomosis; Buttons for anastomosis
• • 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
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/005—Ingredients of undetermined constitution or reaction products thereof
• • 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
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L31/16—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/11—Surgical instruments, devices or methods, e.g. tourniquets for performing anastomosis; Buttons for anastomosis
  • A61B17/1128—Surgical instruments, devices or methods, e.g. tourniquets for performing anastomosis; Buttons for anastomosis of nerves
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
  • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
  • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/412—Tissue-regenerating or healing or proliferative agents
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/412—Tissue-regenerating or healing or proliferative agents
  • A61L2300/414—Growth factors
• • 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
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

• the present invention relates to the field of biology, and in particular to medical biology of the nervous system. It relates more particularly to methods of stimulating nerve regeneration, applicable both to the peripheral nerves and to the central system, and in particular the spinal cord. Due to their local and specific character, the methods of the invention can be used to stimulate nerve regeneration in different pathological situations, and in particular in cases of lesions of the spinal cord, peripheral nerves, brachial or lumbar plexus.
• the present invention provides a solution to this problem of the treatment of nerve, traumatic or degenerative lesions
• the present invention relates in fact to a method of stimulating nerve regeneration by means of a biocompatible sleeve and of a composition of nucleic acids coding for neurotrophic factors
• the present invention also relates to a device for stimulating nerve regeneration comprising a biocompatible sleeve into which is introduced a system for expressing a factor for stimulating nerve growth (neurotrophic factor).
• kits for the stimulation of nerve regeneration comprising on the one hand a biocompatible sleeve and on the other hand a composition comprising a system for expressing a factor for stimulating nerve growth
• the present invention also relates to the use , for the preparation of a composition intended to stimulate 1st nerve regeneration, of a biocompatible sleeve into which is introduced a system of expression of a neurotrophic factor
• the present invention is also applicable both to the regeneration of the peripheral nerves and to stimulate axonal regeneration in the spinal cord
• the present invention follows from several observations It follows in particular from the demonstration that it is possible to surgically restore between two sections of a nerve a physical bridge by means of an appropriate device, and to introduce into this device a expression system of a neurotrophic factor It also stems from the demonstration that it is possible to induce a local concentration of trophic factors, for a time sufficient to stimulate neuronal growth
• the present invention thus combines several particularly advantageous properties on the therapeutic level It allows first of all a lasting action, thanks to an effect of prolonged release of the trophic factor
• the biological effect of the neurotrophic factor is further potentiated by the stent effect of the sleeve, which makes it possible to accelerate and guide neuronal growth
• the method of the invention also allows a very local action and therefore very specific, the trophic factors being partitioned in a sealed device, at the site of the trauma or degeneration
• the results presented in the examples show
• the method of the invention more particularly consists in intervening locally at the level of a nerve section.
• the proximal or distal section of the nerve or sectioned bundle is introduced at one end of a biocompatible sleeve, where it is physically held in place
• a composition comprising a system for expressing a neurotrophic factor is then introduced into said machon
• the second section of the nerve or sectioned bundle is then introduced at the other end of the sleeve, where it is also held in place physically
• the latter is advantageously ligated and / or maintained by a biological glue at the ends
• This device can also allow new injections of expression systems
• the presence of both the support and the factor neurotrophic in high concentration and for a prolonged period makes it possible, as illustrated in the examples, to reconstitute a nervous continuity and thus, to restore the corresponding activity
• the method of the invention consists in taking a peripheral nerve or a root underlying the lesion and in putting in place a biocompatible sleeve after resection of a part of the nerve or of the root La proximal part of the section, whether motor or sensitive, is introduced into the sleeve and held in place, for example by suturing or by applying biological glue
• the expression system coding for the active factor is injected into this sleeve, which is left in place
• the distal part of the section is then reconnected at the other end of the sleeve allowing the restoration of an axonal continuity (Figure 1)
• the method of the invention is also particularly suitable for bridging lesions of the spinal cord.
• This type of trauma constitutes, moreover, one of the main applications of the system of the invention.
• Two types of application can be envisaged, either bypassing the peripheral afferents underlying a lesion to the healthy spinal cord overlying this lesion (Fig 5), or bridging of the healthy marrow overlying a lesion, to the marrow underlying this lesion (Fig 6)
• a medullary lesion Fig 5A
• the tutor can then receive expression systems carrying genes capable of stimulating the axonal elongation of the motor neurons, and / or possibly various factors known to stimulate axonal regrowth such as a peripheral nervous graft or cells
• the tutor is then introduced into a longitudinal incision made in the healthy marrow overlying the lesion so that the proximal end of the tube is flush with the anterior horn of gray sustance (location of spinal motor neurons)
• the stake is fixed by one or several arachnoid sutures and biological glue (F ⁇ g.5B)
• Such an assembly can thus restore functionality to certain vital muscles
• the injured part of the cord is excised and a stake is implanted upstream and downstream so as to join the main bundles (pyramidal, corticospinal, etc.) between the sus- and sub-lesional parts ( Figure 6)
• the stake is then filled with the same substances as before
• proximal part of the section, or proximal part of the nerve means the part of the nerve which is in contact with the central nervous system. As it is a peripheral nerve, its proximal part is that connected to the spinal cord As a lesion of the spinal cord, the proximal part is that which is in contact with the central nervous system
• distal part of the section is also understood to mean the peripheral part of the nerve. As it is a peripheral nerve, its distal part is therefore that connected to the motor plate (j ° nc neuromuscular tion). a lesion of the spinal cord, the distal part is that which is disconnected from the central nervous system
• the sleeve can consist of any device compatible with a therapeutic use.
• the structure and the composition of the sleeve are advantageously defined so that (i) it restores an axonal continuity, (M) it can contain a composition comprising an expression system for active factors, (ni) it can serve as a tutor for axonal regrowth, both from the spinal cord to the periphery, from the periphery to the spinal cord, as well as the spinal cord towards the spinal cord
• the guardian property of the sleeve is exercised by the faculty of the nerves to adhere and to push on it, in particular on its internal face Adhesion can result from any form of biological interaction and / or chemical and / or physical causing adhesion and / or fixing of the cells on the sleeve
• the sleeve be of the waterproof or semi-permeable type, but not allowing the passage of the expression system
• the sleeve is a solid, non-toxic and biocompatible support. It may in particular be a sleeve made of synthetic material (s), such as silicone, PAN / PVC, PVFD, polytetrafluoroethylene fibers (PTFE). ) or acrylic copolymers
• s synthetic material
• PTFE polytetrafluoroethylene fibers
• acrylic copolymers it is preferable to use a sleeve made up or based on biomaterials, such as in particular crosslinked collagen, bone powder, polymers based on carbohydrates, polyglycohic / polylactic acid derivatives, hyaluronic acid esters, or limestone-based supports
• collagen or silicone II can be used.
• a sleeve consisting of a bilayer of collagen type I or III or IV, advantageously IV / IVox, or of silicone. Mention may be made, as a specific example a Silastic (Dow-Coming) sleeve, made of silicone.
• the sleeve advantageously has a tubular shape, of cylindrical or angular section. The diameter of the sleeve can be adapted by a person skilled in the art according to the desired applications.
• a relatively small diameter can be used, from 0.05 to 15 mm More preferably, the internal diameter of the sleeve is between 0.5 and 10 mm
• the sleeves used have an internal diameter of up to 15 to 20 mm, depending on the nerve section concerned
• the diameter of the sleeve advantageously corresponds the diameter of the root
• the length of the sleeve is generally determined by the size of the loss of substance to be compensated Sleeve lengths between 0.5 and 5 cm can be used Preferably, the length of the sleeve remains less than 5 cm, substance losses greater than 5 cm being less frequent
• the method of the invention consists, firstly, in introducing a first part of the nerve into the sleeve. It is advantageously the proximal part of the nerve. This is then held in place to ensure ( i) good nerve growth and (n) tightness of the device To do this, it is possible to make a suture between the nerve and the sleeve and / or to apply a biological glue
• the suture can be carried out according to conventional methods of surgery, using appropriate thread
• the biological glue can be any biocompatible glue, applicable to the nervous system II can be in particular any biological glue used in human surgery, and in particular an adhesive consisting of fibrin Biocolle (Biotransfusion, CRTS, Lille ), Tissucol (Immuno AG, Vienna, Austria), etc.
• the method of the invention comprises, as indicated above, the introduction into the sleeve of a composition comprising a system for the expression of neurotrophic factors
• the term “expression system” designates any construct allowing the expression in vivo of a nucleic acid coding for a neurotrophic factor.
• the expression system comprises a nucleic acid coding for a neurotrophic factor. under the control of a transc ⁇ ptionnel promoter (expression cassette)
• This acid nucleic acid can be DNA or RNA
• cDNA, gDNA or hybrid DNA can be used, i.e. DNA containing one or more introns of gDNA, but not all DNA can also be synthetic or semi-synthetic, and in particular DNA artificially synthesized to optimize codons or create reduced forms
• the transc ⁇ ptionnel promoter can be any promoter functional in a mammalian cell, preferably human, and in particular nervous II It can be the promoter region naturally responsible for the expression of the neurotrophic factor considered when this one is likely to function in the cell or the organism concerned II may also be regions of different origin (responsible for the expression of other proteins, or even synthetic) In particular, they may be promoter regions of eukaryotic or viral genes For example, they may be promoter regions derived from the genome of the target cell.
• any promoter or derived sequence may be used which stimulates or represses the transcription of a gene in a specific or non-specific, inducible or not, strong or weak They may in particular be ubiquitous promoters (promoter of the HPRT, PGK, ⁇ -actin, tubu ne genes, etc.), promoters of the f intermediate ilaments (promoter of the GFAP genes, desmin, vimentin, neurofilaments, keratin, etc.), of promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAl genes, etc.) or of promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.) Likewise, they may be promoter sequences originating from the genome of a virus such as, for example, the promoters of the E1A and MLP genes of adenovirus, the promoter CMV, or the RSV LTR promote
• a constitutive eukaryotic or viral promoter is used. It is more particularly a promoter chosen from the promoter of the HPRT, PGK, ⁇ -actin, tubulin genes or the promoter of the E1A and MLP d genes. adenovirus, the early CMV promoter, or the RSV LTR promoter
• the expression cassette advantageously comprises a signal sequence directing the product synthesized in the secretory pathways of the target cell.
• This signal sequence may be the natural signal sequence of the product synthesized, but it may also be any other sequence. functional signal, or an artificial signal sequence
• the expression cassette generally comprises a region located at 3 ′, which specifies a transcriptional end signal and a polyadenylation site.
• the trophic factors which can be used in the context of the invention are essentially classified in the family of neurotrophins, the family of neurokines, the family of TGF beta, the family of fibroblast growth factors (FGFs) and growth factors of insulin type. (IGFs) (review 16)
• BDNF BDNF, NT-3 or NT-4/5
• the neurotrophic factor derived from the brain (BDNF), described by Thoenen (17), is a protein of 118 amino acids and molecular weight 13.5 kD
• BDNF neurotrophic factor derived from the brain
• the DNA sequence coding for human BDNF and for rat BDNF has been cloned and sequenced (19) as well as in particular the sequence coding for pork BDNF (20)
• BDNF brain-derived neurotrophic factor
• the brain-derived neurotrophic factor (BDNF) produced in the context of the present invention may be human BDNF. or an animal BDNF
• Neurotrophin 3 is a secreted protein of 119 aa which allows in vitro survival of neurons even at very low concentrations (21)
• the sequence of cDNA coding for human NT3 has been described (22)
• the TGF-B family includes in particular the neurotrophic factor derived from Glial cells
• the neurotrophic factor derived from Glial cells, GDNF (23) is a protein of 134 amino acids and molecular weight of 16 kD II has the essential capacity to promote in vitro the survival of dopaminergic neurons and motoneurons (16)
• the neurotrophic factor derived from Glial cells (GDNF) produced in the context of the present invention can be human GDNF or animal GDNF
• the cDNA sequences coding for human GDNF and the Rat GDNFs have been cloned and sequenced (23)
• CNTF Ceral NeuroTrophic Factor
• CNTF is a neurokine capable of preventing the death of neurons
• CNTF is a neurokine capable of preventing the death of neurons
• the invention now allows prolonged and continuous in vivo production of CNTF, alone or in combination with other trophic factors.
• the cDNA and the human and mu ⁇ n CNTF gene have been cloned and sequenced (EP385,060 WO91 / 04316
• IGF-1 Lewis et al, 1993
• FGFa Fibroblast Growth Factors
• IGF-1 and FGFa are very interesting candidates
• the sequence of the FGFa gene has been described in the literature, as well as vectors allowing its expression
• the expression system of the invention therefore allows the production in vivo of a neurotrophic factor chosen from neurotrophins, neurokines and TGF II is more preferably a factor chosen from BDNF, GDNF, CNTF, NT3, FGFa and IGF-l Of particular interest is the production of NT3
• the expression system comprises either two expression cassettes, either a single cassette allowing the simultaneous expression of two nucleic acids (bicistronic unit)
• the system comprises two expression cassettes, these can use identical or different promoters
• the expression cassette (s) advantageously form part of a vector II can in particular be a viral or plasmid vector
• the cassettes can be carried by separate vectors, or by the same vector
• the vector used can be a standard plasmid vector, comprising, in addition to the expression cassette (s) according to the invention, an origin of replication and a marker gene.
• Various types of improved vectors have also been described, devoid of marker gene. and of origin of replication (WO96 / 26270) or having for example an origin of conditional repation (PCT / FR96 / 01414)
• the vector used can also be a viral vector.
• Various vectors have been constructed from viruses, having gene transfer properties. Remarkable Mention may more particularly be made of adenoviruses, retroviruses, AAVs and the herpes virus.
• the genome of these viruses is modified so as to render them incapable of autonomous rephcation in a cell. These viruses are said to be defective for rephcation Generally, the genome is modified by substitution of essential regions in trans for viral rephcation by the expression cassette (s)
• Adenoviruses are viruses with linear double strand DNA with a size of approximately 36 (kilobases) kb
• Their genome comprises in particular a repeated reverse sequence (ITR) to at each end, an encapsidation sequence (Psi), early genes and late genes
• ITR repeated reverse sequence
• Psi encapsidation sequence
• the main early genes are contained in the regions E1, E2, E3 and E4 Among these, the genes contained in the region E1 in particular are necessary to viral propagation
• the main late genes are contained in regions L1 to L5
• the genome of the adenovirus Ad5 has been fully sequenced and is accessible on database (see in particular Genebank M73260) Likewise parts, even all of other adenoviral genomes (Ad2, Ad7, Ad12, etc.) have also been sequenced
• constructs derived from adenoviruses have been prepared, incorporating various therapeutic genes. More particularly, the constructs described in the prior art are adenoviruses deleted from the E1 region, essential for viral rephcation at the level from which heterologous DNA sequences are inserted (Levrero et al, Gene 101 (1991) 195 Gosh-Choudhury et al Gene 50 (1986) 161) Furthermore, to improve the properties of the vector, it has been proposed to create other deletions or modifications in the genome of the adenovirus Thus, a heat-sensitive point mutation was introduced in the mutant ts125, making it possible to inactivate the DNA binding protein 72kDa (DBP) (13)
• Other vectors include a deletion of another region essential for rephcation and / or viral propagation, the E4 region The E4 region is indeed involved in the regulation of the expression of late genes, in the stability of late nuclear RNAs, in the extinction of the expression of proteins of the host cell
• recombinant adenoviruses described in the literature are produced from different serotypes of adenovirus II indeed exist different serotypes of adenovirus, whose structure and properties vary somewhat, but which have a comparable genetic organization More particularly, recombinant adenoviruses may be of human or animal origin Concerning adenoviruses of human origin, mention may preferably be made of those classified in group C, in particular adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 ( Ad12) Among the various adenoviruses of animal origin, mention may preferably be made of adenoviruses of canine origin, and in particular all the strains of adenoviruses CAV2 [Manhattan strain or A26 / 61 (ATCC VR-800) for example] Other adenoviruses of animal origin are cited in particular in application W094 / 26914 incorporated herein by reference
• the recombinant adenovirus is a human adenovirus of group C More preferably it is an adenovirus Ad2 or Ad5 Recombinant adenoviruses are produced in an packaging line, that is to say a cell line capable of complementing in trans one or more of the deficient functions in the recombinant adenoviral genome
• an packaging line that is to say a cell line capable of complementing in trans one or more of the deficient functions in the recombinant adenoviral genome
• line 293 is a human embryonic kidney cell line containing the left end (approximately 11-12%) of the genome of adenovirus serotype 5 ( Ad5), comprising the left ITR, the packaging region, the E1 region, including E1a and E1b, the region coding for the protein pIX and part of the region coding for the protein plVa2
• This line is capable of trans-complementing recombinant adenoviruses defective for the
• Recombinant adenoviruses are usually produced by introduction of viral DNA into the packaging line, followed by lysis of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle being of 24 at 36 hours) After the lysis of the cells, the recombinant viral particles are isolated by ce ⁇ t ⁇ fugation in cesium chloride gradient.
• the expression cassette for the therapeutic gene (s) can be inserted at different sites in the genome of the recombinant adenovirus, according to the techniques described in the prior art. It can first of all be inserted at the E1 deletion. It can also be inserted at the level of the E3 region, in addition to or in substitution for sequences It can also be localized at the level of the deleted E4 region For the construction of vectors carrying two expression cassettes, one can be inserted at the level of the region E1, the other at the level of region E3 or E4 The two cassettes can also be introduced at the level of the same region
• the composition comprising the expression system can be formulated in different ways. It can in particular be saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc, or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition as the case may be of sterilized water or physiological saline, allow the constitution of injectable solutes
• saline solutions monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc, or mixtures of such salts
• sterile, isotonic, or dry compositions in particular lyophilized, which, by addition as the case may be of sterilized water or physiological saline, allow the constitution of injectable solutes
• Other excipients may be used such as, for example, stabilizing proteins (human serum albumin, in particular FR96 03074), poloxamer or else a hydrogel
• This hydrogel can be prepared from any
• polymers polylysine type cationics polymers polylysine type cationics, (LKLK) n, (LKKL) n as described in application W095 / 21931, immine polyethylene (WO96 / 02655) and DEAE dextran or also cationic lipids or lipofectants They have the property of condensing DNA and promote its association with the cell membrane
• mention may be made of hpopolyamines hpofectamine, transfectam, as described in application W095 / 18863 or W096 / 17823) different cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.) as well as peptides of nuclear origin (WO96 / 25508), optionally functional to target certain tissues
• hpopolyamines hpofectamine, transfectam, as described in application W095 / 18863 or W096 / 17823
• DOTMA, DOGS, DOPE, etc. different cationic or neutral
• the expression system used in the invention consists of a defective recombinant adenovirus encoding a neurotrophic factor. More particularly, the neurotrophic factor is NT3.
• the adenoviruses are advantageously formulated and administered in the form of doses between 10 ⁇ and 10 ⁇ 4 pfu, and preferably 10 ⁇ to 10 " ⁇ pfu
• the term pfu (“ plaque forming unit ”) corresponds to the infectious power of an adenovirus solution, and is determined by infection of an appropriate cell culture, and measurement, generally after 15 days, of the number of plaques of infected cells.
• the introduction of the expression system into the sleeve can be carried out in different ways, and in particular by means of syringes Injection by means of microsyringes is preferred (Hamilton or Terumo microsyringe)
• One of the particularly interesting applications of the present invention is the stimulation of the regrowth of peripheral nerves.
• This treatment can be applied in different pathological situations, in particular trauma or nerve degeneration II can be applied to any nerve accessible surgically, and in particular to the radial, ulnar, median, colateral nerves of the fingers and interosseous nerves, for the upper limbs, and sciatic nerves (diameter of about 1 cm at birth) or crural nerves (diameter 6-7 mm), for the lower limbs
• the sleeves used preferably have an internal diameter of up to 15 to 20 mm
• the diameter of the sleeve corresponds to the diameter of the root
• the present invention therefore also relates to a product for the local and prolonged release of a neurotrophic substance at the level of a nerve lesion composed of a biocompatible sleeve making it possible to join the sus- and sub-lesional parts, into which is introduced a neurotrophic factor expression system
• the present invention can be used to stimulate nerve regeneration in vivo in both animals and humans. It can also be used in animals to study the properties of a new trophic factor (new protein, mutant , etc.) For this, an animal is subjected to a nervous section, then a system for expressing the factor to be tested is introduced into a device according to the invention. The capacity of said factor to restore nervous continuity is determined as indicated in the examples This device also makes it possible to compare different factors, or to study synergistic associations of different factors
• Figure 2 Micrograph taken at the sacro-lumbar portion of the spinal cord and showing a high production of ⁇ -galactosidase (revealed by the X-Gal substrate) within the spinal motor neurons
• FIG. 3 Macroscopic appearance of tissue regrowth at D12
• A Example observed in a control animal No tissue continuity is observed between the proximal and distal ends of the nerve repair
• B Appearance of the content of the tutor in an animal having received injection of 10 7 pfu Ad-NT3 It is possible to note the presence of a tissue cable joining the proximal and distal ends of the nerve repair
• Figure 7 Description of the motor response observed as a function of time after the intervention on the nerve and the installation of the device according to the invention 1.
• the viral vectors and in particular the adenoviruses, constitute a particularly preferred embodiment of the invention
• the recombinant adenoviruses used were obtained by homologous recombination according to the techniques described in the prior art. In short, they are constructed in 293 cells, by recombination between a fragment of linearized viral genome (dl324) and a plasmid containing the left ITR. , the packaging sequences, the transgene and its promoter and viral sequences allowing recombination Viruses are amplified on 293 cells II are regularly repurified in P3 in our laboratory Viral genomes can also be prepared in a prokaryotic cell according to the technique described in application WO96 / 25506 The following viruses are more particularly used
• Ad- ⁇ Gal Defective recombinant adenovirus derived from an Ad5 serotype comprising (i) a deletion of the E1 region at the level of which is introduced an expression cassette comprising a nucleic acid coding for the ⁇ -galactosidase of E co under control of promoter of the RUS sarcoma virus LTR (designated LTR-RSV or RSV), and (n) a deletion of the E3 region
• LTR-RSV designated RUS sarcoma virus LTR
• An alternative construction includes an additional deletion in the E4 region, as described in application W096 / 22378 - Ad-CNTF Recombinant adenovirus of serotype Ad5 comprising, inserted into its genome in place of the deleted E1 region, a CNTF expression cassette composed of the cDNA coding for CNTF under the control of a transcptional promoter (in particular the LTR of the RSV)
• WO94 / 08026 An alternative construction comprises an additional deletion in the E4 region, as described in application W096 / 22378
• An alternative construction comprises an additional deletion in the E4 region, as described in application W096 / 22378 - Ad-BDNF Recombinant adenovirus of serotype Ad5 comprising, inserted in its genome in place of the deleted E1 region, a BDNF expression cassette composed of the cDNA coding for BDNF under the control of a transcriptional promoter (in particular the RSV LTR) The details of the construction are given in the WO95 / 25804)
• An alternative construction comprises an additional deletion in the E4 region, as described in application WO96 / 22378
• Ad-FGFa Recombinant adenovirus of serotype Ad5 comprising, inserted into its genome in place of the deleted E1 region, an FGFa expression cassette composed of the cDNA coding for FGFa under the control of a transcriptional promoter (in particular the LTR of RSV)
• a transcriptional promoter in particular the LTR of RSV
• the functionality of the constructed viruses is verified by infection of fibroblasts in culture
• the presence of the corresponding neurotrophic factor is analyzed in the culture supernatant by ELISA and / or by highlighting the trophic properties of this supernatant on primary neuronal cultures
• the animals consisted of male Sprague-Dawley rats weighing 320- 340 g (Iffa Credo - Les Oncins - France) Under general anesthesia (intra-peroneal injection of Pentobarbital 1 ml / kg - Sa ⁇ ofi Animal Health) posterior right is incised at the level of the thigh, and the muscular planes are separated in order to expose the right sciatic nerve The nerve is divided halfway between the pophthal fossa and the separation of the sciatic nerve, and a segment of 5 mm is oté (Fig 1 B) A tubular silicone prosthesis (14 mm long, 1 47 mm internal diameter, wall thickness 0 23 mm - Silastic, Dow Corning Corporation, USA) is presented The proximal end of the nerve is introduced in the tube and is held in place using a 9/0 nylon suture connecting the epineuria and the nerve A second suture between the tube and the nerve at the distal level is put in place so as to obtain a loss 10 mm
• ⁇ -Galactosidase activity was visualized using the X-GalC substrate 4 ) Briefly, longitudinal sections of the sacro-lumbar cord 100 ⁇ m thick are incubated for 18 h at 37 ° C in PBS containing potassium hexacyanoferrate (4 mM), potassium fer ⁇ cyanide (4 mM), X-Gal substrate (0 4 mg / ml), and magnesium chloride (4 mM) After incubation, the tissue sections are rinsed in PBS, then mounted in an aqueous medium (Gelatin-Glycerol)
• a lesion was practiced at the level of the sciatic nerve in the adult rat in order to create a loss of substance of at least 10 mm.
• the proximal and distal parts of the lesion were joined by means of a device according to the invention (tube in silicone 14 mm in length, 1 47 mm in internal diameter - Silastic) into which was introduced either a saline solution, or AV-RSV ⁇ gal (10 7 pfu in 10 ⁇ l), or AV-RSVNT 3 (10 7 pfu in 10 ⁇ l), or the protein NT3
• the functional recovery was measured by electromyography the motor response in the gastrocnemius muscle was recorded every two weeks (Fig 7)
• a functional recovery was observed in the group treated with the AV-RSVNT 3 compared to the other groups This increase was statistically significant by comparison over time with the AV-RSV ⁇ gal group beyond day 112, and from day 70 to day 112 with the rNT3 group
• An electromyographic analysis of the individual profiles shows that the treatment

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