MXPA99008649A - Process for stimulating neural regeneration - Google Patents

Abstract

The invention relates to methods used to stimulate neural regeneration, and can be applied to peripheral nerves as well as the nerves of the central nervous system, especially of the spinal cord. The invention notably employs a system comprising a biocompatible sleeve, into which a system of expression of a neurotrophic factor is inserted.

Description

DEVICE AND PROCEDURE FOR THE STIMULATION OF NERVOUS REGENERATION Prese and invention "concerns the domain of biology and, in particular, the medical biology of the nervous system." It concerns, more prudently, methods of stimulating nervous regeneration, They are also applied on the "peripheral nerves and in the central nervous system and, in particular, the spinal cord. On the side of 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 the case of the lesions of the spinal cord, of the peripheral nerves , of the brachial or lumbar plexus. "Lis system injuries"; nervous, both central (CNS), as peripheral _ (SNP) are frequent in rheumatology and are dramatic. Thus, spinal cord injuries are traumatic or degenerative; Peripheral nerve injuries, brachial or lumbar plexus injuries, "currently include injuries or diseases that severely reduce the chances of life." Although the SNP has a strong ability to regenerate spontaneously, the use of classical techniques for The repair of nerves provides disappointing results.These techniques are composed mainly by direct anastomosis or Ref. 31054 placement of an autologous or heterologous nerve graft when it is found that the tensions are very important to allow the suture of the two nerve extremities (in the case of the loss of substance, or of laceration ex ce sva of the nerve, which With this technique, less than 5% of patients "who have received a repair of the median nerve, at the level of the wrist, find a sensation or a normal motor function afterwards" Most recently, the use of tubular prostheses that join the damaged nerve extremities (cuff technique) has offered an alternative for these classic nerve repair techniques (2,3). advantage of simplifying the conditions of realignment of the nerve bundles and has allowed to successfully aim, at the same time experimentally and clinically, small amounts of substance (up to 5-7 mm (1-6)). However, it seems that in order to point out the parts of substance of a larger size, the addition of neurotrophic substances or of cells inside the tubes is indispensable. Experimentally, various factors, such as FGF-1 and -27 or NGF, in application i n vi in the father (7-10); however, the CNTF or IGF II, in systemic application, have been evaluated, without clearly demonstrating, however, their role in nerve regeneration (7-12). On the other hand, there is currently no clinical treatment for spinal cord injuries. The invention presented a solution to this problem of the treatment of nerve, traumatic or degenerative lesions. The present invention concerns, in effect, a method for the stimulation of nerve regeneration by means of a biocompatible cuff and a composition of nucleic acids that co-regulate the neurotrophic factors. The present invention also concerns a device for stimulating nerve regeneration, comprising a biocompatible cuff in which a system for the expression of a nerve growth stimulation factor (ieurotrophic factor) is introduced. Another aspect of the invention relates to a device for the stimulation of nerve regeneration, comprising, on the one hand, a biocompatible coating and, on the other hand, a composition comprising a system for the expression of a stimulation factor of the The present invention also concerns the use, for the preparation of a composition intended to stimulate the nerve regeneration, of a biocompatible sleeve in which a system for the expression of a neurotrophic factor is introduced.The invention and other invention can be applied both to the regeneration of the peripheral nerves and to the stimulation of the axons in the spinal cord, the invention being derived from various observations. In particular, -1 is derived from the fact that it is possible to surgically re-establish, between two sections of a nerve, a physical point by means of a suitable device, and to introduce into this device a system for the expression of a neurotrophic factor. "It also derives from the fact 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 plane. durable, thanks "to a prolonged release effect of the trophic factor. The biological effect of the neurotrophic factor is also reinforced by the protective effect of the cuff, 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 are shared in a watertight device, on the site of the trauma or degeneration. "The results present in the examples show , for this purpose, that the method of the invention allows a repair of the nerves, fast, effective local.

The method of the invention consists, more particularly, in intervening locally, at the level of a nerve section. The proximal or distal section of the sectioned nerve or bundle is inserted into one end of a biocompatible sleeve, where it is held fixed by physical means. A composition comprising a system for the expression of a neurotrophic factor is then introduced into the cuff. The second section of the sectioned beam or beam is then introduced into the other end of the sleeve, where it is also held fixed by physical means. To avoid a diffusion of the expression system outside the sleeve, it is advantageously linked and / or maintained by a biological glue at the end level. This device can also allow new injections of expression systems. The presence, at the same time, of the support and the neurotrophic factor in high concentration and for a prolonged time, allows, as illustrated in the examples, reconstitute a nervous continuity and, thus, restore the corresponding activity. More specifically in the peripheral nervous system, the method of the invention consists in taking a "peripheral" or "root" nerve underlying the lesion and placing a biocompatible cuff after resection of a "part of the nerve or root. The proximal part of the section, either motor or sensory, is inserted into the sleeve and fixed, for example, by means of a suture or by placing the biological glue. The active factor is injected into this sleeve, which is left in that place.Then, the distal part of the section is branched to the other end of the sleeve, allowing the retention of a conti ñ nity of the axons. (Figure 1) • At the level of the central nervous system, and in particular of the medulla, the method of the invention is also particularly adapted to target the spinal cord lesions, on the other hand, this type of trauma it is one of the main applications of the system of the invention and for which, until now, there is no clinical treatment. Two types of applications can be envisioned: either the construction of peripheral pathways underlying an injury to the healthy marrow, underlying this injury (Figure _5), or the construction of the healthy marrow underlying an injury, the marrow underlying this lesion (Figure 6) In the first case, one or more roots underlying a spinal cord injury (Figure 5 ^) are sectioned; inserted in a tubular prosthesis (sleeve) and kept fixed with the help of sutures and biological glue. The tutor can then receive the expression systems that carry the genes capable of stimulating the lengthening of the axons of the motor neurons; and / or, eventually, different known factors to stimulate the growth of the axons, such as a peripheral nervous system; or cells. The tutor is then introduced into a longitudiincision made in the healthy marrow, underlying the lesion, so that in this proximal end of the tube the anterior horn of the gray matter (place of location of the spimotor neurons) appears. The tutor is fixed by one or several sutures to the arachnoid and with the biological glue (Figure 5B). A montage of this type can, thus, redo functioty to certain vital muscles. In the second case, the damaged part of the cord is removed and an upstream and downstream tutor is implanted, so that "Beams of the main beams (pyramidal, cortical, coespi and" tc. and over-injured (Figure 6). The tutor is immediately filled with the same substances, as mentioned above. In the context of the invention, the proximal part of the section, or proximal part of the nerve, is understood to be the part of the nerve that is in contact with the nevi or central system, since it is a peripheral nerve, its The proximal part is the one connected to the spicord, in the case of an injury to the spicord, the proximal part is the one that is in contact with the central nervous system, it is also understood, on the distal part of the section, or part of it. In the case of a peripheral nerve, the distal part of the nerve is connected to the motor plate (neuromuscular junction), which is a spicord injury. distal is the one that is disconnected from the central nervous system For the application of the invention, the sleeve may be "constituted of any device compatible with a use. therapeutic. The structure and composition of the sleeve are defined ventively, in such a way that (i) it restores a continuity of the axon, (ii) it can contain a composition comprising, a system of expression of the active factors, (iii) it can serve as tutor of the response of the axon, as well as of the spicord to the periphery, from the periphery to the spicord, as from the spicord to the "spicord." The property of the sleeve tutor is exercised by the nerve faculty of adhesion and of pushing on it, in particular, on its intersurface.The adhesion may result from any form of biological and / or chemical and / or physical interaction, which causes the adhesion and / or the fixation of the cells on On the other hand, for applications in human therapeutics, it is also desirable that the sleeve is of the impermeable or semipermeable type, but does not allow the passage of the expression system. nguito is a solid support, non-toxic and biocompatible. It can be, in particular, a sleeve made of material (s) int é ti co (s), such as silicone, PAN / PVC, PVFD, fibers of pol 11 et raf 1 uoroe ti 1 eno (PTFE) or, still, of acrylic copolymers. In a particular embodiment of the invention, it is preferred to use a sleeve consisting of, or from, biological materials, such as, in particular, cross-linked collagen, bone powder, polymers from carbohydrates, acid derivatives polyglycolic / polylactic, esters of hyaluronic acid or limestone-based supports. Preferably, collagen or silicone is used within the framework of the present invention. It may be collagen of human, bovine or murine origin. More preferably, a sleeve consisting of a collagen bilayer of type I or III or IV is used, advantageously IV / IVox, or silicone. One can cite, as a precise example, a Silastic sleeve (Do -Corning), consisting of silicone. On the other hand, the sleeve advantageously has a tubular shape, of cylindrical or angular section. The diameter of the sleeve can be adapted by the person skilled in the art depending on the applications sought. In particular, when it comes to stimulating the regeneration of a peripheral nerve, a relatively small diameter can be used, from 0.05 mm to 15 mm. More preferably, the inner diameter of the sleeve is between 0.5 and 10 mm.For the application of spinal cord regeneration, the most important inner diameter sleeves can be chosen, particularly for these applications, the sleeves used for The two have an internal diameter that can reach 15 to 20 mm, depending on the nervous section in question. For the construction of a bridge of an avulsed root (extirpated by start) at the level of the brachial plexus, the diameter of the sleeve advantageously corresponds to the diameter of the root. The length of the sleeve is generally determined by the size of the loss of the substance to be compensated. The sleeves of a length between 0.5 and 5 cm can be used, preferably the length of the sleeve remains less than 5 cm, the losses of substance greater than 5 cm are less frequent. The invention consists, in a first stage, in introducing a first part of the wound into the sleeve Advantageously, the proximal part of the nerve is added, which is then fixed, to ensure (i) a good nerve rotation and (ii) ) a tightness of the device.To achieve this, it is possible to make a suture between the nerve and the sleeve and / or place a biological glue.The suture can be made according to the classic methods of surgery, using the appropriate thread.The biological glue can be any biocompatible glue that can be applied to the nervous system, it can be especially any biological glue used in human surgery, and in particular a glue constituted by fibrin: Biocolle (Biotransfusion, CRTS, Li la), "Ti s sucol (Immuno AG, Vienna, Aus tria), 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 neurophilic factors. In the sense of the invention, the term "expression system" refers to any construct that allows the expression of a nucleic acid encoding a neurotrophic factor. Advantageously, the expression system comprises a nucleic acid encoding a neurotrophic factor under the control of a transcription promoter (expression package). This nucleic acid can be a DNA or an RNA. In the case of DNA, a cDNA, a gDNA or a hybrid DNA can be used, that is, a DNA containing one or several gDNA introns, but not all. -DNA can also be synthetic or intimate and, in particular, a DNA artificially synthesized to optimize codons or create reduced forms. The transcription promoter can be any functional promoter in a mammalian, preferably human, and especially nervous cell. It may be the promoter region naturally responsible for the expression of the neurotrophic factor considered when it is capable of functioning in the cell or in the organism in question. They may also be regions of different origin (responsible for the expression of other proteins, or synthetic by themselves). Especially, it can be - regions - promoters of the eukaryotic or viral genes. For example, it can be promoter regions extracted from the genome of the blancor cell. Among the eukaryotic promoters, any promoter or sequence derived that stimulates or represses the transcription of a gene in a specific manner can be used., or not; inducible, or not; strong or weak It can be, in particular, of ubiquitous promoters (promoter of the HPRT, PGK, a-actin, tubulin genes, etc.); of the promoters of the intermediate filaments (promoter of the GFAP, desmin, vimentin, neurofilament, keratin, etc. genes); of the promoters of the therapeutic genes (for example, the promoter of the MDR, CFTR, Factor VIII, ApoAI, etc. genes); or still, of the promoters that respond to a stimulus (receptor of the "steroid hormones, receptor of the retinoic acid, etc.) Also, it can be promoter sequences extracted from the genome of a virus, such as, for example, the promoters of the E1A and MLP genes of the adenovirus, the early promoter of the CMV, or still, the promoter of the LTR of the RSV, etc. In addition, these promoter regions can be modified by the addition of sequences of activation, of regulation or that allow an expression It is used, visually, within the framework of the invention, to use a eukaryotic or viral constitutive promoter, more particularly a promoter selected from among the genes HPRT, PGK, α-actin, tubulin or the promoter of the E1A and MLP genes of the adenovirus, the early promoter of the CMV, or still, the promoter of the LTR of the RSV On the other hand, the expression package advantageously comprises a sequence "" signal that directs the prod ucto synthesized in the pathways of the white cell. This signal sequence can be the natural signal sequence of the synthesized product, but it can also be any other functional signal sequence, or of an artificial signal. Finally, the expression package generally comprises a region located at the 3 'position, which specifies an end-of-transcription signal and a polyadenylation site. The trophic factors which can be used in the context of the invention are classified essentially into the family of neurotrophins, the family of neuroqulins, the TGF beta family, the family of fibroblast growth factors (FGFs). and of the insulin-like growth factors (IGFs) (reference 16) .More preferably, "" in the neurotrophin family, it is preferred to use, within the framework of the invention, BDNF, NT-3 or "the NT-4/5. The brain-derived neurotrophic factor (BDNF), described by Thoenen (17), is a protein of 118 amino acids and with a molecular weight of 13.5 kD. In addition, BDNF stimulates the formation of neurites and the survival, in culture, of the ganglion neurons of the retina, of the cholinergic septal neurons, as well as the dopaminergic neurons of the mesencephalon (reference 18). The sequence of the DNA encoding the human BDNF and for the rat BDNF has been cloned and sequenced (19), as well as especially the sequence encoding the pig BDNF (20). Although its properties are potentially interesting, the therapeutic application of BDNF faces different obstacles. In particular, the absence of biodi sponibi 1 idad_ of the BDNF, limits any use, snuff. The urotropic factor of the brain (BDNF), produced within the framework of the present invention, can be the human BDNF or an animal 3DNF. The "ne ur other fine 3 (NT3) is a secreted protein, of 119 amino acids, which allows the survival of the neophytes even at very low concentrations (21). The sequence of the ADJ? C encodes the human NT3 has been described (22) "- The ^" family of TGF-B co-renders, especially the neurotrophic factor derived from the G 1 i to 1 e cells The fact or Tieuro t roi "co derived from the dials cells, GDNF (23), "is a protein of 134 amino acids and molecular weight of 16 kD." It has an essential capacity to promote in vi t ro, the survival of dopaminergic neurons and motor neurons (16). The dials cells (GDNF), produced within the framework of the present invention, can be GDNF, human or an animal GDNF The sequences of the cDNA encoding the human GDNF and the rat GDNF have been cloned and sequenced (23). Another neurotrophic factor that can be used within the framework of the present invention is, "especially, the CNTF ("Cilliary Neuro Trophic Factor" "Neuro Trophic Factor Ciliar ")". CNTF is a neurokin that can prevent the death of neurons. As indicated above, clinical trials that have been interrupted prematurely lack results. The invention now allows the prolonged and continuous production, in vi ve, of CNTF, alone or in co-ribin with other trophic factors.The cDNA and the human and murine CNTF gene have been cloned and sequenced ( EP385 060; O91 / 04316). Other neuro trophic factors that may be used in the context of the present invention are, for example, IGF-1 (Lewis et al., 1993) and the Growth Factors of Fibroblasts. (FGFa, FGFb) In particular, IGF-I and IGFa are very interesting candidates.The geGTe sequence of FGFa has been described in the literature, as well as the vectors that allow its expression in vi vo (WO95 / 25803) Preferably, the expression system of the invention then allows the production in vi ve of a neuro-optic factor chosen from neurotrophins, neurokinins and TGFs. It is, more preferably, a factor chosen from BDNF, GDNF, CNTF, FGFa and IGF-I. Of particular interest is the production of NT3. On the other hand, according to a variant of the invention, it is also possible to carry out "the practice of an expression system that allows the production of two neurotrophic factors." In this embodiment, the expression system comprises either two expression packages or, a single package that allows the simultaneous expression of nucleic acid dcrs (tronic bicis unit) When the system comprises two expression packages, these can use identical or different promoters, eg, the expression systems of the invention, The expression vector (s) are advantageously part of a vector, it can be, in particular, a viral or plasmid vector, in the case of an expression system comprising several expression packages. , the packets can be "" carried by separate vectors or by the same vector The vector used to be a standard plasmid vector comprises, in addition to the expression pack (s), according to the invention, an origin of the replication and a "marker gene. On the other hand, different types of improved "" "vectors, devoid of marker gene and origin of replication (O96 / 26270) or possessing, for example, a conditional origin of replication (PCT / FR96 / 01414), have been described. These vectors are advantageously usable within the framework of the present invention. "" The vector used can also be a viral vector, different vectors have been constructed from viruses, which have remarkable properties for the transfer of genes. , more particularly, adenoviruses, retroviruses, AAVs and herpes viruses.For use as transfer vectors, the genome of these viruses is modified so that they become incapable of autonomous replication in a cell. that these viruses are defective for replication. Generally, the genome is modified by substituting the essential regions during viral replication by the expression (s). Within the framework of the invention, it is preferred to use a viral vector derived from the adenoviruses. Adenoviruses are viruses with linear double-stranded DNA, approximately 36 kb (kilobases) in size. Its genome includes, especially, a repeated inverted sequence (ITR) at each end, a sequence of encapsulation (Psi), early genes and late genes.The main early genes are contained in the El, E2, E3 and E4 regions. Among these, the genes contained in the El region are especially necessary for viral propagation.The main late genes are "contained in regions Ll to L5. The Ad5 adenovirus genome has been completely sequenced and is accessible in databases (see especially Genebank M73260). Likewise, the parties, seeing the totality of other adenoviral genomes (Ad2, Ad7, Ad2, etc.), have also been sequenced. For use as transfer vectors, different constructions derived from adenoviruses have been prepared. More particularly, the constructs described in the prior art are deleted adenoviruses of the El region, essential for viral replication, at which level the heterologous DNA sequences are inserted (Levrero, et al., Gene 101 (1991 195; Gosh-Choudry et al., Gene 50 ~~ (1986) 161). On the other hand, to improve the properties of the vector, it has been proposed to create other deletions or modifications in the adenovirus genome. Thus, a point-sensitive thermosensitive mutation has been introduced into the tsl25 mutant, allowing inactivation of the 72 kD DNA binding protein (DBP) (13). Other vectors purchased a deletion of another region essential for replication and / or for viral propagation, the E4 region. The E4 region is involved, in effect, in the regulation of the expression of late genes, in the stability of late nuclear RNA molecules, in the extinction of the expression of host cell proteins and in the efficacy of the replication of viral DNA. The adenoviral vectors in which the regions El and E4 are deleted, possess "then a background noise of the transcription and an expression of very reduced viral genes." These vectors have been described, for example, in the W094 / 28252 patents, WO95 / 02697, W096 / 22378 In addition, vectors carrying a modification to the level of the Iva2 gene have also been described (096/10088).

The recombinant adenoviruses described in the literature are produced from different adenovirus serotypes. In fact, there are different serotypes of adenoviruses, of which their structure and properties vary "a little, but they have a comparable genetic organization. More particularly, the recombinant adenoviruses may be of human or animal origin. As regards the adenoviruses "of human origin, it is possible to cite, preferably, those classes in group C, in particular the adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad1). Among the various adenoviruses of "animal origin", it is possible to cite, preferably, the "canine source" adenoviruses and especially all strains "of CAV2 adenovirus [strain Manhattan or A26 / 61 (ATCC VR-800), for example] "Other adenoviruses of animal origin" are cited especially in the patent 094/26914, incorporated herein by reference In a preferred embodiment of the invention, the recombinant adenovirus is a human adenovirus "of group C. preferable, it is an Ad2 or Ad5 adenovirus. The "recombinant adenoviruses are produced in an encapsulation line, that is, a line of cells capable of complementing one or more of the deficient functions in the recombinant adenoviral genome, one of these lines being, for example, the line" 2" 93, in which a part of the adenovirus genome has been integrated.More precisely, line 293 is a line of human embryonic kidney cells that contain at the extreme left (approximately 11-12%) of the adenovirus gerioma. serotype 5 (Ad5), comprising the left ITR, the encapsulation region, the El region, including Ela and Elb, the region encoding the pIX protein, and a part of the region encoding the pIVa2 protein. is capable of trans-complementing defective recombinant adenoviruses for the El region, ie, those devoid of all or part of the El region, and of producing viral deposits having high titers. It is also capable of producing, at a permissible temperature _ (32 ° C), virus deposits comprising, in addition, the thermosensitive E2 mutation. Other cell lines capable of "supplementing the El region, have been described, especially supported on the cells" of human lung carcinoma, A549 (W094 / 28152) or on human retinoblastomas (Hum. Gen. Ther. 1996) 215). On the other hand, lines capable of trans-complementing various adenovirus functions have also been described. In particular, the lines that complement the regions "El and E4" (Yeh et al., J. Virol. 70 (1996) 559; Cancer Gen. Ther.2 (1995) 322; Krougliak et al., Hum. Gen. Ther.6 (1995) 1575) and the lines complementing the El and E2 regions (W094 / 28152, WO95 / 02697, WO95 / 27071). Recombinant adenoviruses are usually produced by the introduction of DNA viral in the encapsulation line, followed by a lysis of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle is from 24 to 36 hours.) After the cell lysis, the recombinant viral particles are isolated by cesium chloride gradient centrifugation Alternative methods have been described in the application FR96 08164, incorporated herein by reference.The expression package of the gene (s) te rapéHtico (s), can be inserted in different sites of the recombinant adenovirus genome, according to the techniques described in This technique can be inserted from the beginning at the level of the deletion El. It can also be inserted at the level of the E3 region, in addition to, or in replacement of the sequences. It can also be located at the level of deleted region E4. For the construction of vectors carrying two expression packages, one can be inserted at the level of the El region, the other at the level of the E3 or E4 region. The two packages can also be introduced at the level of the same region. In order to carry out the present invention, the composition comprising can be formulated in different ways. It can be, in particular, saline solutions (monosodium, disodium, sodium, potassium, calcium or magnesium chloride, etc.).; or mixtures of these salts), sterile, isotonic or of dry compositions, in particular, those which, by the addition, as the case may be, of sterilized water or of physiological serum, allow the constitution of solutions and nyectables. Other excipients may be used, such as, for example, stabilizing proteins (serum-human albumin, especially: see FR96"03074), poloxamer, or" still, a hydrogel. This hydro g "e 1 can be prepared from any polymerase (homo or hetero) that is biocompatible and non-cytotoxic These polymers have been described, for example, in the patent O93 / 08845. A certain type of them, especially such as those obtained from ethylene oxide and / or propylene oxide, are "commercial. On the other hand, when the expression system is composed of plasmid vectors, it may be advantageous to add to the composition one or more chemical or biochemical agents that favor gene transfer. In this regard, mention may be made, for example, of the cationic polymers of the polylysine type, (LKLK) n, (LKKL) n, such as those described in the patent WO95V21931; polyethylene imine (WO96 / 02655) and DEAE dextran, or still, cationic lipids or 1 ipof ect ant e s. They have the property of condensing DNA and promoting its association with the cell membrane. Among the latter, mention may be made of lipopgliamines (lipofectamine, transfectam, such as "those described in patent 7. W095 / 18863 or W096 / 17823), different cationic or neutral lipids (DOTMA, DOGS, DOPE," "etci), as well as the peptides of nuclear origin (W096 / "25508), which are optionally subjected to an addition of functional groups in their structure (functionalized) to be directed towards certain tissues The preparation of a composition according to the invention, using a chemical vector of this type is carried out according to any technique known by the art, generally by simple contact between the different components. Particularly preferably, the expression system used in the invention is constituted by a recombinant, -defective adenovirus, which encodes a neurotrophic factor. Still more particularly, the "neurotrophic factor" is NT3 For use in the invention, adenoviruses are "advantageously formulated and administered in the form of doses comprised between 10 4 and 10 14 pfu and, preferably, 10 to 10 10 pfu. The term pfu ("plaque forming unit" "plate forming unit") corresponds to the disinfection capacity of an adenovirus solution and is determined by the infection of a suitable cell culture, measuring, generally after 15 days, the number of areas of infected cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature, the examples mentioned above show, quite remarkably, that doses of 109 and 10"allow (i) an efficient transfer of genes in sectioned neurons; (ii) a durable expression of the transgene in the neurons; and (iii) a restitution of the continuity of the ax The introduction of the expression system in the cuff can be carried out in different ways and, in particular, by means of syringes.The injection by means of my e eringas is the preferred (my cr oje ringa Hamilton or Te rumo "). One of the particularly interesting applications of the "present invention" is the stimulation of the response of the peripheral nerves. This treatment can be applied in different pathological situations, especially in the trauma or degenerative states of the "nerves." It can be applied to any nerve that is surgically accessible and, in particular, to the radial, ulnar, middle, collateral nerves of the fingers. and inter-bony, for the upper extremities, and to the sciatic nerves (diameter of approximately 1 cm at its origin) or crural (diameter of 1-7 mm), for the lower extremities.

Another particularly advantageous application of the invention is the restitution of a nerve continuity at the level of the roots of the brachial plexus (diameter of 5-6 mm), or. in the very bosom of the spinal cord, after truncatism. So far there is no known treatment for this type of injury. The method of the invention allows a bridging construction between the section underlying a section of the medulla and the underlying section thereof, so that the main beams are joined and a nervous continuity is regenerated. For these applications, the sleeves used, preferably have an internal diameter that can achieve 15 to 20 mm. In particular, for the construction of a extracted root, at the level of the brachial plexus, the diameter of the sleeve corresponds to the diameter of the root. The present invention also aims, then, at a product for the local and prolonged release of a neurotrophic substance at the level of a nervous lesion, where the product is composed of a biocompatible sleeve that allows the over-and under-ionized parts to be joined together. , in which ._ a system of expression of a neurotrophic factor is introduced. The present invention can be used to stimulate nerve regeneration in both the animal and the human. In addition, it can be used in the animal to study the properties of a new trophic factor (new protein, mutant, etc.). To achieve this, an animal is subjected to a "nervous section, then an expression system of the factor to be evaluated is introduced into a" device according to the invention. The ability of the factor to restore a nerve continuity is determined as indicated in the examples.This device also allows to compare different factors or to study the synergistic association of the differences.The present invention will be described with greater detail with the help of the examples indicated below, which should be considered as illustrative and not limiting.

Description of the Figures Figure 1: Description of the placement, on a peripheral nerve, of a device according to the invention. Figure 2: My skull taken at the level of the sacral-lumbar portion of the spinal cord and showing a strong production of β-galactosidase (revealed by the X-Gal substrate) within the motor neurons of the spine . Figure 3: Macroscopic appearance of the tissue response to J12. (A) ObseTrvado example in a control animal.

No tissue continuity is observed between the proximal and distal ends of the nerve repair. (B) Aspect "of the contents of the tutor within the animal that has received an injection with a concentration of 107 pfu of Ad-NT3 It is possible to notice the presence of a tissue cable that joins the proximal and distal ends of the nerve preparation. Figure 4: Aspects of retrograde marks by HRP, observed at J12. (A) In the control group, some rare motor neurons, marked very weakly, are observed (B) In the first group treated with a concentration 107 pfu of Ad-NT, there is a large number of strongly marked spinal neurons Figure 5: Description of the placement, in accordance with the 1st invention, of a bridge construction, of the peripheral afferent pathways , Underlying an injury at the level of the healthy marrow, underlying the injury. Figure 6: Description of the placement, in the spinal cord, of a device, in accordance with the invention. Figure 7: Description of the motor response observed as a function of time, after the intervention, on the nerve, and the placement of the device, in accordance with the invention. 1. Methodology 1-1. Adenoviral Vectors ~ As indicated above, viral vectors, and especially adenoviruses, constitute a particularly preferred embodiment of the invention. The "s" recombinant adenoviruses used, have been obtained by homologous recombination, in accordance with the techniques described in the prior art. Finally, they are constructed in 293 cells, by recombination between a fragment of the linearized viral genome (dl324) and a plasmid containing the left ITR, the encapsulation sequences of the transgene, as well as its promoter and the viral sequences. that allow recombination. Viruses "are amplified on the cell"! 293. They are repurified regularly in P3 of our "laboratory." Viral genomes can also be prepared in a prokaryotic cell, in accordance with the technique described in WO96 / 25506.The following viruses are the most particularly used: - Ad -ßGal: Defective recombinant adenovirus, derived from an Ad5 serotype, comprising (i) a deletion of the El region, at which level a "expression" package comprising a nucleic acid encoding β-galactosidase is introduced. of E. coli under the control of the LTR promoter, of the Rous sarcoma virus (designated LTR-RSV or RSV, and (ii) a deletion of the E3 region.The construction of this adenovirus has been described in Stratford-Perric aidet et al. (J. Clin. Invest. 90 (1992) 626) .Ad-NT3: Recombinant adenovirus of the Ad5 serotype comprising, inserted in its genome, in the region of the deleted region, an expression package of the NT3, composed of cDNA encoding e l NT3 under the control of a transcription promoter (in particular, the RSV LTR). An alternative construction comprises a supplementary suppression in the E4 region, as described in the W096 / 22378 patent. -Ad-CNTF: Adenovirus re-encoding serotype Ad5 comprising, inserted into its genome, in place of the deleted region, an expression packet of CNTF composed of cDNA encoding CNTF under the control of a transcription promoter (in particular the RSV LTR). The details of the construction are mentioned in patent WO94 / 08026. An alternative construction comprises a supplementary suppression in the E4 region, as described in the W096 / 22378 patent. -Ad-GDNF: Recombinant adenovirus of the Ad5 serotype comprising, inserted in its genome, in place of the deleted region, an expression pack of the GDNF composed of cDNA encoding the GDNF under the control of a transcription promoter (in particular, the RSV LTR). Details of the construction are given in patent WO95 / 26408. An alternative construction comprises a supplementary suppression in the E4 region, such as that described in the patent 096/22378. -Ad-BDNF: Recombinant adenovirus of serotype Ad5 which "comprises, inserted into its genome, in place of the deleted region, an expression pack of BDNF composed of cDNA encoding BDNF under the control of a transcription promoter" (in particular, the RSV LTR). Details of the construction are given in the patent W095 / 2"5804. An alternative construction comprises a supplementary suppression in the E4 region, such as that described in the patent W096 / 22378. -Ad.FGFa: Recombinant adenovirus of the serotype Ad5 comprising, inserted into its genome, instead of the deleted region, an expression package of FGFα, composed of cDNA encoding FGFα under the control of a transcription promoter (in particular the RSV LTR). of the construction are given in patent WO95 / 25803 An alternative construction comprises a supplementary suppression in the E4 region, such as that described in patent WO96 / 22378.The "functionality of the constructed viruses is verified by the infection of the fibroblasts in culture. The presence of the corresponding neurotrophic factor is analyzed in the culture supernatant by means of ELISA and / or by highlighting the trophic properties of this supernatant on "the primary cultures of neurons." It is understood that other constructs derived from neurons can be carried out. the adenoviruses and that can be used within the framework of the invention and, in particular, the vectors that carry the supplementary suppressions and / or the different promoters and / or "that encode other neurophilic factors." 1-2. Surgical Protocol The animals are constituted by male Sprague-Dawl rats and from 320 to 340 g (Iffa Credo - Les Oncins -France "). Under general anesthesia (intotalperial injection of Pentobarbi such, 1 mL / kg - Sanofi, Animal Health), an incision is made in the skin of the right hind paw at the level of the thigh, and the muscle planes are separated so that the right sciatic nerve is exposed.The nerve is divided in half between the popliteal cavity and the separation of the sciatic nerve and a segment of 5 mm is removed (Figure IB) A tubular silicone prosthesis is presented (14 mm long, 1.47 mm internal diameter, wall thickness: 0.23 mm -Silastic, Dow Corning Corporation, USA). The proximal end of the nerve is inserted into the tube and held fixed with the aid of a 9/0 suture with nylon, reattaching the neural tube to the nerve. A second suture between the tube and the nerve at the distal level is placed, so that a part of substance of 10 mm is obtained (distance 1 for which a peripheral, spontaneous nerve regeneration can be observed in the rat under the experimental conditions used) (Figure 1C). The tightness of the assembly at the proximal level is then carried out with the aid of a fibrin glue (Tissucol, I muno AG, Vienna, Austria), before 10 μL of the viral solution, or isotonic saline solution, for the control animals, are introduced into the guardian, in contact with the proximal extremity of the nerve, with the help of a microsyringe (Figure ID). The dead volume of the tutor is used for an isotonic saline solution (0.9% Sodium Chloride solution), before the distal tip of the nerve is introduced, in turn, into the tubular prosthesis, and the hermeticity of this extremity is secured by fibrin glue (Figure 1E). The muscular and skin planes are closed again with the help of a standard nylon suture, 6/0 and 4/0, respectively.The animals are put back in an individual cage, and kept in cycles day / night, 12h / 12h. 1-3. Coitrol of the axon response and retrograde marking of the spinal motor neurons by means of the Peroxidase of 1 Horseradish (Hors eradish) (HRP) Twelve days later, and after the general anesthesia of the animals, it becomes "to expose the The presence of a continuity of the tissue is observed before the assembly is sectioned at a distance of 3 mm under the proximal end of the nerve section of origin, and the adhesions are dissected. The "stump" obtained in this way is washed with isotonic saline solution, before being used with a 30% (w / v) HRP solution (Sigma Chemical, San Luis, MO, USA) After 1 hour of incubation This solution is removed and the nerve end is washed with an isotonic saline solution before the muscle and skin planes are closed and the animals are put back in their cages Forty-eight hours later, the animals are anesthetized again, and they are fixed, after rinsing with PBS, by means of perfusion int racárdiaca of glutaraldehído to 3.6% The spinal cords are then dissected, they are post-fixed during 3 hours in glutaraldehyde 3.6% "and they are placed in sucrose to 30% (p / v), for a time from 48 hours to 72 hours. The parts of the lumbo-sacral region of the marrows "are cut in freezing, in serial longitudinal cuts, of 35 μm in thickness, and the presence of HRP is revealed following the classical technique described by Mesulam (15), and using 3, 3 ', 5, 5' -te ramethyl-benzidine. 1-4. Detection of β-galactosidase The activity of β-galactosidase has been visualized by using the X-Gal substrate (14). Briefly, the longitudinal sections of the sacral-lumbar cord, 100 μm thick, are incubated for 18 h at 37 ° C, in PBS containing potassium hexacyanoferrate (4 M), potassium ferricyanide (4 M), substrate X -Gal (0.4 mg / mL), and magnesium chloride (4 mM). After incubation, the tissue sections are rinsed in PBS, then they are mounted in aqueous medium (Ge 1 at ina-Gl i cerol). 2. Evidence of retrograde transport of non-replicating adenoviruses by spinal motor neurons, axotomized, and verification of transgene expression. This first study has made it possible to demonstrate that axotomized neurons could carry out a retrograde transport of the adenoviral vectors and express a transgene in a time that can be of 4 weeks. The vector (adenovirus ß-galactose, as described in 1-1) was injected at a rate of 109 pfu per tube, and the expression of its transgene was evaluated at day 4, day 14, 4 weeks. After 4 days, a strong expression of β-galactosidase was observed, at the level of the ventral horn of the sacral-lumbar portion of the spinal cord, corresponding to the roots that innervate the sciatic nerve (Figure 2). This was the expression of the transgene by the spinal motor neurons, it was found again after 14 days, with a number- "total medium, of positive β-galactosidase cells, of 63.2 ± 33.6 cells, and only an efficacy of infection of the order of 12.25%, of the total number of spinal neurons that innervate the sciatic nerve. The presence of a β-galactosidase activity in the sacral-lumbar region of the spinal cord has been detected up to 4 weeks, with a decreasing intensity of marking (Table I). 3. Test of the effect of a vector that encodes a neurotrophin (NT3) on the growth of the axon of the sciatic nerve of the rat, at the level of a loss of substance of 10 mm. As a consequence of these results, the possibility of using a gene therapy system in vi ve to stimulate nerve growth after axotomy is shown, we have evaluated the effect of an adenovirus that carries a transgene that encodes neurotrophin 3 (Ad- NT3 described in 1-1) on peripheral nerve regeneration. The results obtained 12 days after performing the axotomy and nerve repair, with a guiding tutor in Silastic, which has received 107 pfu of the vector, or an isotonic saline solution, show that a tissue continuity is only observed in the group of animals treated with Ad-NT3 (Figure 3, Table II) The analysis by means of retrograde HRP marking of the number of espi nai motor neurons is that they have regenerated an axon through their tutor -guide, indicates that this tissue continuity is constituted by a nervous growth, with an average of 182".3 ± 76.5 HRP positive neurons against 24.25 ± 42.7 HRP positive neurons in the control group (Figure 4, Table II). These results allow us to conclude that a device, in accordance with the invention, that uses the adenoviral vectors for poor replication, which the get? Is that encode the neurotrophic factors, can be used to promote axon growth, both central and peripheral . 4. Comparison of the recovery of the nons after the cutting of a peripheral nerve in the rat. An injury has been practiced at the level of the sciatic nerve, in the adult rat, in order to create a loss of the substance of at least 10 mm. The proximal and distal parts of the lesion have been joined by means of a device, according to the invention (silicone tube, 14 mm long, 1.47 mm internal diameter -Silastic) in which either a solution has been introduced saline, either AV-RSVßgal (10 pfu, in 10 μL), or AV-RSVNT3 (107 pfu in 10 μL), or still, "the NT3 protein." The recovery of functions has been measured by means of the ct romi ograf í a: the motor response in the gastronomic muscle has been recorded during the two weeks (Figure 7). A recovery of the functions has been observed in the group treated with AV-RSVNT3, compared with the other groups ^ This increase It has been statistically significant by comparison, in time, with the group of AV-RSVßgal from day 112, and from day 70 through day 112, with the group "rNT3. An analysis of the physical profiles of the individual profiles shows that treatment with AV-RSVNT3 increases the possibility of a given animal initiating the nerve response. Meanwhile, the response rate does not change when growth has started. These results suggest, then, that the transfer of the gene encodes a neurotrophic factor, by means of the technique described in the present invention, is effective to increase the recovery of the functions, after the cutting of a peripheral nerve.

REFERENCES BIBLIOGR FICAS 1- Archibald et al.- J. Comp. Neurol. 1991, 306, 685-696. 2- Luñdborg e "t al.- J. Neuropathol, Exp Neurol, 1982, 41, 412-422 3- Lúndborg et al.- Exp. Neurol, 1982, 76, 361-375 4- Seckel et al. - Plast, Reconstr Surg 1986, 78 793-798 5- Lifpdborg et al.- Scand J7 Plast Surg Surg Hand Surg 1991, 25, 79-82 6- Lundborg et al.- J. Hand Surg, 1994,19, 273-276, 7- Cordeiro et al.- Plast, Surg Reconstruction, 1989, 13, 183-198, 8- Aebisher et al.- J. Neurosci Res., 1989, 23, 282. -289, 9- La uerrie et at- Microsurg, 1994, 15, 203-210, 10-Derby et al.- Exp. Neurol, 1993, 1191, 176-191, 11- Sahenk et al.- Brain Res. 1994,655,246-250 12-Glazner et al.- Neurosci 1993, 54,791-797 13-Van der Vliet et al., 1975 14- Finiels et al.- Neuroreport, 1995, 7, 373-378. Mesulam - J. Histochem, Cytochem, 1978, 26, 106-117. 16-Henderson, Adv. Neurol. 68 (1995) 235 17- Thoenen (Trends in NeuroSci. 14 (1991) 165 18-Lindsay in Neurot rophic _ Factors, Ed, (1993) 257, Academic Press 19- Maisonpierre et al., Genomics 10 (1991) 558- Leibrock et al., Nature 3"41 (1989) 149 - Henderson et al., Nature 63, 266-270 (1993) - Hohn et al., Nature 344 (19" 90) 339 - L. "- F. Lin. et al., Science, 260, .1130-1132 (1993 TABLE I: Determination of the efficacy of the infection to motor neurons axotomized by an adenoviral vector encoding β-Gal act os i-da s a.

TABLE II: Effect of the injection of an Ad-NT3 on axon recovery on day 12.

N. D Not determined * Animal killed during perfusion It is noted that, in relation to this date, the best method known by the applicant to carry out the aforementioned invention is the conventional one for the manufacture of the objects or substances to which it is referred to. Having described the invention as above, property is claimed as contained in the following.

Claims (14)

1. CLAIMS 1. A device for the stimulation of neural regeneration, characterized poique comprises a bi or com-patible man uite in which a system of expression of a neurotrophic factor is introduced.
2. 2. Uni team, for the stimulation of nerve regeneration, where the equipment _ is characterized because it comprises, on the one hand, a biocompatible sleeve and, on the other hand, a composition comprising a system of e prés i.on a neurotrophic factor.
3. 3. One use, for the preparation of a composition intended to stimulate nerve regeneration, a biorocyte sleeve in which a system of expression of a neurotrophic factor is introduced.
4. 4. The use, according to claim 3, for the preparation of a composition intended to stimulate the regeneration of the peripheral nerves.
5. 5. The use, according to claim 3, for the preparation of an omeo ctor. aest paaa a is to resener. axons in 1 spinal cord
6. 6. One use, for the preparation of an omp os c: or. for the treatment of traumatic lesions of the nervous system, of a biocompatible cuff in which a system of expression of a neurotransmitting factor is introduced. fi c o.
7. 7. A product for the local and prolonged release of a neurotrophic substance, at the level of a nerve lesion, where the product is characterized because it is composed of a biocompatible sleeve that allows joining the over- and under-exposed parts, in which introduces a system for the expression of a neurotrophic factor.
8. 8. The use, according to any of claims 3 to 6, characterized in that the sleeve is constituted by a tubular support, of non-toxic "and biocompatible material.
9. 9. The use, according to any of claims 3 to 6, characterized in that the first section of the rib is inserted into an end of the sleeve, where it is held fixed by means of suture and / or glue, where the expression system is introduced into the sleeve; then, the second section of the rib is inserted into the second end of the sleeve where it is held fixed by means of suture and / or glue.
10. 10. The use according to any of claims 3 to 6, characterized in that the expression system is constituted by a vector comprising a nucleic acid encoding the neurotrophic factor.
11. 11. The use, according to claim 10, characterized in that the vector is a viral vector.
12. 12. The use, according to claim 11, characterized in that the vector is an adenoviral vector.
13. 13. The use, according to claim 10, characterized in that the neurotrophic factor is chosen among the factors of the neurotrophic family, the neurokinins, the TGF beta, the FGFs and the IGFs.
14. 14. The use according to claim 13, characterized in that the neurotrophic factor is chosen from BDNF, GDNF, CNTF, NT3, FGFa and IGF-I.