CZ337599A3 - Apparatus for stimulating nerve regeneration - Google Patents

Abstract

The invention relates to the stimulation of nerve regeneration, which is useful in the treatment of nerves Peripheral nerves and nerves of the central nervous system zejménanamíchu. The device contains a biocompatible cuff which introduces a system for expression of a neurotrophic factor.

Description

The present invention relates to the medical biology of nerve biology, in particular to the system. Specifically,. It relates to methods of stimulating the regeneration of the nervous system, which can be used for both peripheral and central nerves, in particular the spinal cord. Due to their local and specific nature, regeneration of the nervous system can be used to stimulate various pathological methods of the present invention in a variety of situations, particularly in the case of spinal cord lesions or lesions of the peripheral or brachial or lumbar plexus nerves.

BACKGROUND OF THE INVENTION

Nervous system lesions, both central (CNS) or peripheral (PNS), are very frequent and serious in traumatology. Spinal cord lesions, whether of traumatic or degenerative origin, peripheral nerve lesions, or brachial or lumbar plexus lesions, cause injured or ill individuals to be severely affected throughout their lives. Although PNS has a considerable capacity to spontaneously regenerate, the use of conventional methods of nerve repair led to results that were only disappointing. These methods consist primarily of direct anastomoses or the insertion of autologous or heterologous nerve grafts if the tension is so large that it does not permit suturing both ends of the nerve (in the event of significant tissue loss or extensive laceration of the nerve • ♦ φ φ · · · · ΦΦ ··· φ φ * ♦ φ · nerve segment). Using these methods, less than 5% of patients who have undergone n. Medianus repair. in the wrist, has acquired normal sensation or motor function after 5 years (1).

There is currently an alternative method to the above-mentioned conventional methods, namely use. tubular prostheses connecting the ends of the damaged nerve, the so-called cuff technique (2,3). This technique provides the advantage that it simplifies the conditions for re-bonding the nerve bundles and allows successful bridging, both experimentally and clinically, of small nerve tissue losses (up to 5 to 7 mm, see 1, 6). However, in order to allow longer bridging at greater tissue loss, it appears necessary to add a neurotrophic agent or a. cells. Several factors have been tested experimentally, such as FGF-1.a 'FGF-2 or NGF applied in vivo to a stent (7-10) or CNTF or IGFII administered systemically, but the results do not clearly demonstrate a significance for nerve regeneration (7 -12). Furthermore, there is no clinically appropriate method of treating spinal cord lesions.

SUMMARY OF THE INVENTION

The present invention provides a solution to the above problems in the treatment of nerve degenerative origin, nerve regeneration of lesions, whether traumatic or the invention relates to a method of stimulation using a biocompatible cuff encoding also relates to a composition comprising nucleic acids of neurotrophic factors. The present invention is a device for stimulating nerve regeneration comprising a biocompatible cuff into which a system for expressing a nerve growth stimulating factor (neurotrophic factor) is introduced.

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Another aspect of the present invention relates to a kit for stimulating neural regeneration comprising a biocompatible cuff as well as a formulation comprising a system for expressing a nerve growth stimulating factor. The invention also relates to the use of a biocompatible cuff into which a neurotrophic factor expression system is introduced for the preparation of a composition intended to stimulate neural regeneration.

In addition, the present invention is useful for regenerating peripheral nerves regeneration in the spinal cord.

The present invention is and also to axon stimulation as a result of several findings of concentration stimulation of neuronal growth, several properties, in particular from the fact that it has been demonstrated that it is possible to surgically carry out a physical bridge between two nerve sections using a suitable device and , introduce a system for the expression of · neurotrophic factor. Further, it is believed that the possibility of inducing a local trophic factor has been demonstrated for a period sufficient for the present invention thus combining which are particularly advantageous from a therapeutic point of view. In particular, it permits long-lasting action due to the sustained-release effect. In addition, the biological effect of the neurotrophic factor is enhanced by the cuff-promoting effect (stent-effect), which accelerates and leads neuronal growth. The invention also allows for highly localized, and hence very specific, action of trophic factors that are enclosed in a tight device directly at the site of trauma or degeneration. The results presented in the examples show that the method of the present invention allows for fast, efficient and local nerve repair.

The method of the present invention consists in local action at the level of nerve breakdown. The proximal or distal portion of the split nerve or nerve bundle is inserted into the flfl flfl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl fl ¹ flfl flfl • flfl one end of the biocompatible cuff where it is physically fixed. A composition comprising a neurotrophic factor expression system is then introduced into said cuff. Then a second section of nerve is inserted into the other end of the cuff or ^! nerve bundle where it is also physically fixed. In order to prevent diffusion of the expression system outside the cuff, it is preferred to ligate the ends and / or join with the biological adhesive. In addition, this device also allows for the injection of an expression system. The presence of reinforcement and neurotrophic factor in high concentration over a long period of time allows, as the examples show, to reconstitute the continuity of the nerve and thus restore the corresponding activity.

The method of the present invention, which is specific to the peripheral nervous system, consists in taking the peripheral nerve or root adjacent to the lesion and, after resection of the nerve or root portion, a biocompatible cuff. The proximal part of the nerve, whether motor or sensory, is inserted into the cuff and fastened, for example, by sewing or by biological adhesive. The expression system encoding the neurotrophic factor is injected into the cuff which is left in place. The distal part of the nerve is then reconnected to the other ends of the cuff, allowing the axon continuity to be restored (Fig. 1).

From the point of view of the central nervous system, especially the spinal system, there is a method. according to the present invention particularly suitable for bridging damage in the spinal cord. This type of trauma, for which there is no method of treatment, represents the main application possibility of the device according to the invention. Two main uses can be envisaged: either bridging peripheral afferent fibers below the lesion to healthy pulp above the lesion (Fig. 5), or bridging from healthy pulp above the lesion to healthy pulp under the lesion (Fig. 6).

In the first case, one or more roots under the spinal cord lesion are interrupted (Fig. 5A), inserted into a tubular prosthesis

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44 (cuffs) and fastened by sewing or biological glue. An expression system is then introduced into the stent. genes capable of stimulating axonal elongation of motoneurons and / or optionally various factors known to stimulate axon growth recovery, such as e.g. peripheral nerve or cell graft. The stent is then inserted into a longitudinal incision made in a healthy pulp above the lesion so that the proximal end of the tube contacts the gray matter of the anterior horn of the spinal cord (instead of locating the spinal motoneurons). The stent is attached with one or more stitches to the arachnoid and glued with a biological adhesive. Such an arrangement can restore the function of certain key muscles.

In the second case, the damaged part of the pulp is excised and the stent is implanted both upstream and upstream to connect the main bundles (pyramidal, corticospinal, etc.) between the sections above and below the damaged site (Fig. 6). The stent is then filled with the same substrate as above.

Within the scope of this disclosure, the proximal section or proximal portion of the nerve means the portion of the nerve that is in contact with the central nervous system. In the case of peripheral nerves, the proximal part is that part which is connected to the spinal cord. In the case of a spinal cord lesion, the proximal portion is meant that portion that is in contact with the central nervous system.

- The distal part of the section or the proximal part of the nerve means the peripheral part of the nerve. Therefore, in the case of peripheral nerves, it is associated with the nerve

In the case of a spinal cord lesion that is separated from the distal part, that part, by the tip (neuromuscular plate) of the proximal part, is the part that connects to the central nervous system. -.

When applied. of the invention, the cuff may be substantially any device that is compatible with therapeutic use. The preferred structure and composition of the cuff are defined in the following points:

I) restores the continuity of the axon, · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

II) may comprise a preparation comprising a system for expressing active factors, III) may serve as a stent for restoring axon growth, from spinal cord to peripheral, peripheral to spinal cord, and also from spinal cord to spinal cord. The ability of the cuff to serve itself as a stent lies in the ability of the nerves to adhere to and grow on its surface, particularly on the inner surface. The adherence may result from any biological and / or chemical and / or physical interaction causing adhesion and / or association of the cells with the cuff. In addition, for use in human therapy, it is desirable that the cuff is of the impermeable or semi-permeable type and does not permit passage of the expression system.

Preferably, the cuff is a solid, non-toxic and biocompatible reinforcement. In particular, it may be a cuff of synthetic material fibers or materials such as silicone, PAN / PVC, PVFD, polytetrafluoroethylene (PTFE) or acrylic copolymers. In the examples they are. Preferred specific embodiments of the invention wherein the cuff is of biomaterials such as cross-linked collagen, bone powder, sugar-based polymers, polyglycolic / polylactic acid derivatives, hyaluronic acid esters or chalk-based reinforcements. Preferably, silicone or collagen is used for the cuff of the present invention. This may be of collagen, of human, bovine or murine origin. Preferably, the cuff is comprised of a bilayer of type I or III or IV collagen, more preferably type IV / IVox collagen or silicone. An example of such cuffs is Sil.astic cuff (DowCorning) of silicone. Preferably, the sleeve has a tubular shape with a circular or even square cross section. The diameter of the cuff will be chosen by the skilled person according to the desired purpose. Specifically, for example, a relatively small diameter, from 0.05 to 15 mm, is used to stimulate peripheral nerve regeneration. More preferably, the inner diameter is • · · · ··· ·· »

• Cuffs between 0.5 and 10 mm. When used for spinal cord regeneration, a larger diameter cuff is used. Specifically for this application, the cuff may have an inner diameter of up to 15-20 mm, depending on the relevant nerve segment. For bridging roots extending at the brachial plexus level ,; the cuff diameter preferably corresponds to the root diameter. The length of the cuff is generally determined by the amount of tissue loss to be compensated. In general, a cuff of 0.5 to 5 cm can be used. Preferably, the cuff length is less than 5 cm, tissue losses representing a span longer than 5 cm are less common.

As mentioned above, the method according to the invention consists in inserting, as a first step, the first part of the nerve into the cuff. This is preferably the proximal part of the nerve. It is then fixed in place to ensure I) good nerve growth and II) the impermeability of the whole device. To achieve this, it is possible to suture between the nerve and the cuff and / or to use a biological adhesive. The suture can be performed in a conventional surgical manner using a suitable fiber. Biological adhesive is any biocompatible 'adhesive' that can be applied to the nervous system. In particular, it may be a biological adhesive used in human surgery, in particular a fibrin-containing adhesive: e.g. Bi.ocolle (Biotransfusion, CRTS, Lily, France), Tissucol (Immuno AG, Vienna, Austria) and the like.

The method of the invention comprises, as mentioned above, introducing the composition into the cuff, the composition comprising a system for expressing neurotrophic factors.

In the context of the present invention, the term expression system means any construct that allows for the in vivo expression of a nucleatic acid encoding a neurotrophic factor. Preferably, the expression system comprises a nucleic acid encoding a neurotrophic factor that is under the control of a transcriptional promoter (expression cassette). This nucleic acid is either DNA or '44 '· 44 · 4 · 4

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RNA. DNA is cDNA, gDNA or hybrid DNA, which is DNA containing one or more introns of gDNA, but not all. DNA can be. also synthetic or semisynthetic, in particular DNA artificially synthesized to optimize codons or to produce reduced forms.

The transcription promoter may be any promoter that is active in a mammalian cell, and in particular in a nerve cell. It can be. a promoter region that is. naturally, responsible for the expression of a neurotrophic factor, provided that it is capable of functioning in the relevant cell or organism. It may also be a region of other origin (which is responsible for the expression of other ´ proteins, or even a synthetic region). Specifically, it may be a promoter region of eukariotic or viral genes. E.g. it is a promoter region derived from the target cell genome. Eukaryotic promoters may be promoters which are gene-specific, inducible or other, weak or strong, or sequences derived therefrom. These include, but are not limited to, conventional non-specific promoters (e.g., the HPRT gene promoter, PGK, alphaactin, tubulin), tubular filament gene promoters (GFAP, desmin, vimentin, neurofilamenta or keratin genes, etc.), therapeutic gene promoters (e.g. MDR promoter) , CTFR, Factor VIII or ApoAI and the like) or alternatively stimulus-responsive promoters (steroid hormone receptor, retinoic acid receptor, and the like). Similarly, they may be promoters derived from viral sequences, such as the promoters of adenoviral E1A genes. and MLP, the CMV early promoter, or alternatively the promoter from the RSV LTR and the like. In addition, these promoter regions can be modified by adding an activating or regulatory sequence or by adding a sequence that allows tissue-specific or predominant expression.

Preferably, a constitutive eukaryotic promoter or a viral promoter is used according to the invention. _. '·· · • *

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99. In addition, the expression cassette preferably comprises a signal sequence (s) which directs synthesis of the product into the secretory pathway of the target cell. The signal sequence is either a natural signal sequence of the synthesized product or it is another functional signal sequence or even an artificial signal sequence.

Thus, an expression cassette is generally a region located at the 3'-end that carries a transcription termination signal and a polyadenylation site.

Trophic factors, which se. they can enjoy according to presented invention they fall in fact to families neurotrophins, neurokines ,, families beta-TGF and roidin type fibroblast growth shop factor. (FGF) and growth f Actuator

insulin-like (IGF) (see review 16).

It is more preferred according to the present invention to use BDNF,

NT-3 or NT-4/5 from the neurotrophin family.

The brain-derived neurotrophic factor (BDNF _Z brainderived neurotrophic factor) described by Thoenen (17) is. a protein of 118 amino acids with a molecular weight of 13.5 kD. BDNF stimulates neurite formation and ganglion neuronal survival in vitro. from retina, cholinergic septum neurons and dopaminergic mesencephal neurons in the in vitro culture (for review see 18). DNA sequences encoding human BDNF and BDNF rats were cloned and sequenced (19) along with a partial sequence encoding pig BDNF (20). Although the properties of this factor are potentially advantageous, the therapeutic use of BDNF encounters many difficulties. In particular, the lack of bioavailability limits the therapeutic use of BDNF. The brain-derived neurotrophic factor (BDNF) used in the present invention is either human BDNF or BDNF of the animal.

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Neurotrophin-3 (NT3) is a secreted protein consisting of 119 amino acids that allows neuronal survival in vitro even at very low concentrations (21). The cDNA sequence encoding human NT3 has been published (22).

The family derived neurotrophic amino acids neurotrophic glial cells derived is a kD protein. It has 134 basic

TGF-β mainly contains glial cells (GDNF, factor). GDNF (23) with a molecular weight capacity to promote in vitro survival of dopaminergic neurons and motoneurons (16). The GDNF used in the present invention is either human GDNF or GDNF of the animal. CDNA sequences encoding human GDNF as well as GDNF rats were. cloned and sequenced (23).

Another neurotrophic factor that can be used according to the invention is in particular CNTF (ciliary neurotrophic factor). CNTF is a neurokine capable of preventing neuronal death. Clinical trials with this factor were discontinued prematurely due to lack of results. But now the invention allows the prolonged and continuous action of CNTF, together in combination with other trophic factors. The human and murine CNTF cDNAs were cloned and sequenced (EP 385060, WO 91/04316).

Other neutrophic factors that may be used according to the present invention are eg IGF-1 (Lewis et al., 1993) and fibroblast growth factor (FGFα, FGFP). In particular, IGF-1 and FGFα are very suitable candidates. The sequence of the FGFα gene has been described in the literature, together with vectors allowing its expression in vitro (WO 95/25803). ..

The expression system of the present invention. the invention therefore advantageously allows for the in vivo production of neurotrophic factors selected from the group consisting of neurotrophins, neurokines and the TGF group. More preferably, it is a factor selected from the group of:

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Containing BDNF, GDNF, CNTF, NT3, FGFα, and IGF-1. Most preferred is NT3 production.

In addition, in one embodiment of the present invention, it is possible to use an expression system that allows the production of two neurotrophic factors. In such an embodiment, the expression system comprises either two. expression cassettes or a single cassette allowing simultaneous expression of two. nucleic acids (bicistronic unit). If the system comprises two expression cassettes, they may use the same or different promoters.

In the expression system of the present invention, the expression cassette is preferably part of a vector. This is particularly a viral or plasmid vector. In the case of an expression system comprising several expression cassettes, the cassettes may be carried by several separate vectors or by one and the same vector.

The vectors of the present invention are standard plasmid vectors containing, in addition to the expression cassette (s), a replication origin and a marker gene. Various types of improved vectors have been published, either without a replication origin and a marker gene (WO 96/26270) or having, e.g., a conditional origin of replication ((PCT / FR96 / 01414). These vectors can advantageously be used according to the present invention.

The vector of the present invention may also be a viral vector. Various vectors have been constructed from viruses having remarkable gene transfer properties. Particular mention may be made of adenoviruses, retroviruses, AAVs and herpes viruses. In order to be used as a gene transfer vector, the genome of these viruses is modified so that they are not capable of autonomous replication in the cell. These viruses are then replication-defective. Generally, their genome is modified so that regions necessary for viral replication in trans are substituted in the expression cassette.

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It is preferred according to the present invention to use viral vectors derived from adenoviruses. Adenoviruses are 36 kb linear double-stranded DNA viruses. Their genome contains inverted terminal repeat (ITR), encapsidation sequence (Psi), early genes and late genes at each end. The major early genes are contained in the E1, E2, E3 and E4 regions. Of these, the genes in the E1 region are essential for virus propagation. The major late genes are contained in regions L1 to L5. The genome of the Ad5 adenovirus has been fully sequenced and is accessible in a database (see, eg, GeneBank M732260). Similarly, parts or all of the genomes of other adenoviruses (Ad2, Ad7, Ad12, and the like) were also sequenced.

Many adenovirus-based constructs containing various therapeutic genes have been prepared for use as gene transfer vectors. In particular, adenoviruses with a deleted E1 region that are essential for virus replication in which a heterologous DNA sequence has been inserted are described in the art (Levrero et al., Gene 101, 1991, 195, Gosh-Ghoudhury et al., Gene 50, 1986) , 161). In addition, other deletions and modifications of the adenoviral genome have been proposed to improve vector properties. For example, a temperature-sensitive point mutation in the ts125 mutant has been introduced, allowing the inactivation of a 72 kD DNA-binding protein (DBP) (13). In other vectors, the deletions are in another region that is essential for virus replication and propagation, namely E4. The E4 region is indeed involved in regulating the expression of late genes, is involved in the stability of late nuclear RNA, is involved in suppressing the expression of host cell proteins, and plays a role in viral DNA replication efficiency. Adenoviral vectors having deleted E1 and E4 regions exhibit reduced baseline levels. noise transcription and expression of viral genes. Such vectors have been described, for example, in patent applications WO94 / 28152.

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WO95 / 02697 and WO96 / 2237. In addition, a vector carrying a modification in the IVa2 gene (WO 96/10088) has also been described. The recombinant adenoviruses described in the literature are prepared from adenoviruses of different serotypes, whose structure and properties differ somewhat but have a comparable genetic organization.

Recombinant adenoiviruses are either of human or animal origin. As regards adenoviruses of human origin, adenoviruses belonging to group C, in particular type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12) adenoviruses, may be mentioned.

Of the adenoviruses of animal origin, adenoviruses of canine, and in particular all CAV2- adenovirus strains (e.g., Manhattan strain or -A26 / 61 strain, ATCG VR-800 strain), are preferred. Other adenoviruses of animal origin are disclosed, for example, in WO 94/26914, which is hereby incorporated by reference. ,

In ^one preferred embodiment, the recombinant adenovirus is a human adenovirus of group C. More preferably, it is an Ad2 or Ad5 adenovirus.

Recombinant adenoviruses are produced in an encapsidation line, a cell line capable of complementing one or more functions in trans. which are missing in the recombinant adenoviral genome. One such line is line 293 into which part of the adenoviral has been integrated. genome. More specifically, line 293 is a human embryonic kidney cell line that contains the left end (about 11-12%) of the serotype 5 (Ad5) adenovirus genome, including the left ITR region, the encapsidation region, the E1 region, including Ela and E1B, the pIX protein coding region and the pIVa2 protein coding region. This line is. able to transcomplement adenoviruses that are defective in the E1 region, i. that lack some or all of the E1 stretch, and produce the virus at high titer. This line is capable of 9 9 9 9 9 9 9999 9999 9 9

999 9 9 99 to produce at permissive temperature. (32 ° C) virus strain containing E2 with a temperature-sensitive mutation. Other lines capable of complementing E1 have been described, particularly those derived from A549 human lung carcinoma cells (WO 94/28152) or human retinoblasts (Hum. Gen. Ther. 1996, 215). In addition, lines capable of transcomplementing several viral functions have also been described. Particular mention should be made of the lines complementing the E1 and E4 regions (Yeh et al., J. Virol 70: 1996, 559, Cancer Gen. Ther. 2, 1995, 322, Krougliak et al., Hum. Gen. Ther. 6, 1995, 1575 and lines complementing the functions of the E1 and E2 regions (WO 94/28152, WO 95/02697 and WO 95/27071) .Recombinant recombinant adenoviruses are usually produced by introducing viral DNA into the encapsidation line, followed by lysis. cells after about 2-3 days (adenoviral cycle kinetics correspond to 24 to 36 hours) After cell lysis, recombinant virus particles are isolated by cesium chloride gradient centrifugation Alternative methods have been described in patent application FR 96/08164, which is hereby incorporated by reference. .

The cassette for the expression of therapeutic genes may be inserted into different sites of the recombinant adenovirus genome by methods known to the skilled person. First, it can be inserted at the 'deletion level in E1. It can also be inserted into the E3 region, either as an additional sequence or as a replacement for the original sequence. It can also be inserted at E4. In constructing a vector containing two expression cassettes, one may be in the E1 region and the other in the E3 or E4 region. It is also possible to insert both cartridges into one section.

A composition of the present invention comprising an expression system can be formulated in various ways. In particular, it may be in a sterile isotonic solution of a salt (disodium hydrogen phosphate and dihydrogen phosphate, sodium, magnesium, potassium and calcium chloride, etc., or mixtures thereof).

Of such salts), or it may be a dry, in particular a lyophilized preparation which: depending on the case, after addition of sterile water or saline, it makes it possible to form an injectable solution. Other excipients such as stabilizing proteins (human serum albumin, see FR 96/03074, polyxamer or hydrogel) can be used. Hydrogel can be prepared from any biocompatible and non-cytotoxic (homo- or hetero-) polymer. Such polymers have been described Some of them, especially prepared from ethylene oxide and / or propylene, are commercially available, however, when the expression system consists of plasmid vectors, it is preferable to add one or more chemical or biochemical transfer promoters to the formulation. In particular, cationic polylysine polymers of the type (LKLK) n or (LKKL) h described in patent application WO 95/21931, polyethyleneimine (WO 96/02655;), DEAE-dextran or cationic lipids or cationic lipids may be mentioned. These substances have the ability to condense DNA and promote its association with cell membranes, including lipopolyamines (lipofectamine). and transfectam described in the 'applications WO 95/18863 and WO' 96/17823), various cationic or neutral lipids (DOTMA, DOGS, DOPE and the like), as well as peptides of a single origin (WO 96/25508) optionally modified to target certain target tissue. The preparation of a composition comprising such chemical vectors of the present invention is carried out by methods known to those skilled in the art, generally simply by bringing the individual components into contact.

In a particularly preferred embodiment of the present invention, the expression system comprises a recombinant adenovirus encoding a neurotrophic factor. . A particular neurotrophic factor is NT3. For use according to the present invention, the adenovirus is preferably, formulated and administered in the form of doses between 10 ⁴

·. fc fcfcfc fcfcfc fc fcfcfc fcfcfc fcfcfc fcfcfc fcfcfc fcfc fcfc ·· fcfc ^--1 and 10 pfu, preferably 10 'to 10''''pfu. The term pfu (plaque forming unit) corresponds to the infectivity of the adenovirus solution and is determined by infection of a suitable cell culture and by measuring, generally after 15 days, the number of infected cell plaques. Methods for determining the titre of a virus solution are well described in the literature. The following examples clearly demonstrate that doses ^of 10 to 10 allows even ⁷⁾ efficient delivery of genes to separate neuronal II) prolonged transgene expression in these neurons, and iii) restore continuity axons.

The expression system can be introduced into the cuff in various ways, in particular by a syringe. Injection using a microinjection syringe (Hamilton or Terumo) is preferred. One particularly preferred embodiment of the present invention is the stimulation of restored peripheral nerve growth. This approach can be used in a number of pathological conditions, particularly trauma and nerve degeneration. It can be applied to a surgically accessible nerve, especially n.

n. ulnaris, n. medianus, the collateral nerves of the fingers and intercostal nerves of the arms and the sciatica nerve (which has a diameter of about 1 cm at the beginning) or the crural nerve (diameter 6 to 7 mm) in the lower limb.

Another particularly preferred embodiment of the present invention is to restore nerve continuity after injury at the brachial plexus root level (5-6 mm diameter) or directly at the spinal cord level. There is currently no method of treating such lesions. The method of the present invention makes it possible to create a bridging between the marrow lying above and below the injured site, thus joining the main nerve bundles and regenerating the continuity of the nerves. For these applications, the sleeve preferably has a diameter of up to 15 to 20 mm. Specifically, to bridge roots extracted at the brachial plexus level, the inner diameter of the cuff corresponds to the diameter of the root.

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Accordingly, the present invention also provides a device for local sustained release of a neurotrophic agent at the level of a nerve lesion, which device comprises a biocompatible cuff that allows the parts above and below the lesion to be joined and into which a neurotrophic factor expressing system is introduced.

The present invention can be used to stimulate nerve regeneration in vivo, both in humans and animals. In addition, the invention can be used in animals to investigate the properties of novel trophic factors (new proteins, mutants, etc.). In such a case, the nerve of the animal is performed and the device of the present invention is applied into which a system expressing the factor to be tested is introduced. The capacity of the test factor to restore nerve continuity is contracted in a way that is. described further in the examples. The device according to the present invention further makes it possible to compare different factors or to examine the synergistic action of different factors.

The present invention is explained in more detail in the following by way of examples, but these are illustrative only and not limiting.

Description of the picture

Giant. 1: Schematic representation of the application of the device of the present invention to the peripheral nerve.

Giant. 2: Micrographs of the sacrolumbal spinal cord section demonstrating high levels of β-galactosidase production (shown by X-gal substrate) inside the spinal cord motoneurons.

Giant. 3: Macroscopic view of tissue growth restoration in D12. A) Control animal: there is no continuous tissue between the proximal and distal ends of the nerve. B) A view of the stent content of an animal that had been injected with 10 ⁷ pfu of AdNT3. Can '00 00 00 00 00 0 00 0 0 00 0 0 0 0 0

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0 0 0 0 0 0 0000 0000 00 00 00 00 to see the presence of tissue connecting the proximal and distal ends of the restored nerve.

Giant. 4: Retrograde appearance, HRP staining for D12. A) In the control group only is visible a few weakly stained m.otoneuronů B) In the group that was' .podáno IQ ⁷ pfu Ad-NT3 is a large number of spinal cord neurons strongly colored.

Giant. 5: Schematic representation of the application of bridging the peripheral afferent fiber under the lesion into the healthy medulla of the spinal cord above said lesion.

Giant. 6: Description of the application of the device of the present invention in the spinal cord.

Giant. 7: Dependence of motor response on time after nerve surgery and application of the device of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

1. Methods

1.1. Adenoviral vectors

As mentioned above, viral vectors, and especially adenoviruses, are particularly preferred embodiments of the present invention.

The recombinant adenoviruses used herein were obtained by homologous recombination as described in the prior art. Briefly, they were prepared in 293 cells by recombination between a linearized fragment of the viral genome (dl324) and a plasmid containing the left ITR, the encapsidation sequence, the transgene and its promoter, as well as the viral. · Fe · fe fefe · fefe · f · fe · f · fe · fefe sequences allowing recombination. The viruses were then expanded in line 293 cells. These are regularly re-purified in our P3-type laboratory. Viral genomes can also be prepared in prokaryotic cells as described in WO 96/25506. Specifically, the following viruses have been used:

AD-β-gal: Defective recombinant adenovirus derived from serotype adenovirus. An Ad5 comprising (I) a deletion of the E1 region where an expression cassette containing a nucleic acid encoding an E. coli β-galactosidase under the control of the Rous sarcoma virus LTR promoter (RSV-LTR or RSV only) is inserted and II) a deletion of the E3 region. This construct has already been described in Stratford-Perricudet et al. (J. Clin. Invest. 90, 1992, 626).

Ad-NT3: A recombinant adenovirus of the Ad5 serotype, which comprises, inserted into the genome instead of the deleted E1 region, an NT3 expression cassette containing cDNA encoding NT3 under the control of a transcriptional promoter (specifically, the RSV LTR). The alternative construct contained an additional deletion in the E4 region as already described in WO 96/22378.

Ad-CNTF: A recombinant adenovirus of the serotype Ad5, which comprises, inserted into the genome instead of the deleted E1 region, a CNTF expression cassette containing cDNA encoding CNTF under the control of a transcriptional promoter (specifically RSV LTR). Details of this construction are given in WO 94/08026. The alternative construct contained an additional deletion in the E4 region as already described in WO 96/22378.

Ad-GDNF: A recombinant adenovirus of the Ad5 serotype, which comprises, inserted into the genome instead of the deleted E1 region, a GDNF expression cassette containing cDNA encoding GDNF under the control of the transcriptional promoter (specifically • 4 '44

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RSV LTR). Details of this construction are given in WO 94/26408. The alternative construct contained a further deletion in the E4 region, as already described in WO 96/22378.

Ad-BDNF: A recombinant adenovirus of the serotype Ad5, which comprises, inserted into the genome instead of the deleted E1 region, a BDNF expression cassette containing cDNA encoding BDNF under the control of a transcriptional promoter (specifically RSV LTR). Details of this construction are given in WO 94/25804. The alternative construct contained a further deletion in the E4 region, as already described in WO 96/22378.

Ad-FGFα: A recombinant adenovirus of the Ad5 serotype, which comprises, inserted into the genome instead of the deleted E1 region, an FGFα expression cassette containing cDNA encoding FGFα under the control of a transcriptional promoter (specifically RSV LTR). Details of this "construction" are given in WO 94/25804. The alternative construct contained a further deletion in the E4 region, as already described in WO 96/22378.

1.2. Surgical protocol

Male rats Sprague-Dawley weighing 320-340 g (Iffa Credo, Les. Oncins, France) were used as experimental animals. Under general anesthesia (intraperitoneal injection of pentobarbital 1 ml / kg - Sané Santé Animalej's skin was cut to the right hind limb at the level of the thigh and the muscles were dissected to reveal the right sciatic nerve. and a 5 mm segment was removed (Fig. 1B) Tubular silicone was used.

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4444 4444 prosthesis (14 mm long, 1.47 mm in diameter, wall thickness: 0.23 mm, Silastic, Dow Corning Corporation, USA). The proximal end of the nerve was inserted into the tube and held in place by a 9/0 nylon stitch that connects the spinal cord and the nerve. The second stitch between the tube and the nerve distally is established in place to achieve a tissue loss of 10 mm (the maximum distance that can be observed in rats, spontaneous peripheral nerve regeneration under given experimental conditions) (Fig. 1C). Proximal impermeability was then achieved with fibrin glue (Tissucol®, Immuno AG, Vienna, Austria), before being introduced into the stent, in contact with the proximal nerve end, 10 μΐ of viral solution · or isotonic saline for control animals by means of a microinjection syringe (FIG. 1D). The dead volume of the stent was filled with isotonic saline (0.9% sodium chloride solution) before the distal nerve end was inserted, in the bend, into the tubular prosthesis, and the impermeability of this end was again secured with fibrin glue (Fig. 1E). The muscles and skin were then sutured using standard 6/0 and 4/0 nylon sewing thread. The animals were individually housed in. cages and maintained for 12 hour day / night cycles.

1.3. Control of axonal regrowth and retrograde labeling of horseradish peroxidase (HRP) spinal motoneurons.

Twelve days later, and after the animals were placed under general anesthesia, the connection is again uncovered and separated from the surrounding adhering tissues. Before the connection is broken at a distance of 3 mm below the proximal end of the original nerve cut, it is monitored to see if continuous tissue is present. The stub thus obtained is flushed with isotonic saline before being filled with 30% (w / v) solution.

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KRP (Sigma Chemical, St. Louis, MO). After incubation for 1 hour, this solution is removed, and the nerve end is rinsed with isotonic saline before the muscles and skin are closed and the animals are returned to the cages. Forty-eight hours later, the animals are re-anesthetized, and after

PBS washes are fixed by intracardial infusion of 3.6% glutaraldehyde. Then the spinal cord is removed, fixed for another hour in 3.6% glutaraldehyde, and placed in a 30% (w / v) sucrose solution for 48 to 72 hours.

Longitudinal serial sections of 35 µm thickness are cut from the sacrolumbal portions of the frozen spinal cord, and the presence of HRP is examined using the conventional technique described by lesulam (15) and using 3,3 ', 5' - tetramethylbenzidine.

1.4. Detection of β-galactosidase

Β-galactosidase activity was visualized using X-Gal substrate (14). Briefly, longitudinal sections of the sacrificial spinal cord of 100 µm thickness are incubated for 18 h at 37 ° C in PBS (4 mM) containing potassium ferrocyanide (4 mM), substrate X-Gal- (0.4 mg / ml) and magnesium chloride (4 mM). After incubation, tissue sections were rinsed in PBS and then embedded in aqueous medium (GelatinGlycerol).

'··

Example 2

2. Demonstration of retrograde transport of non-replicating adenoviruses by axotomized spinal motoneurons and verification of transgene expression

9999 9 9 9 «► · · · · · · · · · · · · · · · · · · ·. · · · • · · · · * * * * * * * *

The first study made it possible to demonstrate that axotomized neurons can retrograde transport of adenoviral vectors and express the transgene for a period of up to 4 weeks. The vector (adenovirus-β-galactosidase described in 1.1) was injected at 10 ³ pfu per tube, and its transgene expression was tested on day 4, day 14 and at 4 weeks.

At 4 days she was. high expression of β-galactosidase was observed in the ventral corner of the sacrolumbal part of the spinal cord, which belongs to the innervating roots of the sciatic nerve (Fig. 2). This high expression of the transgene by spinal motoneurons was found at 14 days, with a total mean number of β-galactosidase positive cells of 63.2 ± 33.6, ie an infection efficiency of 12.25% of the total number of spinal neurons innervating the sciatic nerve. The occurrence of β-galactosidase activity in the sacrolumbal spinal cord section was detected up to 4 weeks with decreasing labeling intensity (Table 1).

Example 3

3. Test of the effect of a vector encoding neurotrophin (NT3) on restoring growth of sciatic nerve oxon in rats at 10 mm tissue loss

Based on these results-based treatment systems showing the possibility of using gene therapy in vivo to stimulate

We tested the effect of adenovirus after the neurotransmission. carrying a transgene encoding neurotrophin-3 (Ad-NT3 described in 1.1) for peripheral nerve regeneration. Results obtained 12 days after axotomy and nerve repair with a guide stent made from Silasic materials into which 10 'pfu vector or isotonic saline were introduced showed that continuous tissue was observed - only in the group of animals treated with Ad-NT3 (Fig. 3). , Table II). Retrograde labeling with HRP of a large number of spinal motoneurons that regenerated the axon through the guiding stent indicates that this continuous tissue is produced by restored nerve growth, with a mean of 182.3 ± 76.5.HRP-positive neurons versus 24.25 ± 42, 7 HRP positive neurons in the control group (Figure 4, Table II).

These results have made it possible to conclude that the device of the invention using replication defective adenoviral vectors carrying genes encoding neurotrophic factors can be used to promote axonal regrowth, both central and peripheral.

Example 4

4. Comparison of restoration of function after peripheral nerve disruption in rats

Lesions at the sciatic nerve level in adult rats were established to produce a loss of at least TO mm of tissue. The proximal and distal portions of the lesion were joined using a device of the invention (silicone tube, 14 mm long, 1.47 mm internal diameter, material Silolastic) into which either isotonic saline or AV-RSVPgal (10 ⁷ pfu at 10 μΐ) was introduced. ), or AV-RSVNT ₃ (10 ⁷ pfu in 10 μΐ), or as • 0 • · 0 · • 0 · ·

0 000

* 0 0 9

0 0 · 0 0 990 9

9 · ·· 90 alternative protein NT3. Function recovery was measured electromyographically; the motor, calf muscle response was recorded every two weeks (Fig. 7).

Restoration of function was observed in the AV-R, SVNT ₃ treated group compared to the other groups. This increase was statistically significant compared over time with the AV-RSV3gal group after day 112, and from day 70 to day 112 with the rNT3 group. Electromyographic analysis of the individual profiles shows that treatment with AV-RSVNT ₃ increases the likelihood of nerve regrowth in the animal being initiated. But after the re-growth began, the level of that growth no longer changes.

These results suggest that the transfer of the gene encoding the neurotrophic factor by the technique described in the present invention is effective for enhancing function recovery after peripheral nerve disruption.

9 9 9 9 9 * * * · * * * * 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 · · · ·

List of references cited

Archibald et al. J. Comp. Neurol. 1991, 306, 685-696.

- Lundborg et al. J. Neuropathol. Exp. Neurol. 1982, 41,

412-422.

- Lundborg et al. - Exp. Neurol. 1982, -76, 361-375.

Seckel et al. - Plastic. Reconstr. Surg. 1986, 78, 793-798.

- Lundborg et al. - Scand. J. Plast. Reconstr. Surg. Hand

Surg. 1991, 25, 79-82.

- Lundborg et al. - J. Hand Surg. 1994, 19, 273-276.

Cordeiro et al. - Plastic. Reconstr. - Surg. 1989, 13, 183198.

- A.ebisher et al. - J. Neurosci. Res. 1989, 23, 282-289.

- Laquerriere et al. - Microsurg. 1994, 15, 203-210.

Derby et al. - Exp. Neurol. 1993, 119, 176-191.

Sahenk et al. Brain Res. 1994, 655,246-250.

- Glazner et al. - Neurosci. 1993, 54, 791-797.

- Van der Vliet et al., 1975

- Finiels et al. Neuroreport, 1995, 7, 373-378.

- Mesulam - J. Histochem. Cytochem. 1978, 26 _: 106-117.

- Henderson, Adv. Neurol. 68 (1995) 235

- Thoenen (Trends in NeuroSci. 14 (1991) 165)

- Lindsay in Neurotrophic Factors', Ed., (1993) 257, Academic

Press

19 Dec - Maisonpierre et al., Genomics 10 (1991) 558 20 May - Leibrock et al., Nature 341 ( 1989) 149 21 - Henderson et al., Nature 363, 266-270 i (1993) 22nd - Hohn et al., Nature 344 (1990 ) 339 23 - L.-F. Lin et al., Science, 260, 1130-1132 (1993)

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Tables

Table 1

Determination of efficacy of motcneuron infection after axotomy with adenoviral vector encoding β-galactosidase

Period Animal Number strongly labeled neurons Number labeled cellular bodies Total number labeled neurons D4 1 10. 12 22nd 2 36 55 91 ‘ 3 17 31 48 D14 1 16 24 40 2 59 48 107 3 24 6 30 4 weeks diffuse designation

Table 2

Effect of Ad-NT3 injection on restoration of axon growth in D12

Group Animal Period Continuity tissue Count positive for HRP neurons' lich control T01 D12 + 0 T02 D12 - 9 T03 D12 - 88 T05 D13 - 0 Ad-NT3 N71 D12 +++ 182 10 ⁷ pfu N72 D12 +++ 106 N73 D12 +++ N.D. * N75 D14 4- + + 259 '

N.D .: not determined * The animal died during the infusion

Claims (11)

PATENTOVÉ REQUIREMENTS. • · ···· · ·· · PV SSte-to · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · A device for stimulation characterized by a biocompatible cuff into which expression of a neurotrophic factor. A kit for stimulation characterized by a biocompatible cuff and a formulation for expression of a neurotrophic factor. nerve regeneration by comprising a system for nerve regeneration comprising containing a system Use of a biocompatible cuff into which a neurotrophic factor expression system is introduced for the preparation of a device for stimulating nerve regeneration. Use according to claim 3 for the preparation of a composition for stimulating peripheral nerve regeneration. Use according to claim 3 for preparing a composition for stimulating axonal regeneration in the spinal cord. 6. Use of a biocompatible cuff into which a neurotrophic factor expression system is introduced for the preparation of a composition for treating a traumatic lesion of the nervous system. A composition for topical and neurotrophic agents for indicating a biocompatible cuff that provides sustained release to the level of a nerve lesion, comprising the ability to join portions above and below the lesion into which a system of neurotrophic factor expression is introduced. • 4 · 4 · 4 · 4 · 4 4 4 4 444 444 444 Use according to any one of the claims. 3 'to 6, wherein the collar is formed by a tubular reinforcement and a biocompatible material. from non-toxic Use according to any one of claims 3 to 6, wherein the first part of the nerve is inserted into one end of the cuff where it is fastened with suture and / or glue, an expression system is introduced into the cuff, and then a second stretch of nerve is inserted into the other end of the cuff. fastened with stitch and / or glue. Use according to any one of claims 3 to 6, wherein the expression system comprises a vector comprising a nucleic acid that encodes a neurotrophic factor. Use according to claim 10, wherein the vector is a viral vector. Use according to claim 11, wherein the vector 'is an adenoviral vector. The use of claim 10, wherein the non-neurotrophic factor is selected from the group consisting of factors from the neurotrophin, neurokine, beta-TGF, TGF, and IGF families. The use of claim 13, wherein the neurotrophic factor is selected from the group consisting of BDNF, GDNF, CNTF, NT3, FGFα.