MXPA99000533A - Device for mixing an audio sequence with a video sequence - Google Patents

Abstract

This device comprises means (A1, A2,.....An) for acquiring in real time said video sequence in digitised and compressed form;at least one computer (S) and a digital data medium (17) for storing said digitised and compressed video sequence, and at least one mixing station (M1, M2,.....Mp) connected to said computer and comprising:means (9) for decompressing and converting to analog said digitised and compressed video sequence, means for displaying said video sequence, means for acquiring said audio sequence, means for digitising and compressing in real time said audio sequence, and means (R2) for transmitting to the computer (S) the digitised and compressed audio sound for storing on said medium (17) associated with said digitised and compressed video sequence.

Description

PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF LARYNAL SCLEROSIS AHIOTROPHIC Field of the Invention The present invention relates to a new method for the treatment of motor neuron diseases3 and in particular amyotrophic lateral sclerosis. It also refers to vectors and pharmaceutical compositions that allow prolonged expression of therapeutic factors, usable for the treatment of SLA. More precisely, the present invention relates to the treatment of SLA by the systemic administration of therapeutic genes.

Background of the Invention Amyotrophic lateral sclerosis (SLA), also known under the name of Charcot's disease and Lou Gehrig's disease, was first described by Charcot in 1865. SLA is a fatal disease resulting from the degeneration of motor neurons and the corticospinal pathways. With an incidence currently of 2.5 / 100 000 and in constant increase, a prevalence Ref. 28969 of 6-10 / 100,000, the SLA affects 90,000 people in developed countries, mostly adults who are still young (between 50 and 60 years old). The disease is accompanied by a progressive paralysis, which leads to the total loss of motor and respiratory functions, then to the dead one time interval of two to eight years after the appearance of the first symptoms (three years on average). 5% of SLA cases are of family origin and 95% of cases are sporadic. The pathophysiological origin of the sporadic forms of the SLA is still unknown. Several hypotheses have been proposed. Motoneuronal degeneration could result from an alteration of glutamate metabolism, which leads to an increase in the concentrations of this excitatory amino acid in the motor cortex and in the spinal cord ("excitotoxic" hypothesis, analyzed in Rothstein, 1995). The possibility of an autoimmune component has also been invoked based on the presence of autoantibodies against the voltage sensitive calcium channels in certain patients (analyzed in Appel et al., 1995). The involvement of environmental factors such as exposure to certain viruses (analyzed in Gastaut, 1995), or aluminum (Yase, 1984) is also possible. The studies carried out on the hereditary forms of SLA have shown that point mutations in the superoxide dismutase gene in copper and zinc, located on chromosome 21q22-l, are responsible for the pathology in 20% of the familiar forms (Rosen et al., 1993, analyzed in Rowland, 1995). These mutations do not cause a decrease in the activity of the SOD dismutase (analyzed in Rowland, 1995). Mutated enzymes produce potentially cytotoxic hydroxyl radicals that are not produced by wild-type SOD (Yim et al., 1966). The deep study of the functional effect of the mutations on the enzymatic activity of the SOD and on the cellular viability should allow finally understand the physiopathology of the familiar forms of the SLA, and by extension clarify the pathophysiology of all the forms of SLA. The work carried out on the factors likely to have an influence on the survival of motoneurons has made it possible to demonstrate a potential neuroprotective role of several neurotrophic factors (analyzed in Windebank, 1995, Henderson, 1995). Thus, in vitro motoneuronal protection effects have been observed especially with BDNF (Opperheim et al., 1992, Yan et al., 1992,? endtner et al., 1992, Henderson et al., 1993, Vejsada et al., 1995), NT-3 (Henderson et al., 1993), GDNF (Henderson et al., 1994, Oppenheim et al., 1995), three cytokines, CNTF, LIF (analyzed in Henderson, 1995) and cardiotrophin-1 (Pennica et al., 1996), with IGF-1 (Lewis and collaborators, 1993) and the elements of the FGF family (Hughes et al., 1993). All these data suggest that the aforementioned neurotrophic factors reinforce the survival of motoneurons in the various experimental conditions. However, the use of neurotrophic factors in animal models of SLA or in human clinical practice has not yet yielded convincing results. This use has never demonstrated a therapeutic effect and is always accompanied by undesirable side effects such as weight loss, inflammation, fever, etc., which limit the interest of trophic factors in the treatment of SLA and has led to the premature discontinuation of the first clinical trials of SLA-CNTF by Regeneron (systemic administration) (Barinaga et al., 1994). It has not been possible until now to confirm the interest of the neurotrophic factors for the treatment of SLA, or exploit its properties for an eventual therapeutic approach. Due to this, there is currently no means to cure the SLA and very few drugs have a therapeutic effect. Rilutek® is the only treatment available so far. The administration of riluzole (Rilutek®) can reduce the progress of the disease, but has not shown a therapeutic effect on motor function. On the other hand, clinical trials based on the administration of CNTF have been interrupted prematurely, in the absence of results (Barinaga et al., 1994). There is thus in our days a real and important need to have a method that allows to treat the problems of the motor neurons, and in particular of the SLA.

Detailed description of the invention The object of the present invention is especially to propose a novel approach for the treatment of motor neuron pathologies, such as SLA, based on gene therapy. More particularly, the present invention describes vector systems that allow directly promoting the survival of motor neurons involved in these pathologies, by the effective and prolonged expression of certain trophic factors. A first aspect of the invention relates to a method of treating SLA comprising the systemic administration of a nucleic acid encoding a neurotrophic factor. Another aspect of the invention relates to the use of a nucleic acid encoding a neurotrophic factor, for the preparation of a pharmaceutical composition intended for the treatment of SLA. Another aspect of the invention lies in the construction of particular vectors, which allow the expression of effective therapeutic amounts against the SLA of the trophic factors. Another aspect of the invention relates to the administration of expression systems that allow the production of one or more trophic factors, as well as to pharmaceutical compositions comprising said expression systems. It also refers to the creation of novel vectors that allow the coexpression of trophic factors in vivo. The present invention relates more precisely to a new method of treating LSA, based on the continuous in vivo expression of trophic factors.

The present invention now shows that it is possible to obtain in vivo a therapeutic effect particularly pronounced by the in vivo production of neurotrophic factors. The applicant has shown especially that the in vivo injection of neurotrophic factor expression systems, through the systemic route, allows obtaining a continuous production of the therapeutic factor, and that this production is sufficient to obtain a therapeutic benefit in motor neuron diseases, in particularly the SLA. Thus, the Applicant has shown that the systemic administration of these expression systems leads to a very significant increase in the duration of life, accompanied by an improvement in the motor evoked response such as that determined by electromyography. The results described show that this route of administration allows obtaining an appropriate bioavailability of neurotrophic factors, without the effect of toxicity. This therapeutic approach thus allows producing therapeutically active amounts of the molecules, leaving this side of the toxicity threshold of these molecules. Thus, as long as a protein the size of a neurotrophic factor, administered systemically, does not penetrate the nervous system more than with a reduced efficiency due to the existence of the blood barrier.

In the brain, the method of the invention makes it possible to obtain, unexpectedly, an important therapeutic effect. On the other hand, the method of the invention makes it possible to use doses of therapeutic factors that are on this side of the threshold of toxicity, and do not induce side effects. A first object of the invention thus lies in a SLA treatment process comprising the administration, by the systemic route, of a system of expression of a neurotrophic factor. Another object of the invention is also the use of a system for the expression of a neurotrophic factor for the preparation of a pharmaceutical composition for the treatment of SLA, by administration by the systemic route. The invention also relates to a method for prolonging the lifespan of mammals suffering from SLA, which comprises the administration, by the systemic route, of a system for the expression of a neurotrophic factor. In the sense of the invention, the term "expression system" designates any construct that allows the expression in vivo 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 transcriptional promoter (expression cassette). This nucleic acid can be a DNA or an RNA. When it comes to DNA, a cDNA, a gDNA or a hybrid DNA can be used, that is, a DNA that contains one or more introns of the gDNA, but not all. The DNA may also be synthetic or semi-synthetic in nature, and in particular an artificially synthesized DNA to optimize codons or create reduced forms. The transcriptional promoter can be any functional promoter in a mammalian, preferably human, cell. It can be the promoter region responsible naturally for the expression of the neurotrophic factor considered because it is capable of functioning in the cell or organism involved. It may also be regions of different origin (responsible for the expression of other proteins, or even synthetic). In particular, these may be promoter regions of eukaryotic or viral genes. For example, it may be promoter regions that exit the genome of the target or target cell. Among the eukaryotic promoters, any promoter or derived sequence that stimulates or represses the transcription of a gene in a specific manner or not, inducible or not, strong or weak can be used. It can be, in particular, ubiquity promoters (promoter of the genes HPRT, PGK, -actin, tubulin, etc.), promoters of the intermediate filaments (promoter of the genes GFAP, desmin, vimentin, neurofilaments, keratin, etc.), of the promoters of the therapeutic genes (for example the promoter of the MDR genes, CTFR, Factor VIII, ApoAI, etc.), tissue-specific promoters (promoter of the pyruvate kinase gene, villin, intestinal protein of fatty acids binding, a-actin of the smooth muscles, etc. or even promoters that respond to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.) Similarly, the promoter sequences can be reacted from the genome of a virus, such as, for example, the promoters of the ElA and MLP genes of the adenovirus, such as for example the promoters of the ElA and MLP genes of the adenovirus, the early promoter of the CMV, or even the promoter of the LTR of the RSV, etc. In addition, these promoter regions can be modified by the addition of sec activation, regulation, or that allow a specific expression for the tissue or majority. A eukaryotic or viral constitutive promoter is advantageously used in the context of the invention. It is more particularly a promoter chosen from the promoter of the HPRT, PGK, a-actin, tubulin genes or the promoter of the ElA and MLP genes of the adenovirus, the early CMV promoter, or even the RSV LTR promoter. On the other hand, the expression cassette advantageously carries a signal sequence which directs the synthesized product towards the secretion pathways of the target cell. This signal of the sequence can be the sequence of the natural signal of the synthesized product, but can also be any other functional signal sequence, or of an artificial signal sequence. Finally, the expression cassette generally comprises a 3 'region, which specifies a transcriptional end signal and a polyadenylation site. The trophic factors that can be used in the context of the invention are essentially classified into three families: the neurotrophin family, the neurocins family, and the TGF beta family (for a review, see Henderson Adv. Neurol., 68 (1995)). 235). More preferably, in the neurotrophin family, it is preferred to use BDNF, NT-3 or NT-4/5 within the framework of the invention. The neurotrophic factor derived from the brain (BNDF), described by Thoenen (Trends in NeuroSci. (1991) 165), is a protein of 118 amino acids and molecular weight of 13.5 kD. In vitro, BNDF stimulates neurite formulation and survival in the culture of ganglionary neurons of the retina, cholinergic neurons of the septum as well as dopaminergic neurons (analyzed by Lindsay in Neurotrophic Factors, Ed., (1993) 257, Academic Press). The DNA sequence encoding the human BDNF or the BDNF of the rat has been cloned and sequenced (Maisonpierre et al., Genomics 10 (1991) 558), as well as especially the sequence encoding the BDNF of the pig ( Leibrock et al., Nature 341 (1989) 149). Although its properties are potentially interesting, the therapeutic application of BDNF faces different obstacles. In particular, the lack of bioavailability of BDNF limits any therapeutic use. The brain-derived neurotrophic factor (BDNF) produced within the framework of the present invention can be human BDNF or an animal BDNF. Neutrophin 3 (NT3) is a secreted protein of 119 aa that allows in vitro survival of neurons even at very low concentrations. (Henderson et al, Nature 363, 266-270 (1993)). The cDNA sequence encoding human NT3 has been described (Hohn et al., Nature 344 (1990) 339).

The family of TGF-B especially comprises the neutrophic factor derived from glial cells. The neurotrophic factor derived from glial cells, GNDF (L.-F. Lin et al., Science, 260, 1130-1132 (1993)) is a protein of 134 amino acids and molecular weights of 16 kD. It has the essential capacity to promote in vitro the survival of dopaminergic neurons and motor neurons (analyzed in Henderson, 1995). The glial cell-derived neurotrophic factor (GDNF) produced within the framework of the present invention may be human GDNF or an animal GDNF. The cDNA sequences encoding human GDNF and mouse GDNF have been cloned and sequenced (L.-F. Lin, D. Doherty, J. Lile, S. Besktesh, F. Collins, Science, 260, 1130 -1132 (1993)). Another neurotrophic factor usable in the context of the present invention is especially CNTF ("Ciliary NeuroTrófico Factor"). CNTF is a neurocin capable of preventing the death of neurons. As indicated above, clinical trials have been interrupted prematurely due to lack of results. The invention makes it possible to maintain the prolonged and continuous production of CNTF in vivo, alone or in combination with other trophic factors, for the treatment of SLA. The cDNA and the human and murine CNTF gene have been cloned and sequenced (EP385 060, WO91 / 04316. Other neurotrophic factors usable in the framework of the present invention are for example IGF-1 (Lewis et al., 1993) and Fibroblast Growth Factors (FGFa, FGFb) In particular, IGF-I and FGFa are very interesting candidates The sequence of the FGFa gene has been described in the literature, as well as the vectors that allow its expression in vivo ( O95 / 25803) The genes coding for BDNF, CNTF and NT3 are all particularly interesting for the implementation of the present invention According to a first embodiment, the expression system of the invention allows the production of A single neurotrophic factor in vivo In this case, the expression system carries only one cassette of expression.Preferably, the expression system of the invention allows the in vivo production of a neurotrophic factor chosen from the neurotrophins, the neurocinas and the TGF. It is more preferably a factor chosen from BDNF, GDNF, CNTF, NT3, FGFa and IGF-I. According to another embodiment, the expression system of the invention allows the production of two neurotrophic factors in vivo. In this embodiment, the expression system carries either two expression cassettes, or a single cassette that allows the simultaneous expression of two nucleic acids (bicistronic unit). When the system comprises two expression cassettes, they can use identical or different promoters. Preferably, the expression system of the invention allows the in vivo production of combinations of the following neurotrophic factors: BDNF and GDNF; BDNF and NT3; GDNF and NT3, CNTF and BDNF, CNTF and NT3, CNTF and GDNF. Advantageously, the applicant has indeed shown that the administration of 2 systems of expression of neurotrophic factors results in an important therapeutic effect. In 2-neurotrophic factor expression systems, promoters of identical or similar strength, and an identical or similar nucleic acid copy number are generally used. In general, the respective amount of the two factors produced in vivo is quite close. However, it may be preferable in certain situations to produce different amounts of each factor. In this case, either different force promoters or a system in which they are present can be used numerous copies of different genes, or vary the doses administered. In the expression systems of the invention, the expression cassette (s) advantageously form a part of the vector. It can be in particular a viral or plasmid vector. In the case of an expression system carrying several expression cassettes, the cassettes may be carried by separate vectors, or by the same vector. The vector used can be a standard plasmid vector, which carries, in addition to the expression cassette (s) according to the invention, an origin of replication and a marker gene. Different types of improved vectors have been described on the other hand, devoid of the marker gene and the origin of replication (PCT / FR96 / 00274) or possessing for example a conditional origin of replication (FR95 10825). 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 the virus, which have remarkable gene transfer properties. More particularly, adenoviruses, retroviruses, AAVs and herpes viruses can be cited. For use as vectors of Transfer of the genes, the genome of these viruses is modified so that they become incapable of autonomous replication in a cell. These viruses are called defective for replication. In general, the genome is modified by the substitution of the essential trans regions for viral replication by the expression cassette (s). Within the framework of the invention, it is preferred to use a viral vector derived from the adenoviruses. Adenoviruses are linear double-stranded DNA viruses with a size of approximately 36 kb (kilobases). Its genome includes, in particular, a repeated inverted sequence (ITR) in each extremity, a sequence of encapsidation (Psi), of early genes and of late genes. The main early genes are contained in the El, E2, E3 and E4 regions. Among them, 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 on the basis of data (see especially Genebank M73260). In the same way the parts, to see the totality of the other adenoviral genomes (Ad2, Ad7, Adl2, etc.) have been sequenced equally.

For their use as gene transfer vectors, different constructions derived from adenoviruses, which incorporate different therapeutic genes, have been prepared. More particularly, the constructs described in the prior art are adenoviruses deleted from the El region, essential for viral replication, at which level the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Choudhury 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 thermosensitive point mutation has been introduced in the tsl25 mutant, which allows inactivating the 72 kDa DNA binding protein (DBP) (Van der Vliet et al., 1975). Other vectors comprise a deletion of another region essential for the replisation and / or for viral propagation, the E4 region. The E4 region is indeed involved in the region of late gene expression, in the stability of late nuclear RNAs, in the extinction of the expression of host cell proteins and in the efficiency of viral DNA replication . The adenoviral vectors in which the regions El and E4 are deleted, thus possess a resonance of the background of the transcription and an expression of viral genes greatly reduced. Such vectors have been described, for example, in applications 094/28152, WO95 / 02697, 096/22378. In addition, vectors carrying a modification to the level of the Iva2 gene (O96 / 10088) have been described. The recombinant adenoviruses described in the literature are produced from different serotypes of the adenovirus. There are in effect different serotypes of adenovirus, whose structure and properties vary very little, but which present a comparable genetic organization. More particularly, the recombinant adenoviruses may be of human or animal origin. With respect to adenoviruses of human origin, those classes given in group C, in particular adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Adl2) may be mentioned. Among the different adenoviruses of animal origin, adenoviruses of canine origin, and especially all CAV2 adenovirus strains [strain Manhattan or A26 / 61 (ATCC VR-800) for example], can be cited preferentially. Other adenoviruses of animal origin are cited especially in the application W094 / 26914 incorporated herein by reference. In a preferred mode of putting the invention into operation, the adenovirus Recombinant is a human adenovirus of group C. More preferably, it is an Ad2 or Ad5 adenovirus. The recombinant adenoviruses are produced in an encapsidation line, that is, a line of cells capable of trans-complementing one or more of the deficient functions in the recombinant adenoviral genome. One of these lines is for example line 293 in which a part of the adenovirus genome has been integrated. More precisely, line 293 is a line of human kidney embryonic cells containing the left extremity (approximately 11-12%) of the serotype 5 adenovirus (Ad5) genome, comprising the left ITR, the encapsidation region. , the El region, including Ela and Elb, the region encoding the pIX protein and a part of the region encoding the pIVa2 protein. This line is capable of trans-complementing the defective recombinant adenoviruses for the El region, that is to say devoid of all or a part of the El region, and of producing viral raw materials having high concentrations. This line is also capable of producing, at a permissible temperature (32 ° C), virus raw materials that also carry the thermosensitive E2 mutation. Other cell lines capable of complementing the El region have been described, based especially on human lung carcinoma cells A549 (W094 / 28152) or on human retmoblast cells. { Hum. Gen. Ther. (1996) 215). On the other hand, lines capable of trans-complementing various functions of the adenovirus have also been described. In particular, lines can be cited that complement the El 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 the lines complementing the El and E2 regions (094/28152, WO95 / 02697, WO95 / 27071). Recombinant adenoviruses are usually produced by the introduction of viral DNA into the encapsidation line, followed by lysis of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle is 24 to 36 hours). After lysis of the cells, the recombinant viral particles are isolated by centrifugation in a gradient of cesium chloride. Alternative methods have been described in the application FR96 08164 incorporated herein by reference. The expression cassette of the therapeutic gene or genes can be inserted in different sites of the genome of the recombinant adenovirus, according to the techniques described in the prior art. It can be inserted first at the level of the El deletion.

The cassette can also be inserted at the level of the E3 region, in addition or in substitution of the sequences. It can also be located at the level of the deleted E4 region. For the construction of the vectors carrying two expression cassettes, one can be inserted at the level of the El region, the other at the level of the E3 or E4 region. The two cassettes can also be introduced at the level of the same region. As indicated above, in the case of expression systems carrying several expression cassettes, the cassettes can be carried by separate vectors, or by the same vector. The present invention more specifically contemplates the development of efficient vectors particularly for delivering, in vivo and in a localized manner, therapeutically active amounts of GDNF, BDNF, NT3 and CNTF. More precisely, the present invention relates to the systemic injection of an expression system comprising two transfer vectors of the genes each carrying a gene coding for a neurotrophic factor. The invention also relates to the systemic injection of an expression system comprising a bicistronic vector that allows the coexpression of two genes. Preferably, the present invention relates to injection by the systemic route, of an expression system comprising two vectors, one carrying the gene coding for CNTF and the other gene coding for NT3, or one of the genes coding for CNTF and the other gene coding for BDNF, or a gene that codes for GDNF and the other gene that codes for NT3. More preferably, the transfer vectors used are the adenoviral vectors. The Applicant has indeed shown the efficacy of the use of the adenovirus which codes for the neurotrophic factors injected via the i.v. at the time of treatment of different animal models of the SLA. In particular, the results presented in the examples show, for the first time on an animal model of a family form of the SLA, the mouse FALSS93a, a significant increase in the life span, accompanied by better electromyographic performances. The only treatment proposed so far for patients suffering from SLA is riluzole (Rilutek®) which increases the life expectancy of patients by a few months. It has also been shown that riluzole administered to FALSß93A mice can increase their average lifespan by 13 days (Gurney et al., 1996). It is thus possible to predict that any treatment that increases more than 13 days the lifespan of FALSG93A mice is susceptible to provide patients with a therapeutic benefit superior to that of riluzole. The results presented in the examples show that the therapeutic approach according to the invention allows to increase the average lifespan of FALSG93A mice up to about 30 days. This constitutes a very significant improvement in the duration of life, and represents the demonstration of an important therapeutic benefit over the models of the SLA. The pmn mice are another model of the SLA, characterized by an earlier and faster degeneration of the motor neurons and by an average life time of approximately 40 days. The results presented in the examples show that the therapeutic approach according to the invention allows prolonging the average lifespan of the pmn mice from 40 to 53 days, which constitutes a significant improvement of more than 30%. This prolongation of the treated pmn mice is also accompanied by a significant reduction in their mononeuronal degeneration. The set of results obtained by this new therapeutic approach demonstrates for the first time an important improvement of different clinical, electromyographic and histological parameters, in two different models of the SLA.

According to the invention, the in vivo production of trophic factors is obtained by the systemic administration. The results presented in the examples show that this mode of administration allows obtaining a regular and continuous production of a trophic factor by the patient's own organism, and that this production is sufficient to generate a significant therapeutic effect. The systemic administration is preferably an intravenous or intraarterial injection. Intravenous injection is particularly preferred. This injection mode is also advantageous in terms of tolerance and ease of access. This allows larger volumes to be injected than in the intramuscular injection, and repeatedly. The present invention also relates to any pharmaceutical composition comprising a system for the expression of two neurotrophic factors. The pharmaceutical compositions of the invention advantageously contain pharmaceutically acceptable carriers for an injectable formulation. In particular, it can be salty solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, especially lyophilized, which, by the addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutes. Other excipients may be used such as for example stabilizing proteins (serum-human albumin especially: FR 03074) or a hydrogel. This hydrogel can be prepared from any polymer (homo or hetero) bioccmpatible and non-cytotoxic. Such polymers have been described, for example, in the application WO93 / 08845. Certain of them, as especially those obtained from ethylene oxide and / or propylene are commercial. On the other hand, when the expression system is composed of plasmid vectors, it may be advantageous, in the pharmaceutical compositions of the invention, to add chemical or biochemical agents that favor the transfer of the genes. In this respect, mention may be made more particularly of cationic polymers of the polylysine type, (LKLK) n, (LKKL) n such as those described in application 095/21931, polyethylene imine (O96 / 02655) and DEAE dextran or else cationic or lipofectant lipids. They possess the property of condensing the DNA and promoting the association with the cell membrane. Among the latter, mention may be made of lipopolyamines (lipofectamine, transfectama, such as those described in the application).

W095 / 18863 or 09S / 17823) different cationic or neutral lipids (DOTMA, DOGS, DOPE, etc.), as well as peptides of nuclear origin (O96 / 25508) eventually functionalized to target certain tissues. The preparation of a composition according to the invention using one of such chemical vectors, is carried out according to any technique known to the person skilled in the art, generally by the simple putting in contact of the different compounds. The doses of the administered expression system depends on several factors, and especially on the vector used, on the neurotrophic factor (s) involved, the type of promoter used, the stage of the pathology or even the duration of the desired treatment. In general, the expression system is administered in the form of doses comprising 0.1 to 500 mg of DNA per kilogram, preferably 1 to 100 mg of DNA per kilogram. Dosages of 10 mg DNA / kg approximately are generally used. In the case of recombinant adenoviruses, they are formulated and administered advantageously in the form of doses comprised between 10 4 and 10 1 pfu, and preferably 10 to 10 10 pfu. The term pfu ("plaque forming units") corresponds to the infectious power of an adenovirus solution, and is determined by the 2 infection of an appropriate cell culture, and the measurement, usually after 15 days, of the number of pests of the infected cells. The techniques for determining the pfu concentration of a viral solution are well documented in the literature. The injection can be done by means of different devices, and in particular by syringes or by perfusion. Injection by means of syringes is preferred. On the other hand, repeated injections can be practiced to still increase the therapeutic effect. According to a variant of the invention, this treatment can also be applied in combination with riluzole. The invention thus relates to a pharmaceutical composition comprising an expression system according to the invention and a pharmacologically effective amount of riluzole, for the purpose of simultaneous administration or spaced out over time. The results presented below illustrate the present invention without limiting it by this. They demonstrate the particularly advantageous properties of the method of the invention that constitute, according to the knowledge of the applicants, the first disclosure, on an animal model, of one such therapeutic benefit for the SLA.

LEGENDS OF THE FIGURES Figure 1: Comparison of the electromyographic performances of FALSM3A mice with or without the administration of an expression system of a combination of CNTF-GDNF.

Figure 2: Comparison of the electromyographic performances of FALSS93A mice with or without the administration of an NT3 expression system.

Figure 3: Comparison of the survival of pmn mice with or without the administration of a CNTF expression system. The survival of the pmn mice treated by the administration of a CNTF expression system: 100%, n = 7 (curve with thick lines); untreated pmn mice 100%, n = 14 (normal curve).

Figure 4: Comparison of motoneuronal degeneration in pmn mice with or are the administration of a CNTF expression system, the number of myelinated fibers in the phrenic nerve of mice is examined at 25 days of age. Results: pmn mice with CNTF expression system (145, n = 10); pmn mice without "untreated" CNTF expression system (122, n = 8); pmn mice treated with AdlacZ (111, n = 8); "normal" Xt mice (263, n = 4). The vertical bars represent the standard error of the averages (SEM).

EXAMPLES 1. Material and methods The set of experiences described below (construction of adenovirus, injection to the mice, functional measurements) were carried out in the confinement laboratory L3. 1-Animals.

Several lines of transgenic mice that express the mutated forms of SOD responsible for the familiar forms of SLA have been constructed to try to obtain a murine model of the pathology. Transgenic mice overexpressing mutated human SOD that carry a substitution of glycine 93 in alanine (FALSS93A mice) present a progressive motoneuronal degeneration that results in paralysis of the limbs, and performing the measurement at the age of 4-6 months (Gurney et al., 1994). The first Clinical signs consist of a tremor of the limbs to approximately 90 days, then a reduction in the length of the steps to 125 days (Chiu et al., 1995). Histologically, vacuoles of mitochondrial origin are observable in motoneurons after approximately 37 days, and a motoneural loss can be observed after 90 days (Chiu et al., 1995). Scopes of myelinated axons are observed mainly in the ventral medulla and a little in the dorsal region. The phenomena of compensatory collateral reinnervation are observed at the level of the motor plates (Chiu et al., 1995).

For examples 2 to 10, it has been chosen to use mice FA SG 3A.

FALSß3A mice constitute a very good animal model for the study of the pathophysiological mechanisms of SLA as well as for the development of therapeutic strategies. They share in effect with the familiar forms of the SLA, a common pathophysiological origin (SOD mutation), a large number of histopathological and electromyographic characteristics.

Thus, in the laboratory the electromyographic functions of the FALS 93A mice have been characterized in the laboratory, and it has been shown that the FALSG93A mice meet the Lambert criteria for the SLA (Kennel et al., 1996): (1) reduction of the number of motor units with a concomitant collateral reinnervation; (2) the presence of a spontaneous activity of denervation (fibrillation) and fasciculation in the posterior and anterior limbs; (3) modification of the motor conduction velocity correlated with a decrease in the motor evoked response; (4) sensory range step. In addition, the inventors have shown that the reaches of the facial nerves are rare, even in aged FALSS93A mice, which is also the case in patients.

FALSßg »mice that come from Transgenic Alliance (L'Arbresle, France). Pregnant females are released every week. They stop before the animalist of the laboratory. The heterozygous mice that develop the disease are identified by PCR after taking a sample from a piece of the tail and extracting the DNA. There are other animal models that present motoneuronal degenerations (Sillevis-Smitt &De Jong, 1989; Price and collaborators, 1994), either immediately after an acute neurotoxic lesion (treatment with IDPN, excitotoxins) or due to genetic failure (wobbler, pmn, Mnd, Chien HCSMA mice). Among the genetic models, the pmn mice are particularly well characterized on a clinical, histological (Schmalbruch 1991) and electromyographic (Kennel, 1996) level. The pmn mutation is transmitted in the autosomal recessive mode and has been located on chromosome 13. The homozygous pmn mice develop muscular atrophy and paralysis that manifest in the limbs after the age of two to three weeks and that are generalized at once. All untreated pmn mice die before six to seven weeks of age. The degeneration of their motor neurons starts at the level of the nerve endings and ends with a massive loss of the myelinated fibers in the motor nerves and especially in the phrenic nerve, which ensures the innervation of the diaphragm (? Chmalbruch 1991). Contrary to FA SGSJ ?, this muscular denervation is very rapid and is practically not accompanied by signs of reinnervation due to the growth of axonal collaterals. On the electromyographic plane, the process of muscular denervation is characterized by the appearance of fibrillations and by a significant reduction in the amplitude of the muscular response evoked after supramaximal electrical stimulation of the nerve (Kennel et al 1996). A line of Xt / pmn transgenic mice has also been used as another murine model of the SLA. These mice were obtained by a first cross between the female mice C57 / B156 or DBA2 and the male mice Xt pmn * / Xt * pmn (strain 129), followed by a second between the heterozygous females Xt pmn * '/ Xt' pmn * descending (NI) with the initial males. Among the descending mice (N2), the double-heterozygotes Xt pmn * / Xt * pmn (referred to as "Xt pmn mice") carry an Xt allele (evidenced by the Extra-doigt (finger) phenotype) and a pmn allele ( determined by PCR) has been chosen for future growth. 2. Expression systems 2. 1. Plasmidic vectors The different plasmid vectors that allow the expression of one or two neurotrophic factors can be used. Mention may be made, for example, of the plasmids pCRII-BDNF and pSh-Ad-BDNF, which carry a cassette for the expression and secretion of BDNF (WO95 / 25804). Plasmids p-LTR-IX-GDNF can also be mentioned containing a nucleic acid encoding GDNF under the control of the LTR promoter (WO95 / 26408) as well as the plasmid p-LTR-IX-preNGF / CNTF containing the rear CNTF gene sequence and the signal sequence of the betaNGF as well as the repeated inverse sequences (ITR) of the adenoviral genome, the LTR sequences of the Rous Sarcoma virus (RSV) promoter, the encapsidation sequences as well as the adenoviral sequences necessary for homologous recombination. It is understood that any plasmid carrying an origin of replication and a marker gene can be used to construct an expression system according to the invention, by the insertion of one or more cassettes for the expression of a neurotrophic factor. Plasmids can be prepared in a eukaryotic or prokaryotic cell host. 2. 2. -Adenovirus.

As indicated above, viral vectors, and especially adenoviruses, constitute a particularly preferred embodiment of the invention. The recombinant adenoviruses used hereinafter have been obtained by the recombination of the homologue according to the techniques described in the art. previous. In summary, they are constructed in 293 cells, by recombination between a fragment of the linearized viral genome (dl324) and a plasmid that contains the left ITR, the encapsidation sequences, the transgene as well as its promoter and the viral sequences that allow the recombination. The viruses are amplified on 293 cells. They are repurified regularly in the P3 of the inventors' laboratory. Viral genomes can also be prepared in a prokaryotic cell according to the technique described in the application O96 / 25506. The following viruses have been used more particularly: - Ad-CNTF: Recombinant adenovirus of the Ad5 serotype comprising, inserted in the genome in the region of the deleted region, a cassette for expressing the gene CNTF composed of the cDNA encoding the CNTF under the control of a transcriptional promoter (in particular the RSV LTR). The details of the construction are given in the application WO94 / 08026.

The alternative constructs comprise a supplementary deletion in the E4 region, such as those described in the W096 / 22378 application or in the region E3.

Ad-GDNF: Recombinant adenovirus of serotype Ad5 comprising, inserted into its genome at the site of the deleted region, a cassette for the expression of GDNF composed of the cDNA encoding GDNF under the control of a transcriptional promoter (in particular the RSV LTR). The details of the construction are given in the application O95 / 26408). An alternative construction comprises a supplementary deletion in region E4, such as that described in application 096/22378.

- Ad-NT3: Recombinant adenovirus of the Ad5 serotype comprising, inserted in its genome in the place of the deleted region, an expression cassette of the NT3 gene composed of the cDNA encoding the NT3 under the control of a transcriptional promoter (in particularly the LTR of the RSV). An alternative construction comprises a supplementary deletion in the region E4, such as that described in the application W096 / 22378.

Ad-BDNF: Recombinant adenovirus of the Ad5 serotype comprising, inserted in its genome in the place of the deleted region, an expression cassette of the BDNF composed of the cDNA coding for the BDNF under the control of a transcriptional promoter (in particular the RSV LTR). Details of the construction are given in the application WO95 / 25804. An alternative construction comprises a supplementary deletion in region E4, such as that described in application 096/22378.

Ad-FGFa: Recombinant adenovirus of serotype Ad5 comprising, inserted in its genome in the region of the deleted region, a cassette for the expression of FGFa composed of the cDNA encoding FGFa under the control of a transcriptional promoter (in particular the LTR of R? V). The details of the construction are given in the application O95 / 25803). An alternative construction comprises a supplementary deletion in region E4, such as that described in application W096 / 22378.

The functionality of the constructed viruses is verified by the infection of the fibroblasts in the culture. The presence of a corresponding neurotrophic factor is analyzed in the culture supernatant by ELISA and / or evidencing the trophic properties of this supernatant on the primary neuronal cultures. 3. -Administration of recombinant adenoviruses.

Adenoviruses that code for neurotrophic factors are administered intravenously in adult or newborn animals. In adult FA1SG93A mice, 109 pfu of each of the adenoviruses (final volume 200 ul) are thus injected into the caudal vein with the aid of a Hamilton® type microsyringe. In the newborn pmn mice (age 2-3 days), identified by the absence of a supernumerary finger, 2 x 109 pfu (final volume 20 μl) of the adenoviral suspension are injected into the retinal vein with the help of a microsyringe of the type of insulin equipped with a 30 G needle. Newborn animals are lightly anesthetized with ether and under hypothermia. 4-Various techniques.

Electromloqrafla As much as their physical condition permits, the animals are anesthetized by the intraperitoneal injection of a mixture of diazepam (Valium®, Roche, France) and ketamine hydrochloride (Kétalar®, Parke-Davis, France) at a rate of 2 μg / g and 60 μg / g of body weight, respectively. The electromyograph used is a device of the latest generation (Keypoint®) that has the set of software necessary for the acquisition and treatment of electromyographic signals. This material is rented to the company Dantec (Les Ulis, France).

Electromyography of stimulation-detection: motor evoked response (REM) When an electric shock is applied to a nerve, the muscles innervated by this nerve are the residence of an electrical response. They survive after a certain interval of time (distel latency) which corresponds to the conduction times of the stimulation to the synapse, to which the time of signal transmission at the synapse is added. The amplitude of the response is proportional to the amount of muscle fibers innervated. For purely practical reasons, the applicants have chosen to stimulate the sciatic nerve by picking up the motor evoked response at the level of the gastro-calf muscle of the calf. Five electrodes with needle shape. { Dantec) are implanted directly and connected to the electromyograph according to the following scheme: (a) 2 stimulation electrodes are placed, one of them (the active electrode on the path of the sciatic nerve, the other of them (the reference electrode) to the base of the tail, (b) 2 detection electrodes are implanted, one in the gastrocnemius muscle (active electrode), the other on the corresponding tendon (reference electrode), (c) finally an electrode is connected to the ground and is implanted or between the 2 active electrodes, on the thigh of the animal. The amplitude and latency of the REM of the muscle is measured to a stimulation of its motor nerve. It lasts 200 ms at an intensity called supramaximal that corresponds to 150% of the intensity that allows to obtain the maximum action potential. In adult mice, if the muscle and nerve studied are healthy, and under the conditions described below, the amplitude of the evoked response is greater than or equal to 80 mV, and the latency time is generally equal to 0.6 ms.

Histological Analysis Animals are killed by overdose of chloroform and perfused by the intracardiac route with a solution of glutaraldehyde. The phrenic nerves are isolated, samples are taken, they are postfixed by osmium tetroxide and included in epoxy. The phrenic nerves are cut close to the diaphragm, the sections of a thickness of 3 μm are colored with paraphenyldiamine and analyzed by optical microscopy.

. Administration of an expression system that expresses the CNTF gene Injection of the adenoviral vector: Homozygous mice Xt pmn * / Xt * pmn ("pmn mice") aged 2 to 3 days, identified by the absence of the supernumerary finger, have been used for the injection of the adenoviral vector. A suspension of adenoviral CNTF has been prepared by dilution of the adenoviral raw material in a saline-phosphate buffer (PBS) at 2xl09 pfu / μl and administered according to the conditions described in point 3. The AdlacZ coding in E. coli for β-galactosidase (Stratford-Perricaudet, 1992), has been used as the control adenoviral vector.

Results: Northern blot analysis of human fibroblasts infected by AdCNTF demonstrates the presence of two recombinant transcripts of a respective size of 1.1 and 1.6 kb. ELISA analysis reveals the presence of recombinant proteins in the supernatants after infection of the different cell types. All the pmn mice not treated in the experimental sera are deceased before the age of two months and the average of their survival has been 40.4 + 2.4 days (n = 14). The administration of the AdlacZ control vector has not modified the survival of pmn mice. In contrast, pmn mice treated by intravenous injections of AdCNTF have survived up to 73 days (Figure 3). The average survival of the pmn mice treated by the AdCNTF has been significantly improved and represents 52.7 + 3.9 days (n = 7, p <0.011) (The differences between the results of the healthy mice Xy / pmn, of the mice untreated homozygotes and treated pmp mice have been analyzed by Student's t test, values are given on average + standard error of averages (SEM)).

In order to determine if the prolongation of the survival of the pmn mice treated by the AdCNTF reflects an increase in the number of phrenic nerve fibers, an optical microscopy on day 25 has been performed and has shown that in the untreated pmn mice and in the pmn mice that have received the AdlacZ intravenously, the number of myelinated fibers in the phrenic nerves has decreased respectively to 122 + 13 (n = 6) and 111 + 11 (n = 8) compared to the 263 + 8 myelinated fibers in healthy mice (n = 4). The number of myelinated fibers in the phrenic nerves of the pmn mice to which the AdCNTF has been injected is significantly higher than that of the control animals (145 _ + 11, n = 10, p <0.05. of the pmn mice by the AdCNTF induces a 20% reduction in the loss of the myelinated fibers (Figure 4). 6. Administration of an expression system that produces a combination of CNTF-GD F 109 pfu of each of the Ad-CNTF adenovirus and Ad-GDNF have been injected (caudal vein) with the help of the microsyringe in a final volume of 200 μl to 4 FALSS93A mice aged 99 days. In the course of time, the electromyographic workings of the animals have been followed and compared in a control group. The average life span has also been recorded.

Electromyography The results obtained are presented on Figure 1. A reduction of the amplitude of the motor evoked response (REM) in the gastrokemic muscle of the treated FALSG93A mice (AdCNTF + AdGDNF) as well as the untreated FALSG93A mice is observed. This reduction reflects the process of progressive denervation that is one of the characteristics of the SLA. However, the treated mice have an amplitude of REM systematically higher than that of the controls, which shows a decrease in functional range following the treatment.

Longevity The life span of the animals is indicated in the following tables.

Treated animals Animals not treated: The results show that all the animals of the treated group are deceased at an age greater than or equal to the age of the oldest living animal in the control group. These results also show an increase in the duration of life in the treated animals of 30 days on average, with respect to the control animals. These results are particularly unexpected and, compared to 13 days obtained with Rilutek®, demonstrate the therapeutic potential of the method of the invention. 7. Managing an expression system that produces NT3 7 (a) - Administration of an expression system that produces NT3 (mice aged 99 days) 109 pfu of Ad-NT3 adenovirus have been injected (caudal vein) with the help of a microsyringe in a final volume of 200 μl to 4 FALSG93A mice aged 99 days. Over time, the electromyographic workings of the animals are followed and compared with a control group. The results obtained are presented on Figure 2 and show that the treated mice have an amplitude of REM superior to that of the controls, which demonstrate a decrease in functional range following the treatment. 7 (t >) - Administration of an expression system that produces NT3 (mice aged 3 days) . 108 pfu of the adenovirus Ad-NT3 have been injected (temporary vein) with the help of the microsyringe in a final volume of 20 μl to the FALSG93A mice aged 3 days. The life span of the animals is indicated in the following tables.

Treated animals Animals not treated: The results show an increase of 16.1 days of the average lifespan between animals that have been treated with Ad-NT3 intravenously and untreated animals. 8. Administration of an expression system that produces a combination of CNTF-NT3 Ad-CNTF and Ad-NT3 have been injected (caudal vein) with the help of a microsyringe in a final volume of 200 μl to 4 animals aged 99 days. Over time, the electromyographic workings of the animals are followed and compared with a control group. The average lifespan is recorded equally. 9. Administration of an expression system that produces a combination of BDNF - NT3 109 pfu of each of the Ad-BDNF adenovirus and Ad-NT3 have been injected (caudal vein) with the help of a microsyringe in a final volume of 200 μl to 4 animals aged 99 days. During the course of time, the electromyographic functions of the animals are followed and compared with a control group. The average life span is recorded equally.

. Administration of an expression system that produces BDNF 109 pfu of Ad-BDNF adenovirus have been injected (caudal vein) with the help of a microsyringe in a final volume of 200 μl to 4 animals aged 99 days. During the course of time, the electromyographic workings of the animals are followed and compared with a control group. The average lifespan is recorded equally.

Bibliography - AKLI S., et al., Nature genet., 2, 224-228, 1993. - APPEL S.H .. et al. Autoimmunity as an etiological factor n sporadic amyotrophic lateral sclerosts. In Serratrice G.T. and Munsat T.L. eds. Pathogenesis and therapy of amyotrophic lateral sclerosis. Advances in Neurology, 6_ &, pp. 47-58, 1995. Lippincott-Raven publishers, Philadelphia. -BAJOCCHI G. et al. Nature genet., 2, 229-234, 1993. -BARKATS M., et al. Neuroreport, 7, 497-501, 1996. -BARINAGA M. Science 2 =, 772-774, 1994. -CASTEL BARTHE M.N. et al. Neurobiology of Disease _2, 76-86, 1996. -CHIU A. Y., et al. Mol. Cell. Neurosci., =, 349-362, 1995. -DAVIDSON B.L., et al. Nature genet., 2, 219-223, 1993. -DITTRICH F., Ann. Neurol., 21, 151-163, 1994. -FINIELS F. et al., Neuroreport, 7, 373-378, 1995. -GASTAUT JL The viral hypothesis In Serratrice GT and Munsat TL eds.

Pathogenesis and therapy of amyotrophic lateral sclerosis. Advances in Neurology, 68. pp. 135-138, 1995. Lippincott-Raven publishers, Philadelphia. -GURNEY M.E., PU H "CHIU A. Y. et al. Science, 2 = á, 1772-1775, 1994.-GURNEY M.E., CUTTING F.B., ZHAI P. et al. Ann. Neurol., 22, 147-157, 1996.

-HENDERSON CE., CAMU W "METTLING C. et ai. Nature, 2 = 2, 266-270, 1 93.

-HENDERSON C.E. et al. Science, 2 = £ 1062-1064, 1994. -HENDERSON C.E Neurotrophic factors as therapeutic agents in amyotrophic lateral sclerosis: potential and pitfalls. In Serratrice G.T. and Munsat T.L. eds.

Pathogenesis and therapy of amyotrophic lateral sclerosis. Advances in Neurology, = S, pp. 235-240, 1995. Lippincott-Raven publishers, Philadelphia. -HORELLOU P., VIGNE E., CASTEL M.N. et al. Neuroreport, =, 49-53, 1 94.

-HUGHES R.A. et al. Neuron, lfi, 369-377, 1993. -K ENNEL P.F., FI IELS F., REVAH F. et al. Neuroreport, 7, 1427-1431, 1996a.

-K ENNEL P.F. etal. Neurobiol Disease: 137-147, 1996.

-La GAL LA SALLE G. et al Science, 2 = 2, 430-433, 1993. -LEWIS M E. et al. Exp. Neurol., 12a, 73-88, 1993. -OPPENHEIM RW, YIN QW, PREVETTE D . et al. Natura, 2SQ, 755-757, 1992.

-OPPENHEIM R.W. et al Nature, 212, 344-346, 1995. -PENNICA D., ARCE V., SWANSON T.A. et al. Neuron, H, 63-74, 1996. -PRICE D.L. et al., Neurobiol. Disease, 1, 3-1 1, 1 94. -ROSEN D., SIDDIQUE T., PATTERSON D. et al. Nature, 262, 59-62, 1993.

-ROTHSTEGN J D. Excitoto mechanisms ín the pathogenesis of amyotrophic lateral sclerosis. In Serratpce G.T. and Munsat T.L. eds. Pathogenesis and therapy of amyotiophic lateral sclerosis. Advances ín Neurology, é8_, pp. 7-20, 1995. Lippincott-Raven publishers, Philadelphia. -ROWLAND L.P. Proc. Nati Acad. Sci. USA, 22, 1251-1253, 1995. -RUBIN B.A. and RORKE L.B. Vaccine adenoviruses. In Plotkín and Mortimer eds, Vaccmes, pp. 492-512, 1988. W.B. Saunders, Philadelphia. -SCHMALBRUCH H., JENSEN H.S., BJAERG M, KAMIENIECKA Z. et K.URLAN L. J. Neuropathol Exp Neurol. iQ: 192-204, 1991. -SENDTNER M. et al. Nature, 21i, 502-504, 1992a. -SENDTNER M, HOLTMANN B., KOLBECK R. Nature, 2 = O, 757-759, 1992b.

-SILLEVIS SMITT P A E. et al., J. Neurol. Sci., 21, 231 -258, 1989. -STRATFORD-PERRICAUDET L.D., MAKEH I., PERRICAUDET M., BRIAND P. J. Clin Invest. 211, 626-630, 1992. -VEJSADA R., SAGOT Y. and KATO A.C. Eur. J. Neurosci., 7, 108-1 15, 1995.

-WINDEBAN A.J. Use of growth factors in the treatment of motor neuron diseases. In Serratrice G.T. and Munsat T.L. eds. Pathogenests and therapy of amyotrophic lateral sclerosis. Advances in Neurology, = S, pp. 229-234, 1995.

Lipp cott-Raven publishers, Philadelphia-YAN Q, ELLIOTT J. and SN1DER W.D. Nature, 2 = Q, 753-755, 1992. -YANG Y, ERTL H.C.J., and WILSON J M. Immumty, 1, 433-442, 1994. -YANQ, MATHESON C, LÓPEZ O.T. et al. J. Neurosci., 14, 5281-5291, 1994.

-And ASE Y. Metal metabolism in motor neuron disease. In Chen K.M. and Yase Y. eds. Amyotrophic lateral sclerosis m Asia and Oceania, Taipei, pp. 337-356, 1984. Taiwan: National Taiwan University Press. -YEH P., DEDIEU J F., ORSINI C. et al. I Viral., 2Ü, 559-565, 1996. -YIM M.B., et al. Proc. Nati Acad. Sci. USA, 22, 5709-5714, 1996.

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 to which it relates.

Having described the invention as above, the content of the following is claimed as property

Claims (25)

1. The use of a system for the expression of neurotrophic factors for the preparation of a pharmaceutical composition intended for the treatment of SLA by systemic administration.

2. The use according to claim 1, wherein the expression system comprises an expression cassette composed of a nucleic acid encoding a neurotrophic factor under the control of a transcriptional promoter.

3. The use according to claim 1, wherein the expression system comprises two expression cassettes each composed of a nucleic acid encoding each for a different neurotrophic factor, under the control of a transcriptional promoter.

4. The use according to claim 1, wherein the expression system comprises an expression cassette composed of two nucleic acids encoding a neurotrophic factor different, under the control of a single transcriptional promoter (bicistronic unit).

5. The use according to claim 2, wherein the neurotrophic factor is chosen from GDNF, CNTF, BDNF and NT3.

6. The use according to claims 3 or 4, wherein each nucleic acid encodes a different neurotrophic factor chosen from GDNF, CNTF, BDNF and NT3.

7. The use according to claim 6, wherein the expression system comprises a nucleic acid encoding CNTF and a nucleic acid encoding GDNF.

8. The use according to one of claims 2 to 4, wherein the expression cassettes are part of a vector.

9. The use according to claim 8, wherein the expression cassettes are part of a plasmid vector.

10. The use according to claim 8, wherein the expression cassettes are part of a viral vector.

11. The use according to claim 10, wherein the viral vector is an adenoviral vector.

12. The use according to one of the precg claims, wherein the promoter is a constitutive eukaryotic or viral promoter.

13. The use according to one of the precg claims, wherein the systemic administration is an intravenous administration.

14. A pharmaceutical composition for the treatment of degenerative diseases of motoneurons, characterized in that it comprises a system that allows the expression of two neurotrophic factors.

15. The composition according to claim 14, characterized in that said system comprises two gene transfer vectors that Each one carries a nucleic acid that codes for a different neurotrophic factor.

16. The composition according to claim 14, characterized in that said system comprises a gene transfer vector carrying a cassette that allows the concomitant expression of two different neurotrophic factors.

17. The composition according to claim 15 or 16, characterized in that the vectors are viral vectors.

18. The composition according to claim 17, characterized in that the vectors are adenoviruses.

19. The composition according to claim 15 or 16, characterized in that the vectors are plasmid vectors.

20. The composition according to claim 14, characterized in that the neurotrophic factors are chosen from GDNF, BDNF, CNTF, and NT3.

21. The composition according to claim 20, characterized in that it contains two defective recombinant adenoviruses, one carrying a nucleic acid encoding CNTF and the other carrying GDNF.

22. The composition according to claim 20, characterized in that it contains two defective recombinant adenoviruses, one carrying a nucleic acid encoding GDNF and the other carrying NT3.

23. The composition according to claim 20, characterized in that it contains two defective recombinant adenoviruses, one carrying a nucleic acid encoding BDNF and the other carrying NT3.

24. The composition according to claim 14, characterized in that it is injected intravenously.

25. A pharmaceutical composition, characterized in that it comprises a system for expressing the neurotrophic and riluzole factors, for simultaneous administration or spaced out over time.