MXPA00007617A - Use of negative regulation elements for nerve-specific expression of transgenes - Google Patents

Abstract

The invention concerns novel methods and constructs for controlling nucleic acid expression, in particular methods and constructs using NRSE sequences for obtaining a targeted expression of transgenes in the nerve cells in vivo or ex vivo. The invention is particularly adapted for in vivo gene transfer applications, for instance for therapeutic or scientific approach.

Description

UTILIZATION OF NEGATIVE REGULATION ELEMENTS FOR THE NEUROSPECIFIC EXPRESSION OF TRANSGENES The present invention relates to new methods, constructions as well as vectors containing these constructions, which allow to control the expression of a nucleic acid. In particular, the invention relates to methods, constructs and vectors that allow obtaining a directed expression of transgenes in nerve cells in vi v or ex vi ve. The invention is particularly adapted to the applications of transferring genes in vi, for example, therapeutic or scientific methods.

Gene therapy in vi ve or ex vi ve allows local transfer and efficiency of genes encoding parafactors of interest (transgenes), particularly in the nervous system of a host organism. In this regard, different types of vectors have been successfully used for the transfer of different types of transgenes in nerve cells in vi v or ex vi ve. Mention may be made in particular of viral vectors of the adenovirus, AAV or HSV type, or certain non-viral vectors of the cationic polymer type (for example polyethylene imine). Thus, these vectors have allowed a transfer REF .: 121592 efficient and stable transgenes in cells of the nervous system in vi vo (WO 94/08026, WO 93/09239, WO 96/02655). The possibility of carrying out this type of transfer offers numerous applications, particularly in the medical field. Thus, these vectors are used in gene therapy methods in vi ve or ex vi ve. In this regard, different pre-clinical studies are currently underway that refer to the transfer of trophic factors in the nervous system. These vectors are also used for the creation of transgenic animals that allow compounds to be tested or even for different labeling or bioavailability studies.

For all these applications, even when good efficiency and good transfer stability seem to be achieved today, control of the expression of transgenes is imperative. Therefore, different systems have been described to try to restrict the expression of transgenes in certain tissues, only, for example, using promoters called "tissue specific" or complex chimeric systems that require the use of many constructions and / or regulatory factors. As examples of tissue-specific promoters, mention may be made in particular of the Glial Fibrillary Acidic Protein promoter, which is particularly active in glial nerve cells. However, the regulation systems available or valid at that time have certain drawbacks, and particularly a force and / or selectivity not always satisfactory. Thus, in the vast majority of the known systems, selectivity is obtained in general to the detriment of the strength of the promoter, and therefore it is difficult to avoid an expression that is both important, stable and specific.

The present invention provides methods, constructions as well as vectors containing these constructions, which allow to solve the drawbacks of the prior art. In particular, the present invention describes a chimeric promoter that allows the neuronal direction of expression. The invention uses in this aspect, elements of negative regulation, which prevent the expression of genes in non-neuronal cells. This type of construction has many advantages compared to previous systems. A part of expression sequences and ubiquitous promoters that are always short and well characterized can easily be associated with other regulatory elements. On the other hand, these regulatory elements can be associated in theory no matter what ubiquitous promoter and in this way they represent an astute system that allows to easily obtain a neurospecific expression of transgenes of interest even when the expression vector uses a little or no tropism for nerve cells.

Numerous papers describe cis sequences that repress the transcription of neuronal genes in non-neuronal cells. A particular type of sequences endowed with this property is constituted by NRSE ("Neuronal Restrictive Silencing Element"), still called RE-1 (element-1 repressor). The NRSE is a short sequence, approximately 21 bp, evidenced before many neuronal genes: SCG10 [1], sodium channel II [2] and synapsin I [3] for example. On the other hand, some analogous sequences have been rediscovered above other neuronal genes, even though their functionality has not always been demonstrated so far [4]. These NRSE sequences repress the non-neuronal expression of genes, by binding a specific transcription factor, the NRSF protein (Factor Neuronal Restrictive Silencer), still called REST (transcription factor silencer RE-1), present only in non-neuronal cells [5]. This protein belongs to the family of transcription factors in amounts of zinc. It intervenes in the course of development to initiate the repression of neuronal genes in non-neuronal cells after adulthood in the maintenance of this repression.

The teams that have characterized the presence of the NRSE sequences over the neuronal genes, verified the ability of these sequences to reduce the expression of reporter genes after the transfection of non-neuronal cells. They have in this target their NRSE sequence associated with a minimal heterologous promoter, that is, a promoter that does not belong to a basal transcript. Thus, placed, for example, above the thymidine kinase promoter, a copy of the NRSE sequence of the calcium channel II reduces the expression of the reporter gene of the radical in non-nervous cells [9]. Likewise, two copies of the NRSE sequence of synapsin I associated with the minimal c-fos promoter allow the expression in non-nervous cells to be reduced to 75% [3]. On the other hand, the presence of an NRSE sequence has also allowed to regulate SV40 promoter activity in non-neuronal cells [10].

However, NRSE sequences have never been used to direct the expression of a strong ubiquitous promoter, nor in a gene therapy perspective. Thus, j ama 's has been shown if such sequences were capable of suppressing expression induced by a strong promoter. Likewise, it has never been shown if these sequences, placed in a chimerical environment, were always active in vi. More generally, the activity of these sequences combined with heterologous promoters has never been demonstrated until now.

The invention relates to recombinant nucleic acids that allow the neuronspecific expression of genes. It also refers to the vectors comprising these nucleic acids, particularly of viral origin, as well as the cells containing these nucleic acids and / or vectors. The invention also relates to particular chimeric promoters, adapted to the neurospecific expression of genes in the nervous system in vi. Furthermore, the invention relates to compositions comprising these elements and their use for the transfer of genes.

A first aspect of the invention resides in a recombinant nucleic acid comprising: a promoter - one or several NRSE sequences, and - a therapeutic gene.

The constructions according to the invention thus comprise a region of repression in the non-neuronal cells of the active expression of the therapeutic gene, induced by the promoter. This repression region is made up of one or more NRSE regions.

The NRSE region used in the framework of the present invention advantageously comprises all or part of the following 21 bp sequence: TTCAGCACCACGGAGAGTGCC (SEQ ID n ' This sequence corresponds to the NRSE sequence of the SCG10 gene [2]. Furthermore, it is understood that the NRSE region used in the context of the invention may comprise certain variations in relation to this sequence., to the extent where it retains the repression properties indicated above. Thus, sequences of the NRSE type that present certain variations have been observed in different genes, such as those described in the article by Schoenherr et al. [4], incorporated in the present application by reference. Based on the observed variants, a consensus NRSE sequence has been defined, corresponding to the sequence most frequently found. This sequences is: TTCAGCACCACGGACAGCGCC (SEQ ID No. 2). Preferred variants in the sense of the invention comprise substitutions about 1 to 5 base pairs of the sequence SEQ ID No. 2 above. More preferably, they comprise variations on 1 to 3 base pairs. In this aspect, the variations are preferably located on residues 1, 2, 10, 11, 15, 18, 19 and / or 20 of the previous sequence. The substitutions may correspond to the suppression or replacement of the base referred to by all the other base. Thus, an NRSE portion may be represented in the general, all or part of the following sequence: NNCAGCACCNNGGANAGNNNC (SEQ ID No. 3) in which N designates a base chosen from A, T, C or G. Particular examples of portions NRSEs used in the context of the present invention are particularly the following: TTCAGCACCACGGACAGCGCC (SEC ID no0 2) TTCAGCACCGCGGACAGTGCC (SEQ ID No. 4) TTCAGCACCGCGGACAGTGCC (SEQ ID no0 5 TTCAGCACCTCGGACAGCATC (SEQ ID no 6 TTCAGCACCGCGGAGAGCGTC (SEQ ID no 7 TCCAGCACCGTGGACAGAGCC (SEC ID no TTCAGCACCGAGGACGGCGGA (SEQ ID no 9) ATCAGCACCACGGACAGCGGC (SEQ ID no 10) TTCAGCACCTAGGACAGAGGC (SEQ ID no 11) In addition, these regions may also comprise, in one or the other or in both extremities, additional bases that do not interfere with the repression activity indicated above. In particular, they are restriction sites, neutral sequences or sequences that come from cloning steps and cin, for example, regions of a plasmid. or a vector, or sequences bordering the NRSE region in the gene of origin.

These different regions can be prepared by all techniques known to those skilled in the art for the preparation of nucleic acids. Thus, they can be prepared by the techniques of automatic synthesis of nucleic acids using commercial synthesizers. They can also be obtained by the screening of DNA banks, for example by hybridization with specific probes or by ligation experiments to an NRSF factor. In addition, all the other variant of the sequence SEQ ID No. 1 can be identified from the DNA libraries, for example by homology investigation.

Once synthesized, the region of the NRSE type can then be tested functionally. Therefore, a first test consists in determining the capacity of the region that links the NRSF factor. This can be done within the different conditions and for example by the experiences of migration delay on gel, according to the technique described by Morí et al. [2] or by Schoenherr et al. [5], incorporated herein by reference. In a second time, the capacity of the region that represses expression can be determined by the insertion of this region into a test plasmid cining a reporter gene (Chloramphenicol, Acetyltransferase, Luciferase, LacZ, etc.) under the col of a promoter, and the comparison of the expression levels of the aforementioned reporter gene in nerve cells and in non-nervous cells. This type of methodology is described, for example in Schoenherr et al. [4,5] as well as the examples. Preferably, the region is considered functional when it generates a difference of expression between the nerve and non-nerve cells of at least 40%. More preferably, this differential is at least 50%, advantageously at least 60%.

As indicated above, the nucleic acids of the invention may comprise one or more NRSE regions as defined above. It is that the same region repeated several times, be of many variants. Preferably, the constructions of the invention comprise the same region repeated many times. The constructions can comprise up to 50 regions. Advantageously, the region is repeated from 2 to 20 times, and preferably from 3 to 15. As illustrated in the examples, interesting results have been obtained with repeats of 3, 6 or 12 regions, the results are particularly important with 6 and 12 repetitions The NRSE regions can be introduced into the constructions of the invention throughout the non-transcribed or untranslated region or in an intron. Advantageously, they are placed in the 5 'non-coding regions, even more preferably in the proximal region of the promoter. On the other hand, the activity, of these regions that are independent of their orientation, can be introduced in the sense of transcription as well as in the opposite orientation. Finally, when many regions are used, they are inserted preferably side by side, in the same region of the construction. It is understood that, however, they can be introduced in different regions.

The second element that goes into the composition of the nucleic acids of the invention is a promoter, which allows the expression of the transgene in the visualized cell. Advantageously, it is an active promoter in nerve cells or tissues, particularly a eukaryotic promoter. In this aspect, it is, for example, a ubiquitous promoter, that is, functional in most cell types. Even more preferably, it is a ubiquitous eukaryotic promoter. The promoter can be autologous, meaning that it comes from the same species as the cell in which the expression is sought or xenogenic (that comes from another species). Eukaryotic ubiquitous promoters of strong promoters, such as the promoter of the phosphoglycerat or kinase 1 (PGK) gene, can be cited as advantageous examples. It is understood by the promoter that is said strong, any promoter in which the activity is comparable with that of the viral promoters. In eukaryotes, PGK is an enzyme that intervenes during glycolysis. In the mouse. The promoter of this gene, of around 500 bp, comprises a region called "enhancer" (-440 / -120) and a promoter region (-120 / + 80) that contain more sites of transcription initiation [6]. The efficiency of this PGK promoter has already been demonstrated in the previous experiences of gene transfer i n vi t ro and in vi vo [7,8].

According to a preferred embodiment, the invention relates to a nucleic acid comprising the PGK promoter and one or more NRSE sequences. As illustrated in the examples, this type of construction allows to direct the neurospecific expression of a transgene. In this regard, the invention also relates to a chimeric promoter comprising a strong ubiquitous promoter and one or more NRSE sequences. The invention shows in effect that these NRSE sequences can be used to efficiently repress the activity of a strong promoter, included in vi. This thus returns the possible use of these new promoters in numerous applications. A particular chimeric promoter in the sense of the invention is represented by the designation xNRSE-PGK wherein x is an integer from 1 to 50 and preferably from 1 to 20.

Of the other ubiquitous eukaryotic promoters used within the framework of the present invention, are for example the promoters that direct the expression of the obligatory cellular metabolism genes (these genes are called "domesticated" or "domesticated") and specify the necessary proteins in functions common to all cells). These are, for example, genes that intervene in the Krebs cycle, in cellular respiration or even in the replication or transcription of other genes. Mention may be made, with or particular examples of this type of promoter, of the promoter of the genes al-antitrypsin, β-actin, vimentin, aldolase A or Efla (elongation factor).

The promoter used in the context of the invention can still be a neuro-specific eukaryotic promoter, which thus makes it possible to improve its neurospecificity. The promoter of the NSE (Neuronal Specific Enolase) genes can be cited as an example.

NF (Neurofilament), TH (Tyrosine Hydroxylase), DAT (Dopamine transporter), chAT (Acetyl choline transferase), DBH. { Dopamine β-hydroxylase), TPH (Tryptophan hydroxylase), GAD (glutamic acid dehydrogenase) and, more generally, all the promoters of synthesis enzymes or transporters of neuromediators or all other gene promoters in which the expression is specific of a type or on the given neuronal or glial type.

Finally, the use of viral promoters can also be visualized, such as, for example, the promoters CMV (Cytomegalovirus), RSV (Rous Sarcoma Virus), TK (Thymidine kinase), SV40 (Simian virus) and LTR (Repetition Long terminal).

On the other hand, the nucleic acids of the invention also comprise a therapeutic gene. The term "therapeutic gene" is understood to mean, in the sense of the present invention, all nucleic acid comprising at least one open reading phase that encodes an RNA or a therapeutic or vaccine polypeptide. The nucleic acid can be a complementary, genomic, synthetic or semi-synthetic DNA. It can be of varied origin, such as mammal, plant, viral, artificial, etc. The product of transcription or translation present in the therapeutic or vaccinal properties. It can be mentioned by way of particular examples of enzymes, crossing factors (trophic factors in particular), neurotransmitters or their precursors, toxic factors (for example thymidine kinase), antibodies or fragments of antibodies, etc.

The use of constructions of the invention can be visualized to direct the expression of one or more genes that encode an RNA or a protein that desired to direct the neuron without the expression of those in non-neuronal cells, with the objective of establishing animal models or in a perspective of etiological or symptomatic, substitutive or suppressive therapy. These are, for example, genes of the family of trophic factors, such as, for example, neurotrophins (NGF, BDNF, NT3, NT4-5, GMF ...), the crossing factors [family of fibroblast growth factors] FGF (a and b), family of vascular endothelial cell growth factors VEGF, family of epidermal growth factors EGF, family of insulin growth factors IGF (I and II)], the superfamily of TGFβ from the families of the TGFß, GDNF / neurturin.

They are also genes encoding cytokines, such as, for example, CNTF, LIF, Onconstatin M, Cardiotrophin 1 or proteins of the interleukin family.

They are still genes that code for the receptors of these different factors or that code for the transcription factors that regulate the expression of these different factors. It also deals with "genes that encode the synthesis enzymes or the degradation of different neurotransmitters and neuropeptides or their precursors or essential cofactors in this synthesis or this catabolism, or even transcription factors that regulate the expression of these proteins as well as genes that encode the neurotransmitter / neuropeptide receptors (or for the subunits of these receptors) and for the proteins that intervene in the transduction pathways.

Other genes of interest in the context of the invention are in particular genes encoding antioxidant agents such as, for example, SOD (Superoxide Dismutase), GPX (Glutathione Peroxidase), Catalase or an enzyme of cellular respiration; the enzymes involved in the cell cycle as p21, or other inhibitory proteins of the dependent kinases as well as the apoptose genes such as ICE, Bcl2, BAX ...

More generally, the entire gene from which the expression abnormality induces a pathology of the nervous system can be expressed in the constructions of the invention, such as the genes in which the mutation is directly at the origin of the pathologies or the genes in which the products intervene in the same metabolic pathway. They are still toxic genes, for anticancer therapy (for example Thymidine Kinase or Cytokine Deaminase), antisense RNAs, ribozymes, even reporter genes for development studies, kinetics and / or bioavailability, such as the ß-Galactosidase genes , Luciferase or GFP (Green fluorescent protein). It is also about the genes involved in conditional recombination systems in the nervous system with the aim of establishing models of animals, such as transgenic animals that comprise the conditional elimination system.

It is understood that the person skilled in the art can conveniently adapt the type of gene according to the use of the research.

In the nucleic acids of the invention, the different elements are grouped so that the promoter controls the expression of the therapeutic gene or the NRSE sequences control the activity of the promoter. In general, the gene is placed under the promoter and in phase with it.

On the other hand, the regulatory region is generally placed under the promoter, although, as indicated above, it is not necessary with the activity. The distance between the regulatory region (NRSE sequences) and the promoter is variable according to the nature of the sequences used and the number of repeats of the NRSE region. Advantageously, the regulatory sequences are placed at a distance less than 2 kb from the promoter, preferably at a distance of less than 1 kb.

According to a particular form of the invention, NRSE sequences are associated with regulatory systems for the purpose of finely controlling the expression of nucleic acids in cells. Among the regulatory systems, mention may be made of the system using a part of the transactivator tTA controlled by the tetracycline and another part of a sensitive tTA promoter, such as that described in the application FR 98/140080 and incorporated in the present application by reference. Briefly, the nucleic acid comprises a first region comprising a nucleic acid encoding the transactivator of the regulation system for tetracycline (tTA) under the control of a moderate promoter, such as a second region comprising a nucleic acid of interest under the control of a promoter sensitive to tTA. This sensitive promoter can be the entire promoter, even strong, in which the activity is increased in the presence of the transactivator. From a structural point of view, this promoter comprises, in its sequence or at a functional distance thereof, at least one ligation site (or operator region Op) of the tTA factor. Preferably, the two preceding regions are separated. For carrying out the invention, the NRSE sequences can be placed under the sensitive tTA promoter although this is not necessary with the activity, comprised between the two regions described above.

This particular mode of the invention makes it possible advantageously to avoid at the same time not only a regulation of the expression of a nucleic acid by tetracycline, but also a specificity of the expression tissue and particularly in the nerve cells, provided by the NRSE sequences. In addition, this fine regulation of the promoter ensures a remarkable efficiency and safety in the expression of transgenes, necessary in gene therapy.

The present invention also relates to vectors comprising a nucleic acid such as defined above. This vector is advantageously capable of the transduction of the nerve cells of mammals, particularly humans, but it is not necessary that it possess a particular tropism for the so-called cells. Thus, the invention allows the advantageous use of all types of vectors. It is a vector of plasmid type (plasmid, episome, artificial chromosome, etc.) or viral. Among the latter, the vectors derived from adenoviruses, AAVs and herpes viruses can be mentioned in a privileged manner, in which the tropism for cells and nervous tissues has been strongly documented in the prior art. Mention may also be made of other viruses such as retroviruses and rhabdoviruses. In this regard, the examples provided below demonstrate that the NRSE sequences introduced into the viral vectors have a very important activity. Thus, unexpectedly, when NRSE sequences and particularly 6 and 12 sequences have been introduced into a viral vector (adenovirus), they induce a reduction of 91 to 98%, respectively, of the expression of a transgene in non-nervous cells in vi t ro and from 90 to 96% in vi vo. These results advantageously show that it is possible to express specifically in nerve cells a nucleic acid of interest without expression in non-nervous cells, according to a simple system, thanks to these regulatory sequences.

Furthermore, these NRSE sequences have the advantage due to their small size, particularly adapted for the regulation of the expression of genes incorporated in a vector in which the insertion space of the regulatory sequences is limited. Also, it is now possible to visualize associating these sequences with other regulatory systems in the same vector.

A particular object of the invention resides in a defective recombinant adenovirus comprising a nucleic acid of interest under the control of expression sequences, characterized in that the said expression sequences comprise a promoter and one or more NRSE sequences.

In a general manner, the recombinant viruses of the invention are defective, ie incapable of autonomous replication in a cell. The production of defective recombinant viruses is known to the person skilled in the art, as illustrated for example in GRAHAM F. and PREVEC L, (In: Methods in Molecular Biology (1991) MURRAY EJ (ed), the Humana Press Inc, Clifton, NJ, chapter 11, pp. 109-128). Particularly, each of these viruses can be manufactured in encapsidation cell lines that have so-called deficient functions. Such lines have been described in the literature (293 and derived for example).

According to a particular embodiment of the invention, the defective recombinant virus is an adenovirus. In particular, for this virus, the different serotypes have been characterized, in which the structure and properties vary a little. Among these serotypes, it is preferred to use human adenovirus type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin within the framework of the present invention (see application WO 94/26914). Among the adenoviruses of animal origin used in the context of the present invention can be mentioned adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), sheep, swine, aviary or even simia (example: SAV).

Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus. [strain anhattan or A26 / 61 (ATCC VR-800) for example]. Preferably, it is used in the context of the invention of adenoviruses of human or canine or mixed origin.

Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence allowing encapsidation and a nucleic acid according to the invention. Even more preferably, in the genome of the adenoviruses of the invention, the El region is at least not functional. The considered viral gene may become non-functional throughout the art known to the person skilled in the art, particularly by total suppression, substitution, partial elimination or addition of one or more bases in the gene (s) considered. Such modifications can be obtained in vi ve (on isolated DNA) or in si t u, for example, by means of techniques of genetic research methods, or even by treatment by means of mutagenic agents. Similarly, other regions can be modified, and particularly the region E3 (WO 95/02697), E2 (WO 94 / 28-938), E4 (WO 94/28152, WO 94/12649, WO 95/02697). and L5 (WO 95/02697). According to a preferred form of implementation, the adenovirus according to the invention comprises a deletion in the El and E4 regions. According to another preferred embodiment, it comprises a deletion in the El region at which the E4 region and the nucleic sequence of the invention are inserted (Cf FR 94 13355). It is also a recombinant adenovirus, defective for example, by the regions El and / or E2 and / or E4. In the virus of the invention, the deletion in the El region preferably extends from nucleotides 455 to 3329 over the Ad5 adenovirus sequence.

Defective recombinant adenoviruses according to the invention can be prepared by all techniques known to the person skilled in the art (Levrero et al. Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia a nucleic acid of the invention or a nucleic acid of interest under the control of expression sequences comprising a promoter and one or more NRSE sequences. Homologous recombination occurs after co-transfection of the adenoviruses and plasmids into an appropriate cell line. The cell line used should preferably be (i) transformable by the elements and (ii) comprise the sequences capable of complementing the defective adenovirus genome part, preferably under the integrated form to avoid the risks of recombination. As an example of a line, the line of the human embryonic kingdom can be mentioned 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains, in particular, integrated in its genome, the left part of the genome of an Ad5 adenovirus (12%) or of the lines capable of complementing the functions. and E4 such as those described particularly in publications No. WO 94/26914 and WO 95/02697 or in Yeh et al., J. Virol. 70 (1996) 559.

Next, the multiplying adenoviruses are recovered and purified according to the classical techniques of molecular biology.

According to another particular embodiment of the invention, the defective recombinant virus is an AAV. Adeno-associated viruses (AAV), are viruses with DNA of relatively small size, which are integrated into the genome of cells that are infected, stably and site-specific. They are capable of infecting a large spectrum of cells, without inducing effect on the crossing, morphology or cell differentiation. On the other hand, they do not seem to be involved in man's pathologies. The genomes of AAV have been cloned, sequenced and characterized. They comprise approximately 4700 bases, and contain in each extremity an inverse repeat region (ITR) of approximately 145 bases, which serve as the origin of replication for the virus. The rest of the genome is divided into 2 essential regions that carry the functions of encapsidation: the left part of the genome, which contains the rep gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene that encodes the proteins of the virus capsid (virus coating).

The use of vectors derived from AAV for the transfer of genes in vi tro and in vi vo has been described in the literature (see particularly WO 91/18088, WO 93/09239, US 4,797,368, US 5,139,941, EP 488 528). These applications describe different constructs derived from AAV, in which the rep and / or cap genes are deleted and replaced by a gene of interest, and their use for intraviral transfer (on cells in culture) or in vi vo ( directly in an organism) of the gene of interest. The defective recombinant AAVs according to the invention are defective for all or part of the Rep and / or Cap regions. They can be prepared by co-transfection, in a cell line infected by a human helper virus (for example an adenovirus), a plasmid containing a nucleic sequence or a combination of nucleic sequences of the invention bordered by two reverse repeating regions (ITR) ) of AAV, and of a plasmid carrying the encapsidation genes (rep and cap genes) of AAV. A cell line used is, for example, line 293. Other production systems are described, for example, in publications WO 95/14771; WO 95/13365; WO 95/13392 or WO 95/06743. The recombinant AAVs produced are then purified by the classical techniques.

According to another particular embodiment of the invention, the defective recombinant virus is a retrovirus, a rhabdovirus or even an HSV. With respect to retroviruses and herpes viruses, the construction of recombinant vectors has been widely described in the literature: see particularly Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al. génet Eng. 7 (1985) 235; McCorm'ick, BioTechnology 3 (1985) 689, etc. In particular, retroviruses are integrative viruses, which selectively infect dividing cells. They thus constitute vectors of interest for cancer applications. The genome of retroviruses essentially comprises two LTRs, one packaging sequence and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses, the gag, pol and env genes are generally inserted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest. These vectors can be made from different types of retroviruses such as, in particular, MoMuLV ("murine Maloney leukemia virus", still called MoMLV), the MSV ("murine Maloney sarcoma virus"), the HaSV ("Harvey's sarcoma virus"); the SNV ("spleen necrosis virus"); the RSV ("Rous sarcoma virus) or even the Friend virus.

To construct recombinant retroviruses according to the invention containing a nucleic acid of the invention or a nucleic acid of interest under the control of the expression sequences comprising a promoter and one or more NRSE sequences, a plasmid containing particularly the LTR, the encapsidation sequence and the nucleic sequence, then it is used to transfer an encapsidation cell line, capable of trans-delivering the retroviral functions deficient in the plasmid. In general, the encapsidation lines are those capable of expressing the gag, pol and env genes. Such encapsidation lines have been described in the prior art, and particularly line PA317 (US 4,861,719); the PsiCRIP line (WO 90/02806) and the GP + envAm-12 line (WO 89/07150). On the other hand, recombinant retroviruses can comprise modifications at the level of LTRs to suppress transcriptional activity, as well as extended encapsidation sequences, which comprise a part of the gag gene (Bender et al., J. Virol 61 (1987) 1639). The recombinant retroviruses produced in addition are purified by the classical techniques.

The present application shows the possibility of using NRSE sequences to regulate the expression of an in vi vo gene. Indeed, the results presented in the following examples show that the sequences are always active in vi and allow an expression of a transgene in neuronal cells without having expression in non-neuronal cells.

The application also shows that the NRSE sequences are capable, according to their arrangement (number of copies), of efficiently regulating the activity of a strong promoter, which allows numerous and particularly advantageous uses. Indeed, the results obtained show that expression in neuronal cells is not only specific but also comparable with that obtained with strong promoters. The invention also shows, surprisingly, that the activity of the NRSE sequences seems to be potentiated when they are introduced into a vector of viral origin. This last type of construction combines particularly attractive properties such as transfer efficiency and expression selectivity.

The invention also relates to all cells comprising a nucleic acid or a vector or a virus such as defined above. Advantageously, this cell is a mammalian nerve cell. This is in particular a cell that comes from an established line or from a cell that comes from a primary culture. These cells can be used, for example, for the production of polypeptides or for testing the activity of genes. Likewise, they can be used in cell therapy methods, by implantation or injection in a subject.

In this aspect, the invention also aims at a composition comprising a nucleic acid or a vector or a virus or a cell such as those defined above and an excipient. Advantageously, it is a pharmaceutical composition. For use according to the present invention, the nucleic acid, the vector or the cells are preferably associated with one or some pharmaceutically acceptable vehicles to be formulated in administration routes topically and particularly stereotaxic, oral, parenteral, intranasal, intravenous , intramuscular, subcutaneous, intraocular, transdermal, etc ... Preferably, the nucleic acid, the vector or the cells are used under an injectable form. These are in particular saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, particularly lyophilized, which, by addition of according to the case of sterilized water or pathological serum, they allow the constitution of injectable solutes.

According to a preferred form, the composition according to the invention is administered intramuscularly, preferably by injection. In fact, the chosen intramuscular route allows to obtain, in an unexpected way, an important therapeutic effect, thanks to the retrograde transport of products that result from therapeutic genes and products in the muscle. In fact, these products, which correspond to the nucleic acids or which result from the vectors according to the invention, are absorbed at the level of the neuromuscular plates (motor plates) and transported to the cell bodies of the motor neurons for retrograde transport of the length of the length of motor neurons. In addition, this form of administration also has the advantage of preventing undesirable effects due to the ectopic expression of therapeutic genes in the treatment of neurological diseases. By this method, neural cells can be easily infected specifically without promoting the expression of transgenes in the injected muscles or the excretion of their products in the blood joint. Indeed, undoubtedly all the disadvantages encountered so far in therapy are due to the limited excess of neurotrophic factors in the motor neurons, to the very short half-life of these proteins as well as to the deleterious effects found during a systemic administration of these proteins. products. In addition, the ease of access of the place to be injected has been donated, this method is advantageously used whatever the type of neuronal pathology or oneuronal ot.

The quantities of these different elements can be adjusted by the person skilled in the art according to the visualized applications and depending on different parameters, such as, in particular, the site of administration considered, the number of injections, the gene to be expressed or even the duration of the treatment investigated.

The invention also relates to the use, for the preparation of a composition intended for the transfer and expression of a nucleic acid of interest in a tissue or nerve cell, of a construction comprising: a promoter - an o. several NRSE sequences, and - the nucleic acid, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

The use of the nucleic acids of the invention for the transfer of genes in vi v or ex vi ve, for example in gene therapy, makes it possible to limit the ectopic expression of the transgenes of interest and to refer the desired therapeutic effect to the populations neuronal Thus, possible deleterious effects are prevented due to the diffusion of the transgene in the organism. The use of this system can be visualized in the different pathologies of the spinal cord (degenerative or traumatic) as well as in all the other central, peripheral or neuropsychiatric neurological pathology that is expected specifically from neuronal populations. In fundamental neurology, this system should also allow to determine the origin (neuronal or glial) of the effects observed in vi t ro and in vivo.

The invention also relates to the use, for the preparation of a composition for the treatment of motoneuronal diseases, by the intramuscular route, of a nucleic acid of interest in a tissue or nerve cell, of a construction comprising: a promoter - one or more NRSE sequences, and - the nucleic acid, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

The invention also relates to the use of vectors as defined above and comprising a nucleic acid of interest under the control of expression sequences, characterized in that the expression sequences comprise a promoter and one or more NRSE sequences.

The use of viral vectors is based on the natural transfection properties of viruses. These vectors are particularly satisfactory on the transfection plan. It is also possible to use the vectors of the invention for the transfer and expression in neuronal cells of the gene of interest, in vi ve, in vi tro or ex vi ve and particularly for gene therapy. Thus, the expression of a nucleic acid of interest on the control particularly of one or more NRSE sequences, is obtained specifically in nerve cells, while ectopic expression is limited. These vectors can be used in the treatment and / or prevention of different neurological central, peripheral or neuropsychiatric pathologies awaiting the nerve cells and particularly the neurodegenerative diseases as well as the different pathologies of the spinal cord. By way of example, mention may be made in particular of Alzheimer's disease, Parkinson's disease, muscular spinal ataxia, Huntington's chorea.

These vectors can be used particularly in the treatment and / or prevention of motor neuron pathologies, such as, for example, amiometropic lateral sclerosis, spinal amyotrophy of type I (Werdnig Hoffman's disease), type II or III (Kugelberg's disease). -Welander), bulbar spinal atrophy (such as Kennedy's disease).

These vectors are particularly adapted to the treatment and / or prevention of these pathologies by the intramuscular route. As explained above, this therapeutic route allows motor neurons to be obtained thanks to retrograde transport.

More precisely, the invention relates to the use, for the preparation of a composition intended for the transfer and expression of a nucleic acid of interest in a tissue or nerve cell, of a vector as defined above and comprising a in which: a promoter - one or more NRSE sequences, and the nucleic acid, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

The invention also relates to the use, for the preparation of a composition for the treatment of motoneuronal diseases, intramuscularly, of a nucleic acid of interest in a tissue or nerve cell, of a vector as defined above and comprising a construction in which: - a promoter - one or more NRSE sequences, and the nucleic acid, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

The invention further relates to a method of regulating expression of genes in vi v that comprises the insertion, above the gene, of the NRSE sequences and the nvve administration of the resulting construct and / or the vector comprising the building.

The present application will be described in detail with the help of the examples that follow, which should be considered as illustrative and not limiting. These examples describe at first time the cloning of a crossing number of NRSE sequences above the PGK promoter in the plasmids containing the luciferase reporter gene and the selection of constructs that allow a decrease in the expression of luciferase after the transfection of non-neuronal cells. In a second time, the corrnding adenoviral constructions have been generated and tested by infection of cell lines and primary cultures, in addition to in vi ve after the intramuscular injection of recombinant adenoviruses in the mouse. The results presented illustrate the advantages of the nucleic acids of the invention.

DESCRIPTION OF THE FIGURES Fissure 1: Schematic representation of plasmids carrying a chimeric promoter of the invention.

Figure 2: Study of the functionality of plasmids by transfection in nerve cells (PC12) (Fig. 2B) and non-nerve cells (3T3) (Fig. 2A) after measurement of luciferase activity.

Figure 3: Study of the functionality of defective recombinant viruses i n vi t ro by measuring luciferase activity after infection: - of non-nervous cells (3T3 rat fibroblasts) (Fig 3a) - of nerve cells (Feocromocit orna of rat PC12) (Fig 3B) - of non-nervous primary cells, infection of primary cultures of kingdoms of newborn rats (Fig 3C) - of cultures of primary nervous cells, infection of primary cultures of upper cervical ganglia that come from newborn rats (Fig 3D) of cultures of primary non-nervous cells, infection of primary cultures of astrocytes that come from rats (Fig 3E) of primary nerve cell cultures, infection of primary cultures of cortical neuronal cells that come from rat embryos (Fig 3F) Figure 4: Functional study of defective recombinant viruses in vivo by intramuscular injection in the mouse and measurement of the activity of luci ferasa.

Figure 5: Functional study of defective recombinant viruses in vivo by intramuscular injection in the lingual muscles in the mouse and measurement of luciferase activity.

Figure 5A: measurement of luciferase activity in the cells of the lingual muscles on days 8 and 35.

Figure 5B: measurement of luciferase activity in cells of the nervous system (bulb) on days 8 and 35.

MATERIALS AND METHOD Cell lines and primary cultures: Cells derived from rat pheochromocytoma (PC12) are cultured in RPMI 1640 medium (Gibco) containing 15% fetal calf serum (SVF) (Boehringer). 3T3 cells from rat-fibroblasts are maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. Superior cervical ganglion (GCS) primary cultures are obtained from dissections of upper cervical ganglia of newly born Winstar rats (Iffa-Credo), aged 1 or 2 days and dissociated by 3 mg / ml dispase (Boehringer ), are then left on the plates previously coated with collagen from the rat tail. Cells are cultured in 0.5 ml of Leibovitz L-15 medium (Gibco) plugged with bicarbonate and containing several factors such as 70 ng / ml NGF (Nerve Growth Factor), 5% adult rat serum and 10 μM cytokine arabinofuranoside The primary cultures of the kingdom are prepared by dissociation in the trypsin from kidney tissue of newly born Winstar rats (Iffa-Credo), aged 1 or 2 days. The cells are then plated in 10% SVF in order to allow cell divisions until infection.

The cortical cultures are derived from rat embryos E17 Sprague-Dawley (Iffa-Credo). Cells are obtained by mechanical dissection and dissociation and cultured in DMEM medium containing 100 μg / ml transferrin, 25 μg / ml insulin, 10 μg / ml putrescine, 5 ng / ml sodium selenite, 6.3 ng / ml of progesterone and 2 mM of glutamine (Sigma).

The primary cultures of astrocytes are derived from rat embryonic cortical tissue (E17 Sprague-Dawley) and are grown in DMEM medium containing 10% SVF in an atmosphere of 10% C02 and 90% air.

Experiences in vi tro: For transient transfection experiences, one million cells are electroporated by means of a Bio-Rad system at 960 μF and 190 V for PC12 or 250 V cells for 3T3 cells with 6 μg of each plasmid tested, 7 μg of Blue Script plasmid (Stratagén) and 2 μg of the pCAT 3-control vector (Promega) which is a plasmid encoding chloramphenicol acetyl transferase (CAT) that allows the control of transfection efficiency.

The cell cultures are infected by replacing the culture medium with the medium without serum containing the viral suspension, at a MOI of approximately 200 pfu and incubating for 45 minutes. For each construction (plasmid or adenoviral) 3 values of luciferase activity are determined on 3 different wells. Experiences of infection are repeated twice with different viral reserves for each viral construct.

The cells are harvested 48 hours after electroporation or infection in 200 μl of a lysis buffer containing 25 mM Tris Phosphate pH 7.8; 0.08 mM Luciferin; 0.1 mM of ATP; 8 mM MgCl2; 1 M of dithiothreitol; 1 mM EDTA; 15% glycerol and 1% Triton. The luciferase activity is measured with the help of a luminometer of the LUMAT type LB9501 (Berthold). For the transfected cells, 1% bovine serum albumin (BSA) is added to the lysis buffer and the activity is normalized on the basis of the CAT activity determined by the scintillation liquid counting method. For recombinant adenoviruses, the luciferase activity is normalized according to the protein concentration of the cell extracts, determined by the Bio-Rad system (Laboratories Bio-Rad).

Experiences in vivo: C57B16 (Charles River) male mice, aged six months, are anesthetized with a mixture of Rompun (Bayer) / Cetamine (UVA) and the Ad-PGK-luc constructs and Ad-NRSE-PGK-luc are injected slowly (2.5 μl / ml) into the tongue (109 pfu / 4x2.5 μl) and / or into the gastronecmium muscle at a dosage of 2,109 pfu / muscle (30 μl).

Mice are sacrificed with pentobarbital (Sanofi) 7 to 35 days after infection. The bulbs, tongues and muscles are extracted to measure luciferase activity. The bulbs are mechanically dissociated in 200 μl of lysis buffer. The muscles and tongues are dissociated with the help of a DIAX 900 polytron (Heidolph) type system in 1 and 2 ml of lysis buffer respectively.

EXAMPLES 1. Construction of chimeric promoters and plasmid vectors The objective of this example is to describe the obtaining of chimeric promoters as well as the constructions of plasmid vectors containing these particular promoters.

Plasmid pPGK-Luc comprises the Luc gene under the control of the ubiquitous eukaryotic promoter PGK. To obtain the plasmid pPGK-Luc, 500 bp of the murine PGK promoter have been inserted into the commercial plasmid pUT 103 (CAYLA FRANCE) which contains the reporter gene luciferase fused with zeocin and a polyadenylation sequence of SV-40 in the "next" configuration (PolyA).

Then, one, two, three, six and twelve copies of the NRSE sequence of the SCG10 gene (TCAGCACCACGGAGAGTGCC, SEQ ID No. 1) [2] have been inserted in an antiparallel configuration above the PGK promoter. For this, a 26 bp fragment containing the NRSE sequence described above framed by 2 Mlul sites has been constructed by hybridization of 2 corresponding synthetic oligonucleotides. This fragment has been inserted in one or more copies at the level of the Mlul site of pPGK-Luc and has allowed obtaining plasmids containing 1, 2 and 3 NRSE copies. The plasmid containing 6 NRSE copies has been obtained from the plasmid containing 3 (pNRSE3-PGK-Luc) according to the following strategy: a Ba Hl / XhoI fragment containing 3 NRSE copies is extracted from pNRSE3-PGK-Luc and is introduced at the Xhol site level of pNRSE3-PGK-Luc, this generates p6NRSE-PGK-Luc. Similarly, the plasmid containing 12 NRSE copies was obtained from the plasmid containing 6 (p6NRSE-PGK-Luc) introducing the BamHI / Xhol fragment in double copy in p6NRSE-PGK-Luc.

Finally, the expression cassette PGK (with or without NRSE) -Luc-PolyA of the different plasmids described above has been introduced into a plasmid vehicle used for the construction of defective recombinant viruses, between the left ITR sequence of the adenovirus (Terminal Repetition). Inverted, origin of replication) and the sequence coding for polypeptide IX (viral capsid protein) (figure 1).

It is understood that, according to the same protocol, all the other plasmid can be constructed incorporating from 1 to 50 copies of an NRSE region, associated with a ubiquitous eukaryotic PGK promoter, under control which can be introduced throughout the transgene of interest.

Functional analysis for cell line transfections The objective of this example is to demonstrate that the plasmid constructs containing the chimeric promoter comprising the NRSE sequences are functional and allow expression in the cell of a gene of interest.

The plasmid constructs described in Example 1 have been tested for the transient transfection (electroporation) of neuronal lines PC12 (rat pheochromocytoma) and non-neuronal 3T3 (rat fibroblasts). The luciferase activity values obtained are normalized in relation to the CAT activity (chloramphenicol acetyl transferase) measured in a solution containing 125 mM Tris phosphate pH 7.8, 125 μM chloramphenicol and 0.12 μM radiolabeled acetyl coenzyme A.

The dosages are made in the presence of a fluid • Econofluor scintillation with the help of a KONTRON counter after one hour of incubation at 37 ° C. For each plasmid construct, 3 values of luciferase activity have been determined and the means and standard errors are presented in figure 2.

The results obtained show that constructs containing 6 and 12 NRSE copies are capable of decreasing luciferase expression of 66 and 82% respectively, in non-nervous cells (3T3), or a loss of about one logarithm of expression with relation to the construction pPGK-Luc. The results also show that the presence of NRSE regions in nerve cells PC12 does not diminish the expression of luciferase, it is even susceptible to increase it. A statistically significant 25% increase is indeed observed, between pl2NRSE-PGK-Luc and pPGK-Luc.

Thus, it is demonstrated by the results obtained that the plasmid constructs containing the chimeric promoter comprising the NRSE sequences are functional and allow the expression in the cell of a gene of interest in the neuronal cells, only inhibiting the expression in a very significant manner. of the gene in the non-neuronal. 3. Construction of defective recombinant viruses The objective of this example is to describe the obtaining of chimeric promoters as well as the constructions of viral vectors that contain these particular promoters.

In order to complete this study in vi tro and in vi, the three corresponding adenoviral constructions have been generated: Ad-PGK-Luc, Ad-6NRSE-PGK-Luc and Ad-12NRSE-PGK-Luc. These recombinant viruses have been constructed according to the known techniques of biology, by co-transfection, in an encapsidation line (293 cells) of the vehicle plasmids described in example 1 and of a defective adenoviral type 5 genome. Vehicle plasmids were first linearized by enzymatic cleavage at the FspI site and co-transfected with the long Clal fragment of the adenovirus in the 293 cell line by the calcium phosphate DNA precipitation method.

The encapsidated recombinant genomes are further purified according to the classical techniques and particularly by the plaque purification technique. The integrated fragment is further verified by analysis of the restriction fragments and by PCR. Virus stocks have been prepared by propagation of the recombinant adenovirus in cell 293 then by ultracentrifugation particularly in cesium chloride gradient and purification on the Sephadex G25M type purification column (Pharmacia). Viral titrations were determined by reading the optical density and verified in addition by the plate method. All virus stocks had a titre around 2,108 pfu / μl. 4. Functional analysis in vi tro The objective of this example is to demonstrate that the viral constructs described in Example 3 and containing the chimeric promoter comprising the NRSE sequences, are functional and allow the control of the promoter in the different cell types.

The three adenovirus constructs in Example 3 above have been tested in vitro for infection of the cell lines and primary cultures at a dosage of 20 and 200 pfu / cell, respectively. The measurements are made as described above. on 50 μl of supernatant and normalized in relation to the amount of total proteins, obtained with the help of a Bio Rad (Bio-Rad) protein test kit system. For each adenovirus, 3 values of luciferase activity have been determined on different wells. The values of the means and the standard errors obtained are illustrated in figure 3 for the lines and figure 4 for the primary crops.

The expression of the reporter gene luciferase is decreased by 97% and 99% respectively with the recombinant adenovirus Ad-6NRSE-PGK-Luc and Ad-12NRSE-PGK-Luc in the non-neuronal line 3T3 In the PC12 neuronal line, the luciferase activity of the two adenoviruses containing the NRSE sequences is superior to that observed with the Ad-PGK-Luc adenovirus.

Also, after infection of primary cervical ganglion (GCS) primary cultures of newborn rats, the luciferase activity is similar for the 3 adenoviral constructs (an analysis of overall variance to an ANOVA factor does not show the significant difference between the three groups ). In contrast, the expression of luciferase is decreased from 91 to 98% after infection of the primary cultures of newborn rat kingdoms with the recombinant adenoviruses Ad-6NRSE-PGK-Luc and Ad-12NRSE-PGK-Luc, and either as a loss of 2 logarithms of PGK expression in the presence of 12 NRSE copies.

Conforming the previous results, the infection for the 3 astrocyte constructions and neuronal cortex cells from embryos of rats aged 17 days, shows how the expected luciferase activity is very significantly reduced (by a factor of 10) in the as- tocitic cells for constructions containing 6 or 12 NRSE sequences (fig 3E). As has also been foreseen, the luciferase activity is comparable in the cells of the cortex, for the three types of viral constructions mentioned above (fig 3F).

These results are particularly surprising and remarkable insofar as they show (i) that the chimeric promoters of the invention are functional on primary cultures, (ii) that the promoters are functional in a viral context and (iii) that the repression activity and larger in the adenoviral context than in the corresponding plasmid vector (Cf figures 2 and 3).

Thus, this example allowed demonstrating that the viral constructs containing the chimeric promoter comprising the NRSE sequences are functional and allow expression in the cell of interest in the neuronal cells only by very significantly inhibiting the expression of the gene in non-neuronal cells .

Functional analysis in vivo The objective of this example is to demonstrate that the viral constructs described in Example 3 and containing the chimeric promoter comprising the NRSE sequences are functional and allow in vitro control of the promoter.

Taking into account the very encouraging results described in Example 4, the activity of the constructs of the invention has been tested in vi, by measuring the expression of luciferase after intramuscular injection (muscle gastronecmium and muscle of the tongue) of these 3 recombinant adenoviruses in the mouse. . 1 Injection in the sastronecmium muscle Each adenoviral construct was injected at 3 points in the right gastronecmium muscle of 7 mice at a dosage of 2,109 pfu / muscle, under a volume of 30 μl. The injected muscles were removed 7 days after injection and dissociated in 1 ml of buffer in view of a determination of the luciferase activity performed on 150 μl of supernatant. The means and standard errors of the 7 values of luciferase activities obtained, reported to the amount of protein, are presented in figure 5.

These results show that luciferase expression is decreased by 90 and 96% with the recombinant adenovirus Ad-6NRSE-PGK-Luc and Ad-12NRSE-PGK-Luc one week after the injections.

The expression of luciferase after intramuscular injection of Ad-12NRSE-PGK-Luc and Ad-PGK-Luc has been compared, in the longer term, in the mouse. A decrease in luciferase activity is always observed with Ad-12NRSE-PGK-Luc one month (95%) and three months (91%) after the injections. . 2 Injection into the muscles of the tongue Retrograde transport has been used for the purpose of two constructions Ad-PGK-Luc and Ad-12NRSE-PGK-Luc in the bulb and in the muscles of the tongue after intramuscular injection into the tongue of the mouse.

In a manner comparable to those already obtained, the luciferase activities of two constructions are similar in the nervous system, one week after administration (Figure 5B). Conversely, the luciferase activity decreases by 90% in the muscles injected with the Ad-12NRSE-PGK-Luc construct in relation to the muscles injected with the Ad-PGK-Luc construct (Fig. 5A). This repression is maintained over time and for at least 35 days after infection. Interestingly, although the level of expression at 35 days is lower than that obtained at 8 days after infection, the difference in expression between the two preceding viral constructions remains very significant.

Once again, these results confirm the functionality of the constructs of the invention in vivo, and make a repression factor even higher than those observed in vitro after the transfection of plasmids in non-neuronal cells.

These results also demonstrate the high efficiency of the constructs of the invention in vi to direct the expression of a gene of interest in neuronal cells. The use of this method in gene therapy allows to limit the ectopic expression of transgenes of interest. and to use the possible deleterious effects due to the diffusion of the transgene in the organism.

BIBLIOGRAPHY [lj Mori N., Stein R., Sigmund O. and Anderson D.J. Neuron, 4: 583-594 (1990). [2] Mori N., Schoenherr C., Vandenbergh D.J. And Anderson D.J. Neuron, 9: 45-54 (1992). [3] Li L., Suzuki T., Mori N. and Greencard P. PNAS USA, 90: 1460-1464 (1993). [4] Schoenherr C.J., Paquette A.J. and Anderson D. J PNAS USA, 93: 9981-9986 (1996). [5] Schoenherr C.J. and Anderson D.J. Science, 267 1360-1363 (1995). [6] McBurney, Sutherland, Adra, Leclair, Rudnicki, Jardine Nucleic Acids Research, Vol. 19, No. 20: 5755-5761 (1991). [7] Moullier P., Marechal V., Danos O. And Heard J.M. Transplantation, 56: 427-432 (1993). [8] McDonald R.J. Lukason M. J. Raabe O G Canfield D.R., Burr E.A., Kaplan J.M., Wadsworth S.C. and St George J.A. Hum. Gene Ther., 8: 411-422 (1997). [9. Maue R.A., Kraner S.D., Goodman R.H. and Mandel G. Neuron, 4: 223-231 (1990).

[10] Bessis A., Champtiaux N., Chatelin L. and Changeux J.P. PNAS USA, 94: 5906-5911 (1997).

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) DECLARANT: (A) NAME: RHONE-POULENC RORER (B) STREET: 20 AVENUE RAYMOND ARON (C) CITY: ANTONY (E) COUNTRY: FRANCE (F) POSTAL CODE: 92165 (G) TELEPHONE: 33155716922 (H) TELEFAX: 33155717291 (ii) TITLE OF THE INVENTION: Use of negative regulation elements for the neurospecific expression of transgenes (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER DESCIFRABLE FORM: (A) TYPE OF SUPPORT: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: Patentln Relay # 1.0, Version # 1.30 (OEB) (2) INFORMATION FOR SEQ ID NO: 1: sequence of the NRSE region (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (• iv) ANTI-SENSE: NO ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: TTCAGCACCA CGGAGAGTGC C 21 (2) INFORMATION FOR SEQ ID NO: 2: consensus sequence of the NRSE region (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple ( D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 'xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 TTCAGCACCA CGGACAGCGC C 21 (2) INFORMATION FOR SEQ ID NO: 3: general sequence of the NRSE region i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 NNCAGCACCN NGGANAGNNN C 21 (2) INFORMATION FOR SEQ ID NO: 4 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTIC: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 TTCAGCACCA CGGAGAGTGC C 21 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO TTCAGCACCG CGGACAGTGC C (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6 TTCAGCACCT CGGACAGCAT C 21 (2) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple "'(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 TTCAGCACCG CGGAGAGCGT C 21 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: TCCAGCACCG TGGACAGAGC C 21 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTIC: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 TTCAGCACCG AGGACGGCGG A 21 (2) INFORMATION FOR SEQ ID NO: 10: '(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO. { iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10 ATCAGCACCA CGGACAGCGG C 21 (2) INFORMATION FOR SEQ ID NO: 11 i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: - (B) SITE: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 TTCAGCACCT AGGACAGAGG C 21 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

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

Claims (37)

1. The recombinant nucleic acid, characterized in that it comprises: - a promoter - one or more NRSE sequences, and - a therapeutic gene, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

2. The recombinant nucleic acid according to the rei indication 1, characterized in that the promoter is a eukaryotic or viral promoter active in the. cells or nervous tissue.

3. The recombinant nucleic acid according to claim 2, characterized in that the promoter is a ubiquitous, neurospecific eukaryotic promoter or specific to a neuronal type.

4. The recombinant nucleic acid according to claim 3, characterized in that the promoter is a domestic promoter.

5. The recombinant nucleic acid according to claim 4, characterized in that the promoter is chosen from the gene promoter PGK, Ef la, β-actin, vimentin, aldolase A or al-antitrypsin.

6. The recombinant nucleic acid according to claim 1, characterized in that the promoter is a strong promoter.

7. The recombinant nucleic acid according to claim 1, characterized in that it comprises from 1 to 20 NRSE sequences, preferably from 3 to 15.

8. The recombinant nucleic acid according to claim 7, characterized in that it comprises 3, 6 or 12 NRSE sequences.

9. The recombinant nucleic acid according to claim 8, characterized in that the NRSE sequence comprises all or part of the sequence SEQ ID No. 3 in which N is A, G, C or T.

10. The recombinant nucleic acid according to claim 9, characterized in that the NRSE sequence is chosen from all or part of the sequences SEQ ID No. 1, 2, 4-11 or variants thereof.

11. The recombinant nucleic acid according to one of the preceding claims, characterized in that it comprises either the same region, or variant regions defined according to claim 9 or 10, repeated several times.

12. The recombinant nucleic acid according to one of the preceding claims, characterized in that the NRSE sequence (s) are placed above the promoter.

13. The recombinant nucleic acid according to claim 1, characterized in that the therapeutic gene is a nucleic acid comprising an open reading frame that encodes an RNA or a therapeutic or vaccinia polypeptide.

14. The recombinant nucleic acid according to claim 13, characterized in that the therapeutic gene is a nucleic acid comprising an open reading frame that encodes a trophic factor.

15. The recombinant nucleic acid according to claim 13, characterized in that the therapeutic gene is a nucleic acid comprising an open reading frame that encodes an antioxidant agent.

16. The recombinant nucleic acid according to claim 1, characterized in that it comprises regulatory elements in more than the NRSE sequences.

17. The regulation elements according to claim 16, characterized in that they are a tetracycline operator / repressor system.

18. The vector, characterized in that it comprises a nucleic acid according to one of claims 1 to 16.

19. The vector according to the rei indication 18, characterized in that it is a viral vector.

20. The defective recombinant virus, which comprises a nucleic acid of interest under the control of expression sequences, characterized in that the expression sequences comprise a promoter and one or more NRSE sequences.

21. The virus according to claim 20, characterized in that it is an adenovirus.

22. The virus according to claim 20, characterized in that it is an AAV.

23. The virus according to claim 20, characterized in that it is a retrovirus.

24. The virus according to claim 20, characterized in that it is a rhabdovirus.

25. The cell, characterized in that it comprises a nucleic acid according to claim 1 or a vector according to claim 18 or a virus according to claim 20.

26. The cell according to claim 25, characterized in that it is a mammalian nerve cell.

27. The use, for the preparation of a nucleic acid intended for the transfer and expression of a nucleic acid of interest in a tissue or nerve cell, of a construction, characterized in that it comprises: - a promoter -. one or more NRSE sequences, and - the nucleic acid, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

28. The use, for the preparation of a composition for the treatment of motoneuronal diseases intramuscularly, of a nucleic acid of interest in a tissue or nerve cell, of a construction, characterized in that it comprises: - a promoter one or more NRSE sequences, and - the nucleic acid, grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

29. The use of a defective recombinant virus, comprising a nucleic acid of interest under the control of expression sequences, characterized in that the expression sequences comprise a promoter and one or more NRSE sequences.

30. The use according to claim 29, characterized in that it is used for the transfer and expression of the gene of interest in neuronal cells in vivo, in vi tro or ex vi vo.

31. The use, for the preparation of a composition intended for the transfer of a nucleic acid of interest in a tissue or nerve cell, of a vector, characterized in that it comprises a construction in which: - one promoter - one or more NRSE sequences, and - the nucleic acid, are grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

32. The use, for the preparation of a composition for the treatment of motoneuronal diseases, intramuscularly, of a vector, characterized in that it comprises a construction in which: a promoter - one or more NRSE sequences, and - the nucleic acid, are grouped so that the expression of the nucleic acid is controlled by the promoter or that the activity of the promoter is controlled by the sequence (s).

33. The use according to one of claims 31 or 32, characterized in that the vector is a virus.

34. The composition, characterized in that it comprises a nucleic acid according to claim 1 or a vector according to claim 18 or a virus according to claim 20.

35. The composition according to claim 34, characterized in that it is formulated in view of being administered by the intramuscular route, preferably by injection.

36. The chimeric promoter, characterized in that it comprises a ubiquitous promoter and one or more NRSE sequences.

37. The chimeric promoter xNRSE-PGK, characterized in that x is an integer from 1 to 50.