FR2781503A1 - New recombinant baculovirus, for use in human gene therapy of nervous system diseases - Google Patents

Abstract

Recombinant baculovirus (A), or its derivative, containing a heterologous nucleic acid sequence (I) that encodes a product (II) useful for treating nervous system disorders. Independent claims are also included for the following: (1) population of nervous system cells (brain, spinal cord, neural, glial or ependymal cells) infected with (A); (2) implants comprising human cells infected with (A), and (3) a composition containing (A) plus a vehicle.

Description

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 The invention relates to new recombinant viruses and their use for the transfer of genes into cells of the nervous system. It also relates to pharmaceutical compositions comprising said recombinant viruses. More particularly the present invention relates to new vectors derived from baculoviruses and their use for the treatment of diseases of the nervous system.

 Baculoviruses are enveloped viruses with circular double-stranded DNA specific to invertebrates. Their prototype, AcNPV, has a genome of 133 kb. This vector is very widely used as a vector for the expression of eukaryotic genes in insect cells, from two strong promoters [polyhedrin (Ph) and P10], (King and Possee, The baculovirus expression system. London: Chapman & Hall, 1992 .).

 The recombinant baculoviruses are obtained by homologous recombination in insect cells (generally Sf9 or Sf21) between the shuttle plasmid containing the transgene under the control of a viral promoter and the genome of the linearized baculovirus at the recombination site. A baculovirus stock can then be prepared by infection of insect cells (SF9 or SF21) with the recombinant baculovirus. Protein is produced by infection of insect cells with baculovirus: the cell medium is harvested and the protein synthesized in vitro is extracted from insect cells. These viruses are episomal (in the insect), up to 100 kb of recombinant DNA can be integrated into them. Baculoviruses thus have many advantages linked to their ease of production (multiplication in insect cells, industrializable culture conditions, obtaining high titers), their high cloning capacity, and the fact that they present a low risk of dissemination because they are not replicative in mammals. These vectors, however, have not been used in the field of gene therapy, because on the one hand and until very recently it was admitted that these viruses do not infect mammalian cells, and on the other hand transfection Baculovirus in vivo in mammals has never been demonstrated.

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 Hoffmann et al. (PNAS USA 92 (1995) 10099-10103) have shown that a recombinant baculovirus containing a reporter gene under the control of the CMV early promoter is capable of infecting and expressing the transgene in hepatocytes with very good efficiency: humans (primary culture, Huh7 and HepG2 line) murine (primary rabbit culture). However, these authors did not observe significant expression of the transgene in a whole series of lines originating from various origins, including the nervous system (mouse neuroblastoma Neuro 2a, human astrocytoma SW 1088 and rat pheochromocytoma PC 12). .

 Boyce FM and Bucher NLR PNAS USA 93 2348-2352 (1996) obtained significant expression of the LacZ gene placed under the control of an RSV promoter in the HepG2 line as well as in the lines 293 (human kidney), A549 (human lung) and PC 12 (rat pheochromocytoma). They did not obtain significant expression in all the other lines tested, including K-562 (human myelocyte) and HL-60 (human promyelocyte).

 Sandig V. et al. (Human Gene therapy 7 1937-1945 (1996)) used a RSV-LacZ recombinant baculovirus for infection of human and murine hepatocytes. They have shown that the virus is sensitive to complement and therefore cannot infect the liver in vivo. However, they obtained an infection of a piece of human liver perfused ex vivo with baculovirus, after elimination of the serum.

 More recently, Shoji et al. (J. Gen. Virol. 1997) used a recombinant baculovirus containing a gene under the control of the CAG promoter (early CMV enhancer and promoter of chicken ss-actin), and obtained significant expression in different cell lines (HepG2 , Huh7, CPK, Cos7, Hela, FS-L3 KATO-III). Low expression was detected in the RGM-1, PC 12, IMR-32 and MT-2 lines.

 Barsoum et al (Human Gene Therapy, 8 2011-2018 (1997)) have shown that it is possible to pseudotyping a baculovirus containing an expression cassette of the

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 8-galactosidase under the control of the RSV LTR. This makes it possible to improve the transduction potential of these vectors, by improving the infection of the cells which can be infected by the non-pseudotyped baculovirus, and also makes it possible to infect cells which were not infected by the non-pseudotyped baculovirus. However, they did not test for infection of neural cells.

 So far, no baculovirus vector has been used for the transfer of genes in vitro or in vivo in cells of nervous tissue. In fact, only the PC 12 line, which can present under special conditions a "pseudo-neuronal" phenotype (rat phechromocytoma: peripheral nervous system), was found to be able to be infected with baculovirus, however the level of expression remained very slightly higher than that of the control. Finally, no use of baculovirus for gene transfer in vivo in mammals has so far been described.

 The Applicant has now shown that it is possible to construct recombinant baculoviruses comprising a heterologous nucleic acid sequence preferentially coding for a product of therapeutic interest for the treatment of diseases of the nervous system, to administer these recombinant baculoviruses in vivo, and that this administration allows stable and localized expression of the transgene in vivo, and in particular in the nervous system. The present invention thus provides viral vectors usable directly in gene therapy, particularly suitable and effective for directing the expression of transgenes of therapeutic interest in vivo in the nervous system or for an ex vivo application for the transfer of genes in cultures of nervous system cells to be transplanted. The present invention thus offers a particularly advantageous new approach for the treatment and / or prevention of neurodegenerative diseases or the treatment of metabolic diseases requiring specific treatment of the nervous system due to the poor accessibility of therapeutic agents which do not cross the blood-brain barrier. -encephalic.

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 A first subject of the invention resides in recombinant baculoviruses comprising a heterologous nucleic acid sequence coding for a product of therapeutic interest for the treatment of diseases of the nervous system.

 In particular, the heterologous nucleic acid sequence may include one or more therapeutic genes.

 The therapeutic genes which can thus be transferred are any gene whose transcription and possibly translation into the target cell generate products having a therapeutic effect.

 The protein product thus coded can be a protein or a peptide. This protein product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter presents no pathology). In this case, the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein. The therapeutic gene can also code for a mutant of a cellular protein, having increased stability, modified activity, etc. The protein product can also be heterologous towards the target cell. In this case, an expressed protein can for example supplement or provide a deficient activity in the cell allowing it to fight against a pathology.

 Among the therapeutic products within the meaning of the present invention, mention may be made more particularly of hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitter synthesis enzymes, trophic factors, in particular neurotrophics for the treatment of neurodegenerative diseases, traumas having damaged the nervous system, or retinal degenerations. For example members of the neurotrophin family such as NGF, BDNF, NT3, NT4 / 5, NT6 and their derivatives, members of the CNTF family such as CNTF, axokine, LIF and

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 their derivatives, IL6, cardiotrophin, GDNF and its derivatives, members of the IGF family, such as IGF-1, IFGF-2, members of the FGF family, such as FGF 1, 2, 3, 4, 5, 6, 7,8, 9 their derivatives, and TGF-ss.

Among the therapeutic products within the meaning of the present invention, mention may also be made of tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), suicide genes: thymidine kinase, cytosine deaminase, etc.,
The nucleic acid sequences coding for products of therapeutic interest within the meaning of the present invention also cover the genes coding for the proteins involved in the metabolism of amino acids, lipids and other constituents of the cell.

 Mention may therefore be made, without limitation, of the genes associated with diseases of the metabolism of carbohydrates such as, for example, fructose-1-phosphate aldolase, fructose-1,6-diphosphatase, glucose-6-phosphatase, [alpha] -1,4-glucosidase lysosomal, amylo-1,6-glucosidase, amylo- (1,4: 1,6) -transglucosidase, muscle phosphorylase, muscle phosphofructokinase, phosphorylase-b-kinase, galactose-1-phosphate uridyl transferase, all enzymes of the pyruvate complex dehydrogenase, pyruvate carboxylase, 2-oxoglutarate glyoxylase carboxylase, D-glycerate dehydrogenase.

 Mention may also be made of: - genes associated with diseases of the amino acid metabolism such as, for example, phenylalanine hydroxylase, dihydrobiopterin synthetase, tyrosine aminotransferase, tyrosinase, histidinase, fumarylacetoacetase, glutathione synthetase, y-glutamylcysteine synthetase, omithine-synthetase aminotransferase, synthase carbamoylphosphate, ornithine transcarbamylase, argininosuccinate synthetase argininosuccinate lyase arginase L-lysine dehydrogenase L-lysine ketoglutarate reductase valine transaminase leucine isoleucine transaminase, decarboxylase 2-keto acids to branched-chain, isovaleryl-CoA dehydrogenase,

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 acyl-CoA dehydrogenase, 3-hydroxy-3-methylglutaryl-CoA lyase, acetoacetyl-CoA 3-ketothiolase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, ATP: cobalamin adenosyltransferase, dihydrofolate reductase, methylene tetrahydrase 0 sarcosine dehydrogenase complex, proteins belonging to the glycine cleavage system, ssalanine transaminase, serum camosinase, cerebral homocarnosinase.

 - Genes associated with diseases of fat and fatty acid metabolism, such as, for example, lipoprotein lipase, apolipoprotein C-II, apolipoprotein E, other apolipoproteins, lecithin cholesterolacyltransferase, LDL receptor, liver sterol hydroxylase, phytanic acid a -hydroxylase.

 - Genes associated with lysosomal deficiencies, such as for example lysosomal aL-iduronidase, lysosomal iduronate sulfatase, heparan lysosomal N-sulfatase, N-acetayl-aD-lysosomal glucosaminidase, acetyl-CoA: aglucosamine N-acetyltrans lysosomal aD-glucosamine 6-sulfatase, lysosomal galactosamine 6-sulfate sulfatase, lysosomal p-galactosidase, lysosomal arylsulfatase B, (3-glucuronidase lysosomal, Nacetylglucosaminyl-phosphotransferase, aD-mannosidase lomidosidase, aD-mannosidase lomidosidase Lysosomal acid lipase, Lysosomal acid ceramidase, Lysosomal sphingomyelinase, Lysosomal glucocerebrosidase and Lysosomal galactocerebrosidase, Lysosomal galactosylceramidase, Lysosomal arylsulfatase, Lysosomalase A, galactosidase A, p-galactosidase A, l-galactosidase A, l-galactosidase A, l-galactosidase A, l-galactosidase A, l-galactosidase A, l-galactosidase A, l-galactosidase A, galactosidase A, galactosidase A, p-galactosidase A, galactosidase A, galactosidase A, l-galactosidase A, galactosidase A, l-galactosidase A-galactosidase A, galactosidase A, galactosidase A, l-galactosidase A, galactosidase A, galactosidase A, galactosidase A, galactosidase lysosomal acid.

 Mention may also be made, without limitation, of the genes associated with diseases of the steroid and lipid metabolism, the genes associated with diseases of the metabolism of purines and pyrimidines, as well as the genes associated with diseases of the metabolism of the porphyrin and Theme.

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 The heterologous nucleic acid sequence may be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.

 Generally, the heterologous nucleic acid sequence also includes a promoter region for functional transcription in the infected cell. It may be a promoter region naturally responsible for the expression of the gene considered when it is capable of functioning in the infected cell. They can also be regions of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect.

Advantageously, it is an active promoter in cells or nervous tissues, in particular a eukaryotic promoter. In this regard, it may for example be a ubiquitous promoter, that is to say functional in the majority of cell types.

Even more preferably, it is therefore a eukaryotic ubiquitous promoter.

The promoter can be autologous, that is to say coming from the same species as the cell in which the expression is sought or xenogenic (coming from another species). As advantageous examples, eukaryotic ubiquitous promoters may be cited as strong promoters such as the promoter of the phosphoglycerate kinase 1 (PGK) gene or of other promoters directing the expression of genes of compulsory cell metabolism (these genes are said to be "domestic" or "household" and specify proteins necessary for functions common to all cells). These are, for example, genes involved in the Krebs cycle, in cellular respiration or in the replication or transcription of other genes.

As particular examples of this type of promoter, the promoter

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 genes [alpha] 1-antitrypsin, p-actin, vimentin, aldolase A or Efla (elongation factor).

 The promoter used in the context of the invention may also be a eukaryotic promoter of the neurospecific type. By way of example, mention may be made of the promoter of the NSE (Neuron Specific Enolase), NF (Neurofilament), TH (Tyrosine Hydroxylase), DAT (Dopamine Transporter), ChAT (Choline Acetyl Transferase), DBH (Dopamine (3-Hydroxylase) genes ), TPH (Tryptophan Hydroxylase), GAD (Glutamic Acid Dehydrogenase), GFAP (Glial Fibrillary Acidic Protein) and, more generally, all promoters of synthetic enzymes or neuro-mediator transporters or any other gene promoter whose expression is specific for a given neural or glial type or subtype.

 It is also possible to envisage the use of viral promoters, such as for example CMV (Cytomegalovirus), RSV (Rous Sarcoma Virus), TK (Thymidine Kinase), SV40 (Simian Virus) and LTR (Long Terminal Repeat) promoters of RSV, MLV (Murine Leukaemia Virus) or HIV (Human Immunodeficiency Virus).

It is also conceivable to use chimeric promoters such as CAG (CMV enhancer and chicken p-actin promoter), NRSE-PGK or inducible promoters such as promoters inducible by tETracycline (Tet-On and Tet- Off), ecdysone inducible promoters, or other inducible promoters (RU486, 1713-Oestradiol ...) and in particular stress inducible promoters, such as heat stress (hsp70)
In addition, these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression.

 Furthermore, the heterologous nucleic acid sequence may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretion pathways of the cell

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 target. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.

 The virus can be used both in an ex vivo or in vivo approach. As part of a gene therapy applicable to humans, an ex vivo approach can be envisaged by grafting genetically modified cells with a recombinant baculovirus. These cells can be of various origins: (human or non-human). The combination of certain drugs can be considered with a view in particular to improving the maintenance of the transplant or the expression of the transgene (immunosuppressants, anti-complement, etc.). One can also consider the implantation of genetically modified cells by a recombinant baculovirus after packaging in an inert system.

 The baculoviruses according to the invention can be prepared by any technique known to those skilled in the art. In particular, they can be prepared by homologous recombination between a shuttle plasmid and the genome of Autographa california in SF9 cells and amplified in these same cells according to the method described by Gruenwald S. and Heitz J., 1993 (Baculovirus expression vector system, procedure & method manual. Pharmingen Eds, San Diego, CA). Then the baculoviruses which have multiplied are recovered and purified according to conventional molecular biology techniques as illustrated in the examples.

 It is also possible to pseudotyping baculoviruses, that is to say to express an envelope protein other than those of baculoviruses on the surface thereof, thus making it possible to modify the host spectrum of the virus. It is thus possible to use envelopes which make it possible to widen the host spectrum to a very wide variety of cell types or one can on the contrary consider restricting the host spectrum to a specific type of nerve cells. Among the envelope proteins which can be used, there may be mentioned in particular: the VSV glycoprotein, the amphotropic envelope protein of MLV. It is also possible to use envelope proteins which

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 specifically improve the entry of the virus into neural cells (CNS and / or PNS) such as the rabies glycoprotein, the rubella envelope, the HSV glycoprotein. Preferred variants of recombinant baculoviruses according to the invention are baculoviruses pseudotyped by the rabies virus glycoprotein (Rabies Virus) or the VSV glycoprotein (Vesicular Stomatitis Virus).

 These recombinant vectors can be used for the transfer of nucleic acids into nervous tissue cells in vitro, ex vivo or in vivo.

 The in vivo application may be made in particular by stereotaxic injection of the recombinant baculovirus into the central nervous system (brain, marrow). In this regard, it is possible to use the combination of complement inhibitors (CVF, sCR1, FUT175 ...) to improve the efficiency of transfection. It is also possible to carry out the administration of the baculovirus, in particular at the level of the muscle, in order to reach the nervous system by retrograde transport of the baculovirus and / or of the therapeutic product. In the latter case, the injection will advantageously be preceded by inhibition of the complement system in vivo.

 As indicated above, the present invention also relates to any use of a baculovirus as described above for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of diseases of the nervous system. More particularly, it relates to any use of these baculoviruses for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple sclerosis epilepsy or other diseases that affect myelinization, as well as diseases that affect metabolism such as lysosomal diseases.

 The present invention also relates to a pharmaceutical composition comprising one or more recombinant baculoviruses as described above.

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 These pharmaceutical compositions can be formulated for administration by various routes and in particular by the intramuscular, subcutaneous, intraocular, transdermal, intra-cerebral, intra-ventricular, intra-medullary route, etc.

Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the nervous system of the patient. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. Direct injection into the patient's nervous system is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues. The direct injection into the central nervous system of the patient is advantageously carried out by means of a stereotaxic injection device.

The use of such a device makes it possible to target with great precision the injection site.

 In this regard, the invention also relates to a method of treatment of diseases of the nervous system, such as for example neurodegenerative or metabolic diseases, comprising the administration to a patient of a recombinant baculovirus as defined above. More particularly, the invention relates to a method of treatment of neurodegenerative diseases comprising the stereotaxic administration of a recombinant baculovirus as defined above.

 The doses of virus used for the injection can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought. In general, the recombinant baculoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu / ml, and preferably 106 to 1010 pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an insect cell culture, and measures, generally after 5 days, the number of

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 ranges of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

 Another subject of the invention relates to any mammalian cell infected with one or more recombinant baculoviruses as described above. More particularly, the invention relates to any population of human neural cells infected with these baculoviruses. It can in particular be glial cells (astrocytic, microglial, oligodendrocyte, Schwann cell), ependymal, neural, etc.

 The cells according to the invention can come from primary cultures.

These can be removed by any technique known to those skilled in the art, then cultured under conditions allowing their proliferation. More particularly with regard to autologous grafts, they may be astrocytes, embryonic cells, nerve or neuronal progenitor cells such as progenitors originating from the telencephalon or from the human midbrain, these cells can be easily obtained from biopsies . It can also be a heterologous graft or xenografts from pheochromocytomas, or embryonic cells or astrocytes. These cells can be used directly for baculovirus infection, or stored, for example by freezing, for the establishment of autologous banks, for later use. The cells according to the invention can also be secondary cultures, obtained for example from pre-established banks.

 The cultured cells are then infected with recombinant baculoviruses, to give them the capacity to produce a substance of therapeutic interest. The infection is carried out in vitro according to techniques known to those skilled in the art. In particular, according to the type of cells used and the number of copies of virus per cell desired, a person skilled in the art can adapt the multiplicity of infection and possibly the number of infection cycles carried out. It is understood that these steps must be carried out under sterile conditions

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 appropriate when the cells are for in vivo administration. The doses of recombinant baculovirus used for infection of the cells can be adapted by a person skilled in the art according to the aim sought. The conditions described above for administration in vivo can be applied to infection in vitro.

 Another subject of the invention relates to an implant comprising human cells infected with one or more recombinant baculoviruses as described above, and an extracellular matrix. Preferably, the implants according to the invention comprise 105 to 1010 cells. More preferably, they include 106 to 108.

 More particularly, in the implants of the invention, the extracellular matrix comprises a gelling compound and optionally a support allowing the anchoring of the cells.

 For the preparation of the implants according to the invention, different types of gelling agents can be used. The gelling agents are used for the inclusion of cells in a matrix having the constitution of a gel, and to promote the anchoring of the cells on the support, if necessary. Different cell adhesion agents can therefore be used as gelling agents, such as in particular collagen, gelatin, glycosaminoglycans, fibronectin, lectins, etc. Preferably, in the context of the present invention, collagen is used. It can be collagen of human, bovine or murine origin. More preferably, type I collagen is used.

 As indicated above, the compositions according to the invention advantageously comprise a support allowing the anchoring of the cells. The term anchoring designates any form of biological and / or chemical and / or physical interaction resulting in the adhesion and / or fixing of the cells on the support. Furthermore, the cells can either cover the support used, or penetrate inside this support, or both. It is preferred to use within the framework of the invention a solid support, not

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 toxic and / or bio-compatible. In particular, polytetrafluoroethylene (PTFE) fibers or a support of biological origin can be used.

 The implants according to the invention can be implanted at different sites in the body. In particular, the implantation can be carried out in a muscle, a tumor, the central nervous system, or even under a mucous membrane. The implants according to the invention are particularly advantageous in that they make it possible to control the release of the therapeutic product in the organism: This is first of all determined by the multiplicity of infection and by the number of cells implanted .

Then, the release can be controlled either by the withdrawal of the implant, which definitively stops the treatment, or by the use of regulable expression systems, making it possible to induce or repress the expression of the therapeutic genes.

 The present invention thus provides a very effective means for the treatment or prevention of diseases of the nervous system. It is particularly suitable for the treatment of Alzheimer's, Parkinson's, Huntington's, and ALS diseases, or even lysosomal diseases. The baculoviruses according to the invention also have significant advantages, linked in particular to their low immunogenicity, due to the absence of expression of the baculovirus proteins in mammalian cells.

 The present invention will be described in more detail using the following examples which should be considered as illustrative and not limiting.

MATERIALS AND METHODS General techniques of molecular biology
Classically used methods in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, protein extraction at

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 phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Escherichia coli, etc. are well known to the skilled person and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F. M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].

 For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.

 The filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase 1 from E. coli (Biolabs) according to the supplier's specifications. The destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends is carried out by a gentle treatment with nuclease S 1.

 Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

 The enzymatic amplification of DNA fragments by the technique called PCR [Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.

 Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5477] using the kit distributed by Amersham.

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EXAMPLES Example 1: construction of different baculovirus vectors 1.1 - Construction and production of Baculovirus RSV-LacZ:
The recombinant baculovirus RSV-LacZ was obtained by homologous recombination between a shuttle plasmid and the genome of Autographa california in SF9 cells and amplified in these same cells according to the method described by Gruenwald S. and Heitz J., 1993 (Baculovirus expression vector system, procedure & method manual. Pharmingen Eds, San Diego, CA).

 The shuttle plasmid was produced by insertion into plasmid pVL 1392 (Pharmingen, San Diego, CA) of a cassette for expression of the nuclear betagalactosidase gene under the control of the RSV LTR, and of the SV40 polyadenylation signal downstream of the LacZ gene. This expression cassette was inserted into the multiple cloning site in reverse orientation relative to the polyhedrin promoter contained in the plasmid pVL 1392.

 The virus is then produced by amplification in Sf9 cells. It was concentrated by a factor of 100 by ultracentrifugation of 180 ml of supernatant (initially titrated at 3 × 107 pfu / ml) at 25,000 rpm at 4 ° C. for 90 minutes in a rotor SW 28, then taken up in 2 ml. of PBS, ultracentrifuged at 25,000 rpm at 4 ° C. for 90 minutes in a rotor SW 41 in a sucrose gradient (10% to 60%, in PBS).

The white band corresponding to the purified viral particles was removed and resuspended in PBS and ultracentrifuged at 25,000 rpm at 4 ° C. for 90 minutes. The pellet was resuspended in 1.5 ml of cold PBS. This concentrated and purified virus stock was kept at 4 C.

1. 2 - Construction and production of Baculovirus CAG-LacZ:

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The recombinant baculovirus CAG-LacZ was obtained using the shuttle plasmid pAcYMI (Matsuura et al, J. of Gen. Virol. 68 (1987) 1233-1250). An expression cassette containing the CAG promoter controlling the expression of the LacZ gene was inserted into the shuttle plasmid. The CAG promoter is a composite promoter consisting of a CMV IE enhancer, the chicken beta-actin promoter and the rabbit beta-globin polyadenylation signal.

 The recombinant baculovirus was obtained by homologous recombination in Sf9 cells, then amplified in these cells (according to the protocol of Matsuura et al., J. of Gen. Virol. 68 (1987) 1233-1250).

 The virus used was obtained by amplification of an aliquot of the recombinant baculovirus in Sf9 cells. The viral solution is titrated at 1x109 pfu / ml. The virus is then concentrated by a factor of 100 and purified under the same conditions as those described for the baculovirus RSV-LacZ.

Example 2: test of the in vitro infection of different cell cultures 2.1 - Infection of primary cultures of adult astrocytes:
The cells are grown in 4-well dishes. The infection takes place at 37 ° C. in culture medium without serum for approximately 2 h. It was done with increasing concentrations of recombinant virus. After 24 h the cells are fixed (PFA 4%) and stained with X-Gal. Uninfected controls do not show a blue cell.

 Observation of the cultures at 24 and 48 hours after infection shows that the recombinant baculovirus is capable of infecting cells of a primary culture of human astrocytes (human telencephalon cultivated in bFGF) and of expressing the LacZ gene ( observed by X-Gal reaction).

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2. 2 - Infection of primary cultures of human embryonic telencephalon:
The cells are cultured in a medium containing bFGF (medium enabling them to be maintained in a state of undifferentiated or very poorly differentiated progenitor cells).

The infection was made in the complete culture medium for 2 h. Uninfected controls do not show blue cells.

 Observation of cultures four days after infection shows that the recombinant baculovirus is capable of infecting human nerve progenitor cells (human telencephalon cultivated in bFGF) and of expressing the LacZ gene (observed by X-Gal reaction).

 All of these results demonstrate that the recombinant vectors derived from baculoviruses are capable of infecting different types of primary cultures of neural cells in vitro and can thus be used as a gene therapy vector for the transfer of genes ex vivo.

Example 3: In Vivo Test 3. 1 - Stereotaxic injection into the rat striatum:
Adult female Sprague-Dowley rats were injected with 9 l of concentrated RSV-LacZ virus. The injection was made by slow injection of 1 l of virus (0.25 l / min) into three injection sites with 3 sub-sites for each of the sites.

Stereotaxic coordinates with respect to Bregma:
A: +1.8, L: + 2.5, VO: -5, VI: -4.6, V2: -4.2
A: +0.6, L: + 3.2, V0: -5, V1: -4.6, V2: -4.2
A: + 0.6, L: +2, VO: -5, V1: -4.6, V2: -4.2
After one week the rats are fixed by intracardial perfusion of PFA 4% in PBS and the brains post-fixed for 24 h at 4 C in PFA 4%. The brains

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 are then cryoprotected by immersion in 15% sucrose in PBS for 72 h. The brains are then frozen in cold isopentane (-50 C) and preserved at -80 C.

3.2 - Histology on the brains of the injected rats:
The brains are cut at the striatum with a cryostat (cutting thickness: 20 m). The sections are studied in immunohistochemistry to detect the presence of E-coli P-galactosidase. The slides are washed in PBS, incubated in methanol + 0.3% H 2 O 2 for 1 hour, washed in PBS, then preincubated for 2 h with PBS + 10% FCS + 0.1% Triton. Then they are incubated overnight with anti-B-Gal antibody (Cappel: 1: 5000th) washed with PBS, then incubated with the secondary antibody (Vectastain Goat Anti-Rabbit Kit). The revelation is made in DAB + Nickel.

 A fluorescent double labeling B-Gal + GFAP was done using an anti-B-Gal monoclonal antibody (Promega) and a polyclonal anti-GFAP antibody.

Very few cells are doubly labeled.

 Fluorescent double labeling B-Gal + NeuN was done using a polyclonal anti-B-Gal antibody (Cappel) and an anti-NeuN polyclonal antibody. NeuN is a specific marker for differentiated neurons. It is observed that most of the cells are doubly labeled.

 There is a more pronounced transduction and labeling of neurons at the periphery of the corpus callosum as well as neuronal cells in the striatum.

The immunohistochemistry study with the anti-BGal antibody demonstrates that the recombinant baculovirus allows infection and expression of the transgene in a large number of nerve cells. It also shows that the recombinant baculovirus is insensitive to complement inactivation in the CNS.

Claims (18)

1. Recombinant baculovirus comprising a heterologous nucleic acid sequence encoding a product of therapeutic interest for the treatment of diseases of the nervous system. 2. Baculovirus according to claim 1, characterized in that the heterologous nucleic acid sequence is a gene or an antisense sequence. 3. Baculovirus according to claim 2, characterized in that the heterologous nucleic acid sequence is a gene coding for a product of therapeutic interest chosen from hormones, lymphokines, growth factors, enzymes for synthesizing neurotransmitters, trophic factors, proteins involved in the metabolism of amino acids, lipids or carbohydrates. 4. Baculovirus according to claim 3, characterized in that the trophic factors are chosen from members of the neurotrophin family such as NGF, BDNF, NT3, NT4 / 5, NT6, members of the CNTF family such as CNTF, axokine, LIF, IL6, cardiotrophin, GDNF, members of the IGF family, such as IGF-1, IFGF-2, members of the FGF family, such than FGF 1, 2.3, 4.5, 6.7, 8.9 and TGF-p. 5. Baculovirus according to claim 3, characterized in that the gene coding for a product of therapeutic interest is the gene coding for p-glucuronidase. 6. Recombinant baculovirus according to one of claims 1 to 5, characterized in that it is a baculovirus expressing an envelope protein other than that of baculoviruses. 7. Baculovirus according to claim 6, characterized in that the envelope protein is the glycoprotein of the rabies virus or the glycoprotein of VSV (Vesicular Stomatitis Virus). <Desc / Clms Page number 21> 8. Recombinant baculovirus according to one of claims 1 to 7, characterized in that it also comprises promoter sequences allowing the expression of the heterologous nucleic acid sequence. 9. Baculovirus according to claim 8, characterized in that the promoter sequence is chosen from the promoters of the NSE (Neuron Specific Enolase), NF (Neurofilament), TH (Tyrosine Hydroxylase), DAT (Dopamine Transporter), ChAT (Choline Acetyl) genes Transferase), DBH (Dopamine P-Hydroxylase), TPH (Tryptophan Hydroxylase), GAD (Glutamic Acid Dehydrogenase), GFAP (Glial Fibrillary Acidic Protein). 10. Recombinant baculovirus according to one of claims 1 to 9, characterized in that it also comprises signal sequences making it possible to induce secretion of the therapeutic product. 11. Use of a recombinant baculovirus comprising in its genome a heterologous nucleic acid sequence coding for a product of therapeutic interest for the preparation of a pharmaceutical composition intended for the treatment of diseases of the nervous system. 12. Use of a recombinant baculovirus according to claim 11, characterized in that the heterologous nucleic acid sequence is a gene or an antisense sequence. 13. Use of a recombinant baculovirus according to claim 12, characterized in that the heterologous nucleic acid sequence is a gene coding for a product of therapeutic interest chosen from hormones, lymphokines, growth factors, enzymes synthesis of neurotransmitters, trophic factors, proteins involved in the metabolism of amino acids, lipids or carbohydrates. <Desc / Clms Page number 22> 14. Use of a recombinant baculovirus according to claim 13, characterized in that the trophic factors are chosen from members of the neurotrophin family such as NGF, BDNF, NT3, NT4 / 5 and NT6, members of the family CNTF such as CNTF, axokine, LIF, IL6, cardiotrophin, GDNF, members of the IGF family, such as IGF-1, IFGF-2, members of the FGF family, such as FGF 1.2, 3.4, 5.6, 7.8, 9, and TGF-p. 15. Use of a recombinant baculovirus according to claim 13, characterized in that the gene coding for a product of therapeutic interest is the gene coding for P-glucuronidase 16. Population of cells of the nervous system (eg brain, spinal cord, neural, glial, ependymal cells, infected with one or more recombinant baculoviruses according to one of claims 1 to 10. 17. Implant comprising human cells infected with one or more recombinant baculoviruses according to one of claims 1 to 10. 18. Pharmaceutical composition comprising one or more recombinant baculoviruses according to one of claims 1 to 10, in combination with a pharmaceutically acceptable vehicle.