MXPA98009881A - Generation of v replication molecules - Google Patents

Abstract

The present invention relates to circular DNA molecules and replication, usable in gene therapy, as well as a particularly effective method for its generation in situ from a viral vector.

Description

GENERATION OF IN VIVO REPLICATION MOLECULES FIELD OF THE INVENTION The present invention relates to circular and replication DNA molecules, usable in gene therapy. The invention also describes a particularly efficient method for its generation in situ from a corresponding viral vector.

BACKGROUND OF THE INVENTION Gene therapy consists of correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) by introducing a genetic information into the affected cell or organ. This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell is also reintroduced into the organism, or directly in vivo into the appropriate tissue. In this second case, there are different techniques, among which various transfection techniques involving vectors of different natures. It can be chemical and / or biochemical vectors, natural or synthetic on the one hand or viral vectors on the other hand. By way of illustration of REF .: 28713 viral vectors, the DNA and DEAE-dextran complexes (Pagano et al., J. Virol. 1 (1967) 891), DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. However, its use mainly involves the possibility of producing significant quantities of DNA of pharmacological purity. The viral vectors (retroviruses, adenoviruses, adeno-associated viruses, ...) are very effective, comparatively with the chemical and / or biochemical vectors described above, mainly for the passage of the membranes. Among these viruses, adenoviruses have particularly interesting properties for use in gene therapy. Mainly, they have such a large host spectrum, that they are capable of transducing dividing cells or quiescent cells and the adenovirus genome persists under the extra-chromosomal form, and important pathologies in humans have not been associated until this date. . In contrast, the use of retroviruses whose genome is randomly integrated into the genome of the infected cell is limited to dividing cells. Adenoviruses have thus been used to transfer genes of interest in muscular quiescent cells (myotubes; Ragot et al., Nature 361 (1993) 647)), hepatic (Jaffe et al., Nature genetics 1 (1992) 372), nervous (A li et al., Nature genetics 3 (1993) 224), bronchial epithelial (Rosenfeld et al., 1992), etc. However, in rapidly renewing tissues a progressive loss of transgene expression is observed due to dilution effect during cell division. The improvement of adenoviral vectors and the development of new generations of vectors has led to the reduction of the potential risks of pathogenic, residual, as well as immunogenic power linked to the replication of the vector, the recombination of its genome and the expression of proteins. viral To prevent such risks as much as possible, the currently proposed viral vector constructs are modified so as to render the vectors unable to replicate autonomously in the target cell. They are called defective. Generally, the genome of the defective viruses is devoid of at least the sequences necessary for the replication of the virus in the infected cell. Thus in the particular case of the adenoviruses, the constructs described in the prior art are deleterious or deleted adenoviruses of the El and eventually E3 regions at which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 ( 1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Other constructs comprise a deletion at the level of the El region and a non-essential part of the E4 region (094/12649), or a modified genomic organization (FR 94 13355). However, it resides or remains, during the production of these defective viral vectors, the risk of recombinations that generate viral particles of replication or transcomplementations in vivo by cellular functions of type El. It is clear that the use in gene therapy of vectors thus contaminated, can have very harmful consequences such as inducing a viral spread and causing an uncontrolled spread with risks of inflammatory reaction and immune response directed against viral proteins, etc ... On the other hand, the improvement of the stability of the expression of the transgene in the cells transduced by the adenoviral vector, and more particularly in the dividing cells remains an important problem to be solved. Accordingly, it remains to this day in gene therapy, a real need in transfer vectors that would essentially manifest the advantages of each of the above-described vectors namely good transfection properties, calculated for example in those of the viral vectors and in particularly those of the adenoviruses and a perfect innocuousness that is translated in particular by an absence of generation of viral particles of replication in vivo, risk that does not exist with the plasmids or non-viral vectors.

DESCRIPTION OF THE INVENTION The aim of the present invention is precisely to propose a new concept of gene transfer that satisfies the aforementioned requirements. The present invention resides mainly in the generation in situ, via a viral vector, of circular DNA molecules, of replication, therapeutic and advantageous in terms of the stability of transgene expression and of innocuity. In fact, these are devoid of any viral genome sequence capable of inducing an inflammatory-type immune response as well as a specific response directed against viral proteins that can have a deleterious effect on the organism and limit the duration of transgene expression. .

The subject of the present invention is therefore the circular, replication DNA molecules that comprise at least: (i) one or several genes of interest with the sequences necessary for their expression, (ii) a functional origin of replication in the cells of mammals, and, (iii) a sequence that results from site-specific recombination or specific recombination of a sequence between two sequences recognized by a recombinase. The present invention particularly discloses the setting up of a specific process and constructions, particularly effective for the production of these therapeutic DNA molecules. More particularly, the method according to the invention resides in the production of therapeutic DNA molecules defined above from a viral vector. In a pretended manner, the applicant has thus shown that it would be possible to generate in situ from a viral vector and this by specific recombination of a sequence, a circular DNA molecule of therapeutic and replication nature. Furthermore, in an advantageous embodiment, the transgene and the origin of replication are inactive in the viral vector, however its activity is the event of site-specific recombination or specific recombination of a sequence. Even more advantageously, the recombination event is induced conditionally by the expression of the recombinase, which offers a high level of control in the expression of the gene of interest and in the replication of the episomal molecules produced. Such a protocol is particularly advantageous in the therapeutic field: taking advantage of the good transfection capacities expressed in a general way by the viral vectors comparatively with the non-viral vectors, - considerably reducing the risks of viral contamination, of local inflammatory reaction as well as of response immunological antiviral, taking into account the reduced number of viral vectors put into practice. The latter is introduced only in a necessary proportion to the generation of the therapeutic DNA molecule, - it allows to expand the field of application of certain viral vectors: Thus, adenoviral or adenoviral vectors are of limited applications in proliferating cells. such as the cells of hematopoetic strains. The present invention allows to explore its infectious power to generate in the proliferative cells stable circular replication molecules. The DNA molecule as claimed, thus possesses the ability to efficiently assure the transfer in the target cells of or of the therapeutic genes that contain them. For this fact, it contains an origin of replication characterized mainly by the fact that it is functional in mammalian and human cells. Advantageously, the origin of replication used is a conditional origin of replication, that is, whose activity can be regulated. Even more preferably, the origin of replication is arranged in such a way that it is inactive in the viral vector, and active after the specific recombination of a sequence. Advantageously, the origin of replication used is of eukaryote origin, viral or mammal. A preferred example of origin of replication capable of being put into operation in the context of the present invention is, more particularly, the origin of replication of the Epstein Barr virus (EBV). The EBV virus belongs to the Herpesviridae family. Its origin of replication comprises two elements: the sequence oriP (1.7kb) responsible for replication, whose activity is induced by the protein encoded by the EBNAl gene. This sequence can be carried in trans. These elements allow them, at the same time, replication, episomal preservation and the segregation of 5 to 20 copies per cell of a plasmid vector. The oriP sequence is composed of a repetition of 20 30bp patterns or letters, separated from 960bp from the origin of replication that is formed from a repeated inverse word or motive of 65bp and comprises 4 imperfect copies of the word or 30 bp pattern. The EBNAl protein binds 30bp words or motifs at the level of the origin of replication and allows the recruitment of cellular factors at the time of the S phase and the replication, synchronous to cell division, of a plasmid having the sequence oriP in cis In addition EBNAl, certainly by the simultaneous fixation at the level of repeated motifs and chromosomal structures, allow intranuclear conservation and segregation of the episome at the time of cell division. Plasmids containing the oriP origin of replication of the EBV genome and allowing the expression of the viral protein EBNAl (641 amino acids) are maintained in a stable episomal manner in the transfected human cells and their replication is synchronous to cell division (Lupton et al. Levine, 1985).

The origin of replication can also be derived from papillomaviruses. The papillomaviruses use a viral latency system with episomal conservation of the genome analogous to that of the Epstein Barr virus (EBV). This system has been studied particularly for bovine papillomavirus type 1 (BPV-1). The origin of BPV replication is active in the presence of the El and E2 proteins. As in the case of EBV, episomal preservation is independent of replication and is ensured by the fixation of E2 at the level of the MME sequence (minichromosome maintenance element), but it also needs the presence of El. change, contrary to EBV's oriP / EBNAl system, the replication of the episome is not synchronous to cell division (Piirsoo et al., 1996). The origin of replication can also be made up of sequences capable of autonomous replication, or ARS (autonomously replicating sequences). ARSs have been isolated from chromosomes of mammals, mainly man and mouse. Preferentially, the localized ARS sequence can be cited by stacking the c-myc locus in man (Ariga et al., 1988) and the 4kb fragment of the mouse adenosine deaminase gene locus (Virta-Pearlman et al., 1993).

Regarding the gene of interest, it can be a therapeutic gene, vaccinal, agronomic or veterinary. It also contains a promoter region of functional transcription in the cell or target organism, as well as a region located at 3 ', and which specifies an end signal of transcription and polyadenylation. As far as the promoter region is concerned, it can be a promoter region naturally responsible for the expression of the gene considered when it is capable of functioning in the cell or the related organism. It can also be regions of different origin (responsible for the expression of other proteins, or also synthetic). Primarily, it can be promoter sequences of eukaryotic or viral genes. For example, it can be promoter sequences from the target cell genome. Among the eukaryotic promoters, any promoter or derived sequence that stimulates or represses the transcription of a gene in a specific manner or not, inductive or not, strong or weak can be used. One can deal in particular with ubiquitous promoters (promoter of the genes HPRT, PGK, a-actin, tubulin, etc.), of promoters of intermediate filaments (promoter of the genes GFAP, desmin, vimentin, neurofilaments, keratin, etc.), of promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAI, etc. genes), tissue-specific promoters (pyruvate kinase gene promoter, villin, intestinal protein of fatty acid binding, alpha- smooth muscle actin, etc.) or even promoters that respond to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.). Likewise, it can be promoter sequences exiting the genome of a virus, such as, for example, the promoters of the E1A and MLP genes of adenovirus, the early promoter of CMV, or even the promoter of the LTR of RSV, etc. In addition, these promoter regions can be modified by the addition of activation sequences, of regulation, or that allow tissue-specific or majority expression. On the other hand, the gene of interest may also include a signal sequence that directs the synthesized product into the secretion pathways of the target cell. This signal sequence can be the natural signal sequence of the synthesized product, but it can also be any other functional signal sequence, or an artificial signal sequence. Advantageously, a promoter of viral origin chosen between the early promoter of the CMV or the LTR of a retrovirus or a mammalian promoter is used. Another origin of replication and at least one gene of interest, the DNA molecules of the invention comprise a region resulting from the specific recombination between two sequences. This specific recombination of a sequence or site-specific can be obtained from various systems that entrain or carry the specific recombination of a sequence between the sequences. The specific recombination system implemented in the context of the present invention, in view of the in situ generation of the claimed DNA molecules, they may be of different origins. In particular, the specific sequences and the recombinases used can belong to different structural classes, and mainly to the family of the bacteriophage P1 recombinase. More preferably, the specific recombination of a sequence used according to the method of the invention is obtained by means of two specific sequences which are capable of recombining with each other in the presence of a specific protein, generally designated recombinase. It is for this reason that the circular DNA molecules according to the invention further comprise a sequence resulting from this specific site recombination. The sequences that allow recombination used in the field of the invention, generally comprise from 5 to 100 base pairs, and, more preferentially, less than 50 base pairs. Among the recombinases belonging to the integrase family of bacteriophage 1, the lambda phage integrase can be mentioned mainly (Landy et al., Science 197 (1977) 1147), P22 and F80 (Leong et al., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus influenzae (Hauser et al., J. Biol. Chem. 267 (1992) 6859), the integrase Cre of phage Pl, the integrase of plasmid pSAM2 (EP 350 341) or even the FLP recombinase of the plasmid 2 m of the yeast Saccharomyces cerevisiae. When the DNA molecules according to the invention are prepared by recombination by means of a specific site system of the integrase family of the bacteriophage lambda, the DNA molecules according to the invention generally also comprise a sequence resulting from the recombination between two fixation or att binding sequences of the corresponding bacteriophage or plasmid. Among the recombinases belonging to the Tn3 transposon family, we can mainly mention the resolvase of transposon Tn3 or the transposons gd, Tn21 and Tn522 (Stark et al., 1992); the Gin invertase of the bacteriophage mu or even the plasmid resolvase, such as that of the even fragment of RP4 (Abert et al., Mol.

Microbiol. 12 (1994) 131). When the DNA molecules according to the invention are prepared by recombination by means of a specific site system of the Tn3 transposon family, the DNA molecules according to the invention generally also comprise a sequence resulting from the recombination between two sequences of recognition of the resolvase of the considered transposon. According to a preferred embodiment, in the genetic constructions of the present invention, the sequences that allow site-specific recombination are derived from a bacteriophage. More preferably, it is composed of the binding or binding sequences (attP and attB sequences) of a bacteriophage or derived sequences.These sequences are capable of recombining specifically with each other in the presence of a recombinase designated integrase. The derivative includes the sequences obtained by modification (s) of the binding or binding sequences of the bacteriophages, which retain the ability to recombine specifically in the presence of the appropriate recombinase.Thus, it can be reduced fragments of these sequences or on the contrary extended by the addition of other sequences (restriction sites, etc.) It can also be variants obtained by mutation (s), mainly by point mutation (s). It is designated according to the invention, by sequence attP and attB of a bacteriophage or a plasmid sequences of the specific recombination system of the bacteriophage or plasmid or, that is, the attP sequence present in the phage or plasmid and the corresponding chromosomal attB sequence. As preferred examples, mention may be made, in particular, of binding or binding sequences of phages lambda, P22, F80, Pl, HP1 of Haemophilus influenzae or even of plasmid pSAM2, or 2 m. In accordance with a preferred embodiment of the invention, sequences that allow site-specific recombination or specific recombination of a sequence are derived from the phage recombination system Pl. This phage Pl possesses a recombinase of this Cre name which specifically recognizes a nucleotide sequence of 34 base pairs called the lox P site. This sequence is composed of two palindromic sequences of 13 bp separated by a conserved sequence of 8 bp. In a particular variant, the invention thus relates to a circular and replicative DNA molecule comprising (a) a sequence from the specific site recombination between two loxP regions of the bacteriophage Pl, at least one gene of interest and an origin of replication functional in mammalian and human cells and which according to a preferred mode possesses conditional functionality. In this regard, the present invention also provides the particular gene constructs appropriate for the production of the therapeutic DNA molecules defined above. These gene constructs, or recombinant DNAs according to the invention, comprise mainly the gene (s) of interest, the origin of replication and the EBNAl protein gene surrounded by the two sequences that allow site-specific recombination placed in direct orientation. These sequences can be cloned in the form of cassettes in the bacterial plasmids. The plasmid DNA can, in a first period, be transfected into human cells in order to test the functionality of these sequences. These cassettes are then used to construct the viral vectors that possess these same sequences integrated in their genome. As indicated above, another aspect of the present invention resides in a method of in situ production of the therapeutic DNA molecules defined above from a viral vector by site-specific recombination. The use of a vector advantageously makes it possible to optimize the administration of the claimed DNA molecule in the cells to be treated.

In this regard, the present invention also has as an object a viral vector comprising, inserting in its genome, at least one region of DNA surrounded by two sequences that allow a site-specific recombination and placement in direct orientation, the region of DNA that it comprises at least one origin of replication and one gene of interest. In accordance with a privileged mode of the invention, the origin of replication as well as the gene of interest integrated in the viral vector, are present under an inactive form, the promoter is cloned in direct orientation to one of the extremities of the expression cassette (Figure 1) After recombination between the two LoxP sites, the promoter is located before the gene of interest and separated from the latter by a LoxP site, the first ATG of the transcript corresponding to the start codon of the transgene. oriP is not active except in the presence of the EBNAl protein it is expressed under the control of the same promoter as the transgene, in the form of a bicistronic messenger.The translation of EBNAl is initiated by an internal initiation mechanism at the level of a IRES sequence derived from the encephalomyocardite virus (ECMV), from the picornavirus family, expression of the transgene and the EBNAl protein as well as replication of the plasmid are thus directly conditioned by the recombination event between the two LoxP sites. According to another variant of the invention, the region of virus infection is even in the replicon (the region of DNA surrounded by the sequences that allow site-specific recombination). This mode of operation offers a complementary security to the system, as explained later. The viral vector used can be of various origins, since it is capable of transducing the animal cells and preferably the human cells. In a preferred embodiment of the invention, vectors derived from adenoviruses, adeno-associated viruses (AAV), herpes viruses (HSV) or retroviruses are used. It is particularly advantageous to use an adenovirus, for direct administration or for the ex vivo modification of cells to be implanted, or a retrovirus, for the implantation of producer cells. The viruses according to the invention are defective, that is to say that they are unable to replicate autonomously in the target cell. Generally, the genome of the defective viruses used in the field of the present invention are thus devoid, at least of sequences necessary for the replication of the virus in the infected cell. These regions can be either deleted (in whole or in part), or non-functional turns, or substituted by other sequences and mainly by the heterologous nucleic sequence of interest. Preferably, the defective virus nevertheless retains the sequences of its genome which are necessary for the encapsidation of the viral or viral particles. More specifically, it concerns adenoviruses, it is preferable to use human adenoviruses type 2 or 5 (Ad2 or Ad5) or adenoviruses of animal origin within the framework of the present invention (see application W094 / 26914). Among the adenoviruses of animal origin which may be used in the field of the present invention, there may be mentioned adenoviruses of canine, bovine, murine origin (for example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, aviary or even simian (example: SAV). Preferentially, the viral vectors of the invention are defective adenoviruses: > s comprising embedding or grafting into their genome, a gene sequence comprising at least one origin of replication and a gene of interest and surrounded by two sequences placed in direct orientation. These allow to induce a conditional activity of the origin of replication and of the transgene, through the intermediary of the specific site recombination, dependent on the presence of the recombinase. Advantageously, in the genome of these adenoviruses of the invention, the El region becomes at least non-functional. Even more preferably, the El gene and at least one of the genes E2, E4, L1-L5 are not functional. Other regions can also be modified, and mainly the region E3 (WO95 / 02697), E2 (W094 / 28938), E4 (W094 / 28152, 094/12649, WO95 / 02697) and L5 (WO95 / 02697). According to a preferred mode of operation, the adenovirus according to the invention comprises a deletion in the El and E4 regions and the DNA region surrounded by the two sequences that allow a site-specific recombination, is inserted at the region level. The inactive. According to another preferred embodiment, it comprises a deletion in the El and E4 regions and the DNA region surrounded by the two sequences that allow a site-specific recombination is inserted at the level of the inactive E4 region. As indicated below, according to a particular mode of implementation of the invention, the adenovirus also comprises a cassette for the expression of the recombinase gene. The claimed vectors are obtained by recombination with plasmids as defined above, ie characterized in that they comprise between two site-specific recombination regions, at least one origin of replication and one gene of interest of conditional activity. As regards more specifically the definitions of the origin of replication, of two sequences that allow a specific site recombination and of the gene of interest, present in the region of DNA integrated in the viral vector claimed, will be reported to the definitions above. Defective recombinant adenoviruses according to the invention can be prepared by any technique known to those 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 among others the DNA sequences of the invention, or by construction of a viral genome in E. coli. Homologous recombination occurs after co-transfection of the adenoviruses and plasmid into an appropriate cell line. The cell line used should preferably (i) be transformable by the elements, and (ii), comprising 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, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains, in its genome, the left part of the genome of an Ad5 adenovirus (12%) or lines capable of complementing the El and E4 functions as described primarily in applications No. WO 94/26914 and WO95 / 02697. Next, the multiplying adenoviruses are recovered and purified according to the classical techniques of molecular biology, as illustrated in the examples. With regard to adeno-associated virus (AAV), it is DNA virus of relatively small size, which are integrated into the genome of cells that are infected, in a stable and site-specific manner. They are capable of infecting a large spectrum of cells, without inducing an effect on cell growth, morphology or differentiation. On the other hand, they do not seem to be involved in pathologies in man. The AAV genome has been cloned, sequenced and characterized. It comprises approximately 4,700 bases, and contains in each extremity an inverse repeat region (ITR) of approximately 145 bases, which serve as an origin of replication for the virus. The rest of the genome is divided into two 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 coding for the capsid proteins of the virus. The use of AAV-derived vectors for gene transfer in vitro and in vivo has been described in the literature (see mainly 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 or deleted and replaced by a gene of interest, and their use to transfer in vitro (in cells in culture) or in vivo (directly in an organism) the gene of interest. Defective recombinant AAVs according to the invention can be prepared by cotransfection, in a cell line infected by a human helper virus (for example an adenovirus), a plasmid containing the nucleic sequences of the invention surrounded by two inverse repeated regions (ITR) ) of AAV, and a plasmid carrying the encapsidation genes (rep and cap genes) of AAV. The produced recombinant AAVs are then purified by classical techniques. Concerning herpes viruses and retroviruses, the construction of recombinant vectors has been widely described in the literature: see mainly Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al. Genet Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc. In particular, retroviruses are integrating viruses, which selectively infect dividing cells. They thus constitute the vectors of interest for applications in cancer. The retrovirus genome essentially comprises two LTRs, one encapsidation sequence and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted, 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 MoMuLV ("murine moloney leukemia virus", also called MoMLV), MSV ("murine moloney sarcoma virus"), HaSV ("harvey sarcoma virus"); the SNV ("spleen necrosis virus"); the RSV ("rous sarcoma virus") or also the Friend virus. To construct the recombinant retroviruses according to the invention, a plasmid comprising mainly the LTRs, the encapsidation sequence and the sequences of the invention are generally constructed, then used to transfect a so-called encapsidation cell line capable of trans-delivering retroviral or retroviral functions deficient in the plasmid. Generally, the encapsidation lines are thus capable of expressing the gag, pol and env genes. Such encapsidation lines have been described in the prior art, and mainly line PA317 (US 4,861,719); the PsiCRIP line (WO90 / 02806) and the GP + envAm-12 line (WO89 / 07150). On the other hand, recombinant retroviruses can comprise modifications at the LTR level to suppress transcriptional activity, as well as extended or extended encapsidation sequences, comprising a part of the gag gene (Bender et al., J. Virol. 61 (1987 ) 1639). The recombinant retroviruses produced are then purified by classical techniques. As preferred vectors according to the invention, adenoviruses comprising in their genome a region of DNA according to the invention and surrounded by repeated inverse sequences of the bacteriophage Pl (loxP region) placed in orientation can be proposed more particularly. direct By way of illustration of this type of vector, the following constructions with the bgalactosidase gene (LacZ) or the Herpes virus thymidine kinase (TK) gene can be mentioned more particularly. (Figure 1) . The gene of interest can be any gene (cDNA, gDNA, RNA, synthetic or semi-synthetic nucleic acid) that codes for an RNA or a therapeutic or vaccinal protein such as enzymes, blood derivatives, hormones, lymphocytes: interleukins, interferons , TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc; the apolipoproteins: ApoAI, ApoAIV, ApoE, etc. (FR 93 05125), dystrophin or a minidistrofin (FR 9111947), the tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), the genes coding for the factors involved in the coagulation: Factors VII, VIII, IX, etc., or even all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.), a ligand RNA (W091 / 19813) etc. The gene of interest can also be an antisense sequence, whose expression in the target cell allows controlling 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 EP 140 308. The vectors of the invention are particularly adapted to the expression of coding sequences for toxic factors. In particular, it can be cell poisons (diphtheria toxin, pseunomonas toxin, ricin A, etc.) of a product that induces sensitivity to an external agent (suicide genes: thymidine kinase, cytosine deaminase, etc.) or even killer genes capable of inducing cell death (Grb3-3 (PCT / FR94 / 00542), anti-ras ScFv (W094 / 29446), etc.). The system of the invention makes it possible in fact to produce the mainly viral vectors containing these sequences without toxicity to the production cells, then induce the expression of these toxic molecules selectively in the target cells after site-specific recombination. This type of construction is particularly adapted to antitumor therapy strategies, for example, in which the objective is to selectively destroy the affected cells. This system is also particularly interesting for the expression of cytokines, interferons, TNF or TGF for example, whose uncontrolled production can have very remarkable side effects. The present invention is also directed to any eukaryotic cell transfected by at least one viral vector or a DNA molecule, according to the invention as defined above.

Another object of the present invention resides in a method of producing a DNA molecule as defined above, according to which a culture of host cells containing a viral vector according to the invention, is contacted with the recombinase that allows to induce site-specific recombination or specific recombination of a sequence. More precisely, the present invention refers generally to any method of preparation, characterized in that it is carried out in the presence of: (i) modified host cells containing at least one viral vector, the vector comprising in its genome at least one DNA region, surrounded by two sequences that allow a site-specific recombination and placed in direct orientation, the region comprises at least one origin of replication and a gene of interest and (ii) the recombinase that allows site-specific recombination to be induced in situ. specific, in order to generate circular DNA molecules and replication. Different protocols may be proposed within the framework of the present invention for effecting this contacting of the viral vector with the specific recombinase. It can be carried out mainly at the level of the host cell or by cotransfection with a plasmid or coinfection with a viral vector containing the gene of the so-called recombinase; or by induction of the expression of a gene encoding the recombinase directly present in the genome of the host cell. The gene coding for the recombinase may be present in the host cell under the form integrated to the genome, in a plasmid or even in an annexed viral vector of adenovirus type for example. In this case, the viral vector implemented in order to generate the DNA molecule according to the invention, is as defined above. According to another method, the cassette of expression of the gene is carried in the viral vector equally responsible for the expression of the gene of interest. In this particular case, the method according to the invention implements or operates a viral vector comprising in its genome another DNA region delimited by two coding sequences for the specific recombination sites and comprising at least one origin of replication and a gene of interest, a cassette for the expression of the recombinase gene. A vector constitutes an object of the present invention.

In this regard, according to a particular variant, the invention relates to an adenovirus comprising a first deletion in the El region into which the replicon is inserted (the region of DNA surrounded by two site-specific recombination sequences) and a deletion made at E4 and / or at E3 at which level the expression cassette of the recombinase protein is inserted.

Reciprocally to the mode of operation of the described procedure, the replicon can be inserted into the deleterious part corresponding to the E3 or E4 region while the recombinase expression cassette is inserted at the level of the deleterious region. According to another variant, the replicon as well as the recombinase expression cassette are inserted at the level of the defective region. More specifically, as indicated above, the sequences that allow site-specific recombination or specific recombination of a sequence are the LoxP sequences and the recombinase is the Cre protein, whose mode of intervention in the recombination sequences has been described higher. According to a preferred mode of the invention, it will be otherwise desirable to be able to control and in particular induce the expression of this recombinase within the host cell. For these purposes, it is advantageously proposed within the framework of the present invention to control the expression of the gene coding for the recombinase. For this purpose it is proposed to accommodate the expression of the gene under the control of a regulatory element. It can be mainly an inducer promoter, which allows to control the levels and / or the periods of expression of this gene, as for example the promoter of the LTR of MMTV (Pharmacia), which is induced by dexamethasone or a promoter regulated by tetracycline (W094 / 29442; WO94 / 04672). It is understood that other promoters can also be used, and in particular the variants of the MMTV LTR leading, for example, the heterologous regions of regulation (regions "enhancer (enhancers)" mainly). In another embodiment, the expression of the gene encoding the recombinase is under the control of regulated promoters in order to avoid or constitutive accumulation of the protein in the host cell, or to minimize "leakage or leakage" to the nuclear compartment and a certain cytotoxicity. The same can be associated to the elements that work as transcription transactivation fields. Representative of this type of elements, mention may be made, in particular, of hormone receptors which include the steroid, retinoic acid and thyroid receptors, among which those of glucocorticoids, mineral corticosteroids, thyroids, estrogen and aldosterone may be mentioned more particularly. and retinoic acid. This type of construction between Cre recombinase and the DNA binding field of a glucocorticoid receptor has been described, for example, in Feil et al. (PNAS 93 (1996) 10887). The possibility of regulating the expression of the recombinase is particularly important in the case of vectors of the invention which carry both the replicon and the recombinase expression cassette. Indeed, in this embodiment, if the recombinase is expressed for example during the production of viral vectors, the replicon will be excised from the viral genome before its encapsidation. For this reason, it is useful to have a system in which the expression of the recombinase protein is repressed in the cells that produce viruses. This is obtained as indicated above using a regulated promoter (tetracycline or MMTV type), and / or a hormone receptor-like element which, associated with the recombinase, maintains this in the extranuclear compartments in the absence of the hormone (FIGS. 6 and 7). Therefore, in the absence of the hormone, the recombinase can not react, and, in the presence of the hormone, it is also transported to the nuclear compartment where it exerts its activity. An interesting approach to control the expression of the recombinase is to use a recombinase fused to a hormone receptor (DNA binding field) and thus inactive in the absence of the hormone, then to place the gene in the viral vector in such a way that be under the control of the replicon promoter. This embodiment is represented, for example, in Figure 6b. On the other hand, to increase the safety of the system, it is also possible, as indicated above, to include in the replicon the viral vector encapsidation region (Figure 7). This makes it possible to prevent the deposit or stock of virus produced from being contaminated by the vectors that have lost the replicon. Indeed, if in spite of the regulatory systems mentioned above, an expression of the active recombinase intervenes during the production of the virus, this will lead to the cleavage of the replicon from the viral genome, and thus the generation of viral genomes devoid of replicon If the region of encapsidation of the virus is carried by the replicon, the viral genomes thus generated will not be encapsidated. Thus, only the viral genomes that carry both the replicon and the cassette of expression of the recombination, can be encapsidated. The present invention also relates to pharmaceutical compositions comprising at least one viral vector according to the invention, or a transfected cell according to the invention. These compositions can be formulated for the purpose of topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, etc. administration. Preferably, the composition according to the invention contains pharmaceutically acceptable vehicles for an injectable formulation. It can be treated in particular of saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or of dry compositions, mainly lyophilized, which, by addition of as the case may be, sterile water or physiological saline, allow the constitution of injectable solutes. In the case of retroviruses, it may be advantageous to directly use the encapsidation cells or the infected cells ex vivo for their reimplantation in vivo, possibly in the form of neo-organs (W094 / 24298). The doses of vectors used for the injection can be adapted according to different parameters, and mainly depending on the mode of administration used, the aforementioned pathology or even the duration of the treatment sought. In a general manner, their recombinant viruses according to the invention are formulated and administered in the form of doses comprised between 104 and 1014 pfu / ml. For AAVs and adenoviruses, doses of 106 to 1010 pfu / ml can also be used. The term pfu ("plaque forming unit") corresponds to the infectious power of a suspension of virions, and is determined by infection of an appropriate cell culture, and measured, usually after 48 hours, the number of pests of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature. According to the gene of interest, present in the DNA molecules of the invention or the regions integrated in the genome of the viral vectors, they can be used for the treatment or prevention of numerous pathologies, including genetic diseases (myodystrophy, mucoviscidosa, etc.), neurodegenerative diseases (Alzheimer's, Parkinson's, ALS, etc.), cancers, pathologies linked to coagulation disorders or to dyslipoproteinemias, pathologies linked to viral or viral infections (hepatitis, AIDS, etc.), or in the agronomic and veterinary fields, etc. The present invention will be described more fully with the help of the following examples, which should be considered as illustrative and not limiting.

Legend of the figures Table 1: Origin of the sequences used for the construction of the episome Figure 1: Scheme of construction of the episome Figure 2: Representation of the structure of the episome.

Figure 3: Sequence between the promoter (P-RSV) and the expression cassette (TK-CITE-EBNA1) in the plasmid pLoxP-ori-TK-EBNAl (A) and in the episome after recombination (B, C and D) -SEC ID n ° 3 Figure 4: Scheme of the cloning steps of the LoxP-ori-TK-EBNAl cassette Figure 5: Scheme of the cloning steps of the LoxP-ori-LacZ-EBNAl cassette Figure 6: Representation of a adenoviral vector carrying a replicon and a regulated expression cassette of Cre: CRER: Cre regulated, either at the level of the promoter, or by a fusion, or both. In (b), the orientation of the replicon allows the CRER cassette to be placed under the control of the promoter present in the replicon. The gray box corresponds to the LoxP sites. Figure 7: Representation of an adenoviral vector carrying a replicon and a regulated expression cassette of Cre, the region of viral encapsidation Psi (?) Is even in the replicon. The gray box corresponds to the LoxP sites.

General techniques of cloning and molecular biology Classical molecular biology methods such as centrifugation of cesium-ethidium bromide gradient plasmid DNA, restriction enzyme digestions, gel electrophoresis, electroelution of DNA fragments from agarose gels , the transformation in E. coli, the precipitation of nucleic acids etc, are described in the literature (Maniatis et al., 1989, Ausubel et al., 1987). The nucleotide sequences have been determined by the chain termination method following the protocol already presented (Ausubel et al., 1987). Restriction enzymes have been provided by New-England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham Ltd (Amersham).

For ligatures, the DNA fragments are separated according to their size in 0.7% agarose or 8% acrylamide gels, purified by electrophoresis and then electroeluted, extracted from phenol, precipitated in ethanol, then incubated in a Tris buffer. -HCl pH 7.4 50 mM, 10 mM MgC12, 10 mM DTT, 2 mM ATP, in the presence of T4 phage DNA ligase (Biolabs). Oligonucleotides are synthesized using the chemistry of phosphoramidites protected in b by a cyanoethyl group (Sinha et al., 1984, Giles 1985) with the Biosearch 8600 automatic DNA synthesizer using the manufacturer's recommendations. The plasmid DNAs are purified following the alkaline lysis technique (Maniatis et al., 1989).

EXAMPLES Example 1. Description of a vector according to the invention (Figures 1-3) Figure 1 describes the general structure of a vector according to the invention and, more particularly, the region comprised between the sequences that allow site-specific recombination or specific recombination of a sequence. A more detailed construction is given in Figure 2. The orientation of the promoter (P) and the transgene expression cassette (TG) and the EBNAl protein is indicated by the arrows and thus does not allow the expression of these two genes more than after recombination in the presence of Cre recombinase. The detail of the sequences between the promoter (P) and the gene after recombination is present in Figure 3. In this figure, the promoter is the LTR of the RSV virus (P-RSV) and the gene is the gene of the TK thymidine kinase. The first ATG of the messenger RNA, which corresponds to that of the TK gene, is underlined. As illustrated in Figure 1, expression of EBNA-1 is obtained from a polycistronic messenger by internal translation initiation using the IRES (Infernal Ribosome Entry Site) sequence also called the CITE sequence ("Cap Independent Translation Entry") of the Encephalomyocardite virus (ECMV). The signal sequence of the end of transcription and polyadenylation of SV40 virus (pA) has been introduced into the 3 'end of the EBNAl coding sequence. The oriP sequence is placed between the expression cassette TK-EBNA-1 and the RSV promoter. This sequence is not active for replication except in the presence of EBNA-1, therefore, it only works after recombination and episome formation.

Example 2. Construction of plasmids LoxP-oriP-TK-EBNAl and LoxP-ori-LacZ-EBNAl The steps of the construction of plasmids are present in Figures 4 and 5 and the origin of the sequences used is summarized in Table 1. 2. 1. Construction of plasmid pLoxP-oriP-TK-EBNAl (Figure 4) 1. Plasmid pSLori was digested with the restriction enzymes Hpal and EcoRI and the obtained 1817 bp fragment, corresponding to the sequence of oriP, was cloned between the Smal and EcoRI sites of the previously dephosphorylated plasmid pIC19H, to give the pIC plasmid -ori (Figure 4A). 2. Plasmid pIC-ori has been digested to the HindIII and BglII sites, dephosphoryl, and the obtained 1900 bp fragment has been cloned between the HindIII (174) and BamHI (208) sites of vector pBS246 to give the vector pLox- ori (Figure 4A). 3. The 399 bp BamHI-SalI fragment of the pCEP4 vector, which corresponds to the SV40 virus transcription arrest and polyadenylation signal sequence, has been repaired with the Klenow DNA polymerase then cloned between the Smal et Sali sites. (repaired with Klenow DNA polymerase) of the previously dephosphorylated pIC20R vector, to give the vector pICpA (Figure 4A). 4. The IRES sequence of ECMV comprised between nucleotides 16 and 518 of the vector pCITE-2 has been amplified by PCR with the aid of the synthetic oligonucleotides 1 5'-GGCCTCTAGACAGCTGGTTATTTTCC-3 '(SEQ ID No. 1) and 2 5' -GGCCGGATCCCATATTATCATCG-3 '(SEQ ID No. 2) comprising the sites Xbal and PvuII (oligo 1) and BamHl (oligo 2) has its 5' end. The product obtained was digested with the restriction enzymes Xbal and BamHl and cloned between the Xbal and PstI sites of the previously dephosphorylated pCMV-EBNAl vector, to give the vector pCITE-EBNAl. The sequence between the ATG 12 of the IRES, which corresponds to the start codon of the translation, and the serine codon that follows the methionine codon of the EBNAl protein, has been deleted by directed mutagenesis with the help of the PCR technique (former site). PCR). The complete sequence of IRES and EBNAl obtained after PCR has been completely sequenced (Figure 4A). 5. The Xbal-Pstl fragment of the pClTE-EBNAl vector (2546 bp) was cloned between the Xbal and PstI sites of the previously dephosphorylated plasmid pICpA, to give the vector pCITE-EBNAl-pA (Figure 4A). 6. The Clal-EcoRI fragment of the vector pCITE-EBNAl-pA (2945 bp) was cloned into the vector pLox-ori, partially assimilated to the EcoRI site located at the end of ori, then assimilated with Clal and dephosphorylated to give the pLox-ori-CITE-EBNAl-pA vector (Figure 4B). 7. The Clal-BsaWl fragment (1270 bp) of the pCMV-TK vector, obtained by partial digestion with BsaWl and repair of the BsaWl site with the Klenow DNA polymerase, has been cloned between the ClaI and PvuII sites of the pLox-ori- CITE vector. -EBNAl-pA to give the vector pLox-ori-CITE-TK-EBNAl-pA (Figure 4B). 8. The BamHI-SalI fragment (600 bp) of the plasmid pRSV-TK, which corresponds to the promoter sequence of the LTR of RSV, has been cloned between the BamHl and SalI sites of the pLox-ori-CITE-TK-EBNAl-pA vector, previously dephosphorylated, to give the plasmid pLox-ori-pRSV-TK-CITE-EBNAl-pA (Figure 4B). 2. 2 Construction of plasmid pLoxP-oriP-LacZ-EBNAl (Figure 5) The clones 1-6 described in Example 2.1 described above and illustrated in Figure 4A are common to the construction of the vectors pLox-ori-pRSV-TK-CITE-EBNAl-pA and pLox-ori-pRSV-LacZ-CITE- EBNAl-pA. 7. The cloning of the RSV LTR promoter was carried out according to step 7 of example 2.1. (Figure 5). 8. The BamHI-StuI fragment (3205 bp) of the pRSV-GalIX vector, corresponding to the LacZ gene preceded by a nuclear localization signal (NLS), has been cloned between the Smal and BglII sites of the pIC20H vector, previously dephosphorylated, to give the pICLacZ plasmid (Figure 5). 9. The Xbal-NruI fragment (3205 bp) of the pICLacZ vector has been cloned between the Xbal and PvuII sites of the vector pLox-ori-pRSV-CITE-EBNAl-pA to give the vector pLox-ori-pRSV-LacZ-CITE- EBNAl-pA (Figure 5).

Example 3. Validation of the system by cotransfection of human cells (Hela-EBNAl; 143B-TK-) with the help of plasmids CMV-Cre (pBS185) and LoxP-oriP-TK-EBNA. 1 or LoxP-oriP-LacZ-EBNAl. 3. 1. In vitro validation A line of Hela cells expressing the EBNAl gene stably has been transfected with the pLox-oriP-LacZ-EBNAl plasmids and a plasmid expressing the Cre recombinase gene under the control of the cytomegalovirus promoter (pBS185). The recombination efficiency and functionality of the LacZ gene expression cassette have been evaluated by the bgalactosidase activity in the cells, observed only in the presence of Cre recombinase. The results obtained demonstrate the efficiency of generation of the replicon under the action of the Cre recombinase, evidenced by the activation of LacZ gene expression after recombination between the LoxP sites and formation of the episome. In addition, after 3 weeks of culture of the co-transfected cells [passage once a week (1:10 dilution)], a conservation of the proportion of cells expressing the LacZ gene is observed, when LacZ expression has disappeared in the cells transfected with the control plasmid that does not possess the oriP origin of replication, demonstrating the functional nature of oriP in the construct. Similarly, a TK ~ (143TK ~) cell line is cotransfected by the LoxP-ori-TK-EBNAl and pBS185 plasmids. The transfected cells expressing the TK gene are selected in HAT medium. The stability of TK gene expression during cell divisions and thus the activity of the oriP-EBNAl system is verified by immunofluorescence, with the help of a monoclonal antibody specific to the Herpes virus TK protein. The presence of the episome and its replication during cell divisions is evidenced by the Hirt technique, followed by an amplification of the episomal DNA by the PCR technique, with the help of specific baits. 3. 2. In vivo validation The activity of these constructs in vivo is proven by intratumoral injection (electroporation) of the plasmids DNA pLoxP-ori-TK-EBNAl or pLoxP-ori-LacZ-EBNA1 and pBSl85 in tumors induced by subcutaneous injection of Hela or Hela cells. EBNAl in the aligned mouse. The injection of the cells previously cotransfected with these same plasmids is carried out in parallel. The conservation of the transgene (TK or LacZ) in the tumors transduced by these constructions is analyzed by immunohistochemistry, by comparison it has a controlled plasmid that does not possess the origin of replication.

Example 4. Analysis of the recombination and episomal conservation system. 4. 1. Construction of plasmid pCre Plasmid p-Cre contains the Cre recombinase gene fused at 5 'in the nuclear localization signal of the SV40 virus T antigen and expressed from the thymidine kinase (TK) promoter of the virus. herpes. A similar construction is carried out by carrying, in the place of the TK promoter, a promoter regulated by tetracycline or the MMTV promoter. On the other hand, a cassette carrying a Cre-ER fusion is also performed. 4. 2. Construction of plasmid pRep The plasmid replicon (pRep) containing a fusion of the gene of resistance to phleomycin with the gene of the Bgalactosidase (Zeo-LacZ), oriP and the earliest cytomegalovirus promoter (P.CMV), without the EBNAl protein has been constructed as follows. The Stul-clal fragment (1149-4553) of the plasmid pRSV-Gal-IX containing the LacZ gene and the polyadenylation signal of the SV40 virus has been cloned between the ClaI and EcoRV sites (2029-2032) of pLox-ori (example 2 and Figure 4) to give the pLox-ori-LacZ plasmid. The BglII-BglII (473-1226) fragment of pCEP4 containing the early CMV promoter has been cloned into the BamH1 site of pLox-ori-LacZ to give pLX2.

The 5 'end of the LacZ gene in pLX2 (Xbal-Clal fragment) has been replaced by the Ncol-clal fragment of plasmid pUT651 (CAYLA, Table 1). To facilitate cloning, this fragment was previously subcloned between the EcoRV and clal sites of the plasmid pIC20RpA then assimilated or directed by Xbal and clal and cloned between the Xbal and Clal sites of pLX2. The controlled plasmid contains the same structure as pRep devoid of the origin of the replication (OriP). 4. 3. Construction of plasmid pRep-EBNAl The cassette allowing the expression of EBNAl from the IRES of ECMV has been constructed by mutagenesis directed from the plasmid pCITE-EBNAl-pA and cloned into the Adenovirus vehicle vector pAdLX2 (5-2-2) to give pAdLX2-EBNA1 or pRep-EBNAl. The IRES as well as the start of EBNAl have been completely sequenced. The expression of EBNAl from IRES has been analyzed by Western Blot in Hela cells transfected by pRep-EBNAl. The pCMV-EBNAl plasmid which has served for the construction of pCITE-EBNAlpA serves as a control. 4. 4. In vitro validation 4.4.1. Analysis of recombination and episomal preservation in Hela-EBNAl cells In a first stage, the transfection experiences of Hela cells expressing the EBNAl protein constitutively (Hela-EBNAl) with pRep alone or with pCre have shown that the Co-transfection of two plasmids would allow cleavage of the replicon and induction of the expression of the β-galactosidase gene. The control cells transfected with the replicon plasmid alone show a constant but almost negligible background resonance of β-galactosidase activity. The expression of the transgene has been followed during the successive steps of the cells, at the rate of 2 transplants per week for one month, which is equivalent to 24 cell divisions. In cells transfected with pRep + pCre, the expression of β-galactosidase is maintained throughout the duration of the experiment when it has been rapidly lost (after any cell divisions) in the cells cotransfected with the control plasmid that does not carry the origin of replication oriP. In a second step, cells cotransfected by (pRep + pCre) have been selected in the presence of phleomycin. The episomal DNA has been isolated by the Hirt technique, in line or not by digestion at the level of the unique Xhol site then eventually assimilated by Mbol to demonstrate that the DNA has replicated well in eukaryotic cells. The analysis by Southern-Blot of the samples thus obtained allowed to put in evidence, the presence of a replicon of the expected size. The estimate of the number of copies per cell is from 1 to 10. Then the selected cells have been maintained in the presence or absence of selection pressure. In these two conditions, a similar conservation of the expression of β-galactosidase is observed with a slow and constant decrease during cell divisions. After 27 divisions, a β-galactosidase activity is then detected in 25% of cells by direct in vitro staining. These results correspond to a stability of episome segregation of 97% by division. They are in agreement with the published results, in effect a loss rate of 1 to 5% per division has been reported (Simpson et al., 1996). 4. 4.2. Analysis of EBNAl functionality in Hela cells The experiences of cotransfection of Hela cells with pRep-EBNAl and pCre allow us to prove that EBNAl is correctly expressed from the IRES and ensures the conservation of the expression of β-galactosidase during the successive steps. Hela-EBNAl cells transfected with the same plasmids that serve as control. 4. 5. Validation in vivo in a tumor model induced in the aligned mouse. The Hela and Hela-EBNAl cells are tumorigenic in the nude or aligned mouse, subcutaneous injection of 106 cells induce the formation of a detectable tumor in 8 days that grow very rapidly, and can be followed for a month. In a first experience Hela EBNAl cells transfected by pRep and pCre then selected in vitro in the presence of phleomycin have been injected. The analysis of the tumors (3cm diameter) at 21 days, after the coloration in X-gal, demonstrates the conservation of the replicon in vivo in this model. In a second experiment, the Hela-EBNAl cells transfected by pRep then infected by the AdCre (see example 5.2) have been injected. Cells transfected with two plasmids derived from pRep that allow the expression of lacZ in the absence of recombination and that carry or not oriP, injected simultaneously, that serve as control. 21-day tumor analysis demonstrates that Cre third-generation adenovirus allows the excision of the replicon and that its presence does not alter cell viability or the early stages of episome establishment. Note that no ßgalactosidase activity has been detectable in cells transfected with the control plasmid that does not carry oriP. These results clearly show that the constructions according to the invention are functional in vivo in the tumor cells, a stability of episomes is also indicated after 21 days.

Example 5. Construction of recombinant adenoviruses of the first and third generation.

This example describes the construction of viral vectors according to the invention, comprising a region that can generate, by site-specific recombination, a circular molecule and replication in vivo. These viral vectors further comprise a sequence coding for the recombinase that allows recombination. . 1. Preparation of first generation adenovirus (from the plasmids BS185, pLoxP-oriP-TK-EBNAl and 'pLoxP-oriP-LacZ-EBNAl).

The Cre recombinase expression cassette and the complete replicon sequence including the LoxP sites are cloned from vectors pBSl85 and pLox-oriP-TK-EBNAl or pLoxP-oriP-LacZ-EBNAl in the El region of adenoviral vectors which comprise a deletion of all or part of the El region (Addl327;? E1-? E3). Recombinant adenoviruses are isolated by classical techniques of homologous recombination in 293 cells. Viral vectors are also prepared by double recombination in E. coli with the help of plasmids containing the Adeno 5? E1? E3 genome, then the The viral genome is encapsulated in an adenovirus particle in an appropriate line. For reasons of cloning ability, the LacZ gene is advantageously replaced by a smaller marker gene. . 2. Preparation of the third generation adenovirus The use of third generation adenoviruses has certain advantages in relation to those of the first generation; not only on the level of safety but also of the decrease in the inflammatory response and the increase in the stability of the expression of the transgene. These vectors also have an increased cloning capacity and an absence of direct cytopathic effect in vitro and in vivo. The third generation vectors (Adll007;? E1? E3? E4) can also be prepared by the classical techniques of homologous recombination in the appropriate packaging cells (WO 96/22378) or by double recombination in E.coli then packaged. The two adenoviruses have been constructed by double recombination with the help of E. coli skeletons containing a third generation adenovirus genome PXL2811 (pRSV-bGal-? El,? E3,? E4-dll007-SspI) and pXL2789 (p? El,? E3,? E4-dll007-SspI), and suicide plasmids (Kana-SacB) pMA37 or pXL3048, intended to modify the El region according to the strategy described above (Crouzet et al., 1997 PNAS, 94: 1414; WO96 / 25506). . 2.1. Preparation of Cre adenovirus (Ad-Cre! The XhoI-BamHI fragment (451-2057) of plasmid pMC-Cre (Table 1) was repaired with the Klenow and cloned into the EcoRV site of the suicide plasmid pMA37. The genome of the adenovirus obtained by double recombination with the plasmid pXL2811 has been aligned by digestion with Paci and transferred in the IGRP2 cells (W096 / 22378) with the help of Lipofectamine (GIBCO). The Ad-Cre 3.0 thus produced has been amplified in the same cells then purified according to the classical techniques (cesium chloride) or by chromatography (FR96 / 08164).

The structure of the adenovirus Cre genome has been confirmed by enzymatic digestion. . 2.2. Preparation of adenovirus Rep (Ad-Rep) Plasmid pLX2 was digested with BamHl then recirculated to eliminate the polyadenylation signal of SV40, 5 'of the LacZ gene. The Notl-Notl (1-6145) fragment of the thus obtained plasmid was repaired by Klenow then cloned into the EcoRV site of the suicide vector pXL3048 previously digested by BamHl and SalI, repaired by Klenow then recirculated to destroy these two sites, to give the Plasmid pAdLX2. The Xhol-Sall fragment (441-3457) of the pCITE-EBNAlpA-mutagenized plasmid was cloned into the Xhol site of the plasmid pAdLX2 previously obtained to give the plasmid pAdLX2-EBNAl (pRep-EBNAl). Ad-Rep has been obtained by recombination with plasmids pAdLX2-EBNAl and pXL2789, in accordance with the techniques described above for Ad-Cre. . 2.3. Preparation of adenovirus Rep / Cre (Figure 6) The Rep-Cre adenovirus carrying both the replicon and the Cre recombinase gene is constructed. This strategy allows to increase the transfer efficiency of the replicon and especially in vivo. The expression cassette of cre is inserted into the adenovirus genome in the regions, El, E3 or E4. A perfectly regulated expression of the recombinase is sought to prevent the excision of the replicon from the adenovirus genome, during its propagation in the IGRP2 cells. The regulation of recombinase expression at the transcriptional level (specific tissue activated promoter in vivo or inductible promoter) or at the post-transcriptional level (Cre fusion with the receptor binding field to steroid hormones Cre-ER) has been contemplated . To construct these adenoviruses, the replicon is introduced into the plasmid pXL2789 as described in the preceding example. The Cre expression cassette comprising the tet or MMTV promoter, or a Cre-ER fusion is then introduced by double homologous recombination into E. coli in the plasmid to generate the pRep-Crel plasmids (Rep and Cre in El) and pRep-Cre2 (Rep in El and Cre in E4). These plasmids are then treated by Pací to extract the recombinant viral genome, which is introduced into the IGRP2 cells to produce the corresponding viruses. . 2.4. Preparation of Rep / Psi adenovirus (Figure 7) This construction advantageously allows to avoid contamination of Ad-Rep-cre by deleterious or deletion Adenovirus in the case or a perfect regulation of Cre activity could not be obtained. The strategy is based on the insertion of the psi packaging signal in the replicon. The vector pXL3048 is modified by mutagenesis directed to the level of the ITR region in order to delete the encapsidation signal and to introduce a LoxP site to give the plasmid pXL3048-? Psi-LoxP. The deleterious or deleted replicon sequence from the "left LoxP" site is isolated by enzymatic digestion from plasmids pAdLX2 or pAdLX2-EBNAl and cloned into the EcoRV site of plasmid pXL3048-? Psi-LoxP.

Example 6. Validation of the system by co-administration with the two recombinant adenoviruses: The functionality of the tyric vectors of the invention is monitored in vitro and in vivo: 6. 1. Validation in vitro, by infection of different cell lines.

The recombinase activity of Cre adenovirus has been demonstrated in vitro in Hela-EBNAl cells transfected by pRep as well as in a mouse embryonic cell line (LoxP-bgal) in which the expression of lacZ can be activated by recombination between two sites loxP. The efficacy of replicon excision from the adenoviral genome has been studied in IGRP2 cells co-infected by Ad-Rep and Ad-Cre. The direct analysis of the viral DNA isolated by the Hirt technique and digested by Xhol reveals a total disappearance of the fragment corresponding to the unclean replicon of the Ad-Rep genome demonstrating the efficiency of the recombination between the two LoxP sites. The analysis by Southern of the same samples has allowed to put in evidence a fragment of 9.2 kb that correspond to the replicon. In Hela cells coinfected by Ad-Rep and Ad-Cre, a bgalactosidase activity is observed at 48h in 50% of the cells when no bgalactosidase activity is detectable in cells infected with the adeno-Rep alone. At 96h, the number of cells expressing lacZ increases with cell division. After passage of the cells the expression of lacZ is still preserved for at least 20 days after co-infection. These results demonstrate that (i) the replicon can be efficiently released in the cell by co-infection with these two adenoviruses, that (ii) recombination releases the replicon and activates the expression of the transgene and that (iii) the replicon makes it possible to ensure stable expression of the transgene during cell divisions. 6.2. Validation in vivoidation has been demonstrated by the transfer of normal human cells. { keratinocytes, cells in hematopoetic strains (CD34 +), cells in bronchial epithelial strains, myoblasts ...) or cancerous (MDA, HT29, ...) previously co-infected by Ad-Rep and Ad-Cre, in the mouse naked or aligned. The expression of the transgene and the stability of the episome during the cell divisions are verified by the techniques described above.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Rhone Poulenc Rorer SA (B) ADDRESS: 20, avenue Raymond Aron (C) CITY: ANTONY (E) COUNTRY: France (F) POSTAL CODE: 920165 (ii) TITLE OF THE INVENTION: GENERATION OF MOLECULES OF IN LIVE REPLICATION. (üi) SEQUENCE NUMBER: 3 (iv) DESCIFRABLE FORM BY COMPUTER: (A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER: IBM PC compatible (C) EXPLOITATION SYSTEM: PC-DOS / MS-DOS (D) LOGICIAL: Patentln Reeléase # 1.0, Version # 1.30 (OEB) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GGCCTCTAGA CAGCTGGTTA TTTTCC 26 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: simple (D) CONFIGURATION: linear ; ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GGCCGGATCC CATATTATCA TCG 23 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 147 base pairs (B) TYPE: nucleotide (C) NUMBER OF HEBRAS: double (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CAGCTGGACG TCGGTTCGAA CCGACCTGCA TTTGAGGAGA AGTCTGGATT 50 ATTGAAGCAT ATCGTATGTA ATATGCTTCA ATATAATTCC CAAGGCCTAG_100_TAGCGCTCGA GCTCTAGATC TATAGCTAAC GGCGGTGGTA CCGAAGC 147 Table 1: Origin of the sequences used for the construction of the episome 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 (31)

1. A viral vector characterized in that it comprises a region of DNA surrounded by two sequences that allow a specific site-specific recombination or specific recombination of a sequence and placed in direct orientation, the region comprises at least one origin of replication and one gene of interest.

2. A viral vector according to claim 1, characterized in that the origin of replication is chosen between the origin of replication of the EBV virus, the origin of replication of the papilloma virus, and the ARS sequences.

3. A viral vector according to claim 1 or 2, characterized in that the origin of replication is the origin of replication of the EBV virus.

4. A viral vector according to claims 1 to 3, characterized in that the activity of the origin of replication is dependent on site-specific recombination.

5. A viral vector according to claim 4, characterized in that the origin of replication comprises the oriP region of the EBV virus and a coding sequence for the EBNAl protein whose expression is dependent on site-specific recombination or specific recombination of a sequence.

6. The viral vector according to one of claims 1 to 5, characterized in that the expression of the gene of interest is dependent on site-specific recombination.

1 . The viral vector characterized in that it comprises a region of DNA delimited by two sequences that allow a site-specific recombination and placed in direct orientation, the region that successively comprises a functional promoter in the mammalian cells, the oriP sequence of the EBV virus, a cassette of expression comprising üh gene and the EBNAl protein gene separated by an IRES sequence derived from the ECMV virus, the promoter and the expression cassette are oriented in such a way that the expression of two genes is possible only after recombination site-specific or specific recombination of a sequence.

8. The viral vector according to one of claims 1 to 7, characterized in that the sequences that allow site-specific recombination are sequences capable of recombining specifically in the presence of a recombinase.

9. The viral vector according to claim 8, characterized in that the sequences that allow site-specific recombination are derived from a bacteriophage.

10. The viral vector according to claim 9, characterized in that the sequences that allow site-specific recombination are derived from the bacteriophage Pl.

11. The viral vector according to claim 10, characterized in that it is the inverse repeated sequences of the bacteriophage Pl (loxP region), whose recombination is induced by the Cre recombinase.

12. The viral vector according to claim 8, characterized in that it also comprises, outside the region delimited by the two sequences that allow a specific site recombination, the gene coding for the corresponding recombinase.

13. The viral vector according to claim 12, characterized in that the gene coding for the recombinase is placed under the control of an inducer promoter.

14. The viral vector according to claim 13, characterized in that the promoter is chosen from the MMTV promoter induced by dexamethasone and a promoter induced by tetracycline.

15. The viral vector according to claim 13, characterized in that the recombinase gene further comprises a regulating regulatory element for the binding field of a hormone receptor.

16. The viral vector according to claim 15, characterized in that it is a receptor chosen from the receptors of glucocorticoid, mineralcorticoid, thyroid, estrogen, aldosterone and retinoic acid.

17. The viral vector according to one of the preceding claims, characterized in that the region of encapsidation of the virus is even in the region of DNA surrounded by the two sequences that allow site-specific recombination.

18. The viral vector according to one of the preceding claims, characterized in that it is a defective recombinant adenovirus.

19. The viral vector according to claim 18, characterized in that it is an adenovirus comprising a deletion of all or part of the El region.

20. The viral vector according to claim 19, characterized in that it is an adenovirus which also comprises a deletion of all or part of the E4 region.

21. The viral vector according to one of claims 1 to 17, characterized in that it is a defective recombinant retrovirus.

22. The viral vector according to one of claims 1 to 17, characterized in that it is a defective recombinant AAV.

23. A cell modified by insertion of a viral vector according to one of claims 1 to 22.

24. The cell according to claim 23, characterized in that it is a eukaryote cell.

25. A pharmaceutical composition comprising at least one viral vector according to one of claims 1 to 22 or a cell according to claim 23.

26. A method of in situ preparation of circular replication DNA molecules, characterized in that a population of cells containing a viral vector according to claim 8 is placed in the presence of the recombinase that allows site-specific recombination to be induced in situ.

27. The method according to claim 26, characterized in that the presence of the recombinase is carried out by transfection or infection of said cells with a plasmid or a viral vector containing the recombinase gene.

28. The procedure of in situ preparation of replication circular DNA molecules comprising: (i) transformation of cells by means of a viral vector comprising: a region of DNA surrounded by two sequences that allow a site-specific recombination in the presence of a recombinase and placed in direct orientation, the region comprising at least one origin of replication and a gene of interest, and, the gene encoding the recombinase under the control of an induced promoter, and, (ii) the induction of recombinase expression.

29. The use of a viral vector to generate in situ circular replication DNA molecules.

30. A method for the transfer of nucleic acids in a cell comprising (i) the transformation of cells by means of a viral vector comprising: a region of DNA surrounded by two sequences that allow a site-specific recombination in the presence of a recombinase and placed in direct orientation, the region comprises at least one origin of replication and a gene of interest, and, the gene encoding the recombinase under the control of an induced or induction promoter, and, (ii) the induction of expression of the recombinase.

31. The replication circular DNA molecule characterized in that it comprises at least: (i) one or several genes of interest with the sequences necessary for their expression, (ii) the oriP origin of replication of the EBV virus, and, (iii) a region resulting from the specific recombination between two sequences.