KR100537142B1 - In vivo production method of cloned components - Google Patents

Abstract

The present invention discloses circularly replicable DNA molecules useful for gene therapy, and methods that are particularly efficient for generating them in situ from viral vectors.

Description

In vivo preparation of replicable molecules

The present invention relates to annular replicating DNA molecules that can be used for gene therapy. The invention also describes a method that is particularly efficient for in situ production of such DNA molecules from corresponding viral vectors.

Gene therapy involves the introduction of genetic information into diseased organs and cells to correct deficiencies or abnormalities (mutations, abnormal expressions, etc.).

This genetic information can be introduced in vitro into cells extracted from the tissue, and the modified cells can then be reintroduced into the body or introduced directly into the tissue in vivo. For this second direct introduction, various techniques exist, among which are different transfection techniques involving different types of vectors. These are on the one hand natural or synthetic chemical and / or biochemical vectors and on the other hand are viral vectors. In the case of viral vectors, in particular complexes of DNA and DEAE-dextran [Pagano et al., J. Virol. 1 (1967) 891], complexes of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), complexes of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413 ], Liposomes [Fraley et al. J. Biol. Chem. 255 (1980) 10431, and the like. However, their use entails the possibility of producing particularly large amounts of DNA of pharmacological purity.

Viral vectors (retroviruses, adenoviruses, adeno-associated viruses, etc.) are very efficient compared to the chemical and / or biochemical vectors described above, particularly in the passage of membranes. Among these viruses, adenoviruses show the most advantageous properties for use in gene therapy. In particular, they are quite broad in host range, capable of transducing dividing or resting cells and the adenovirus genome persists in extrachromosomal form; Moreover, they are not yet associated with major pathologies in humans. On the other hand, the use of retroviruses whose genome is randomly integrated into the genome of infected cells is limited to dividing cells. Adenoviruses can thus express these genes in resting muscle cells (myotubes; Ragot et al., Nature 361 (1993) 647), hepatocytes (Jaffe et al., Nature genetics 1 (1992) 372), neurons (Akli et al. al., Nature genetics 3 (1993) 224) and epithelial bronchial cells (Rosenfeld et al., 1992) and the like. However, in the case of rapidly regenerating cells, it is observed that the expression of the transgene due to the dilution effect is gradually lost during cell division.

Improvements in adenovirus vectors and the development of new generations of vectors focus on reducing the potential potential risks of pathogenicity and immunogenicity associated with replication of the vector, recombination of its own genome and expression of viral proteins. .

In order to avoid this risk as much as possible, currently proposed viral vectors are modified to produce vectors that are unable to replicate themselves in target cells. This phenomenon is said to be missing. In general, the genome of a defective virus thus lacks the minimum sequence necessary for replication of the virus in the infected cell. That is, in certain cases of adenoviruses, the constructs described in the prior art are adenoviruses in which the E1 and optionally E3 regions are deleted at the level at which the heterologous DNA sequence is inserted. Levrero et al., Gene 101 (1991) ) 195; Gosh-Choudhury et al., Gene 50 (1986) 161]. Other constructs include deletions at the level of the El region and non-essential levels of the E4 region (WO 94/12649), or modified genomic constructs (FR 94 13355).

However, the risk of in vivo trans complementation by recombinant or El cell function to produce replicative viral particles remains during the production of these defective viral vectors. Therefore, the use of contaminated vectors in gene therapy may have enormous consequences, such as, for example, inducing viral proliferation and the risk of inflammatory and immune responses to viral proteins, resulting in uncontrolled transmission. Do.

Moreover, enhancement of the stability of transgene expression in transduced cells by adenovirus vectors, and especially in dividing cells, remains a major problem to be addressed. As a result, inherently the advantages of each of the aforementioned vectors, i.e. complete safety, resulting in excellent transfection properties based on, for example, those of the viral vector and especially those of the adenovirus and in particular the absence of the production of replicable viral particles in vivo There is a real need for delivery vectors to demonstrate the problem, and the risks that do not exist when using plasmids or non-viral vectors remain to date in gene therapy.

TABLE 1 Origin of sequences used in episomal construction.

1: Diagram for Episome Construction.

2: Episomal structure diagram.

Figure 3: Sequence between plasmid pLoxP-ori-TK-EBNA1 (A) and promoter (P-RSV) and expression cassette (TK-CITE-EBNA1) in episomal (B, C and D) after recombination-SEQ ID NO: 3.

Figure 4: Diagram of cassette LoxP-ori-TK-EBNA1 cloning step.

5: Diagram of cassette LoxP-ori-LacZ-EBNA1 cloning step.

Figure 6 is a diagram of adenovirus vectors carrying a replicon and a regulated Cre expression cassette (CRE _R : Cre regulated at the level of the promoter, or by fusion, or by both). In (b), the orientation of the replicon allows the cassette CRE _R to be placed under the control of the promoter present in the replicon. Gray boxes correspond to LoxP sites.

Figure 7 is an illustration of an adenovirus vector carrying a replicon comprised of a replicon and a regulated Cre expression cassette in which the virus capsidation region Psi (ψ) is included in the replicon. Gray boxes correspond to LoxP sites.

General cloning and molecular biology techniques

Centrifugation of plasmid DNA in a cesium chloride-ethidium bromide gradient, digestion with restriction enzymes, gel electrophoresis, electroelution of DNA fragments from agarose gels, e. Conventional molecular biology methods such as transformation into E. coli, precipitation of nucleic acids and the like are described in Maniatis et al., 1989, Ausubel et al., 1987. The nucleotide sequence is determined by a chain termination method following the procedure already presented (Ausubel et al., 1987).

Restriction enzymes are provided by New-England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham Ltd (Amersham).

For ligation, DNA fragments are isolated according to their size in 0.7% agarose or 8% acrylamide gel, purified by electrophoresis after electrophoresis, eluted with phenol, precipitated with ethanol, and then T4 phage DNA ligase. Incubate in 50 mM Tris-HCl, pH 7.4, containing 10 mM MgCl ₂ , 10 mM DTT, 2 mM ATP in the presence of Biolabs. Oligonucleotides are synthesized with a Biosearch 8600 automated DNA synthesizer using the chemistry of phosphoramidite protected at b by cyanoethyl groups (Sinha et al., 1984, Giles 1985) as recommended by the manufacturer.

Plasmid DNA is purified using alkaline lysis techniques (Maniatis et al., 1989).

It is an object of the present invention, to be precise, to propose a new concept of gene transfer that meets the above requirements.

The invention resides in situ generation via viral vectors of therapeutically replicable cyclic DNA molecules, in particular in terms of expression stability and safety of transgenes. In fact, they do not contain any sequence of the viral genome that can elicit specific responses to viral proteins that can have an inflammatory immune response and deleterious effects on the body and limit the duration of transgene expression.

The subject matter of the present invention is, therefore, at least;

(i) one or more genes of interest having sequences necessary for replication,

(ii) a replication source that functions in mammalian cells, and

(iii) is a replicable circular DNA molecule comprising a sequence resulting from site-specific recombination between two sequences recognized by recombinase.

The present invention is based in particular on the development of methods and specific constructs that are particularly efficient for the production of such therapeutic DNA molecules. In particular, the method according to the invention lies in the production of therapeutic DNA molecules as defined above from viral vectors.

Surprisingly, we have demonstrated that it is possible to generate in situ circular cyclic DNA molecules with therapeutic and replication properties from viral vectors and by site-specific recombination. Moreover, in an advantageous embodiment, the transgenes and the origin of replication are inactive in the viral vector and their activity depends on site-specific recombination events. Even more advantageously, the recombination event is conditionally induced by the expression of recombinase, thus providing a high level of control throughout the expression of the gene of interest and throughout the replication of the resulting episomal molecule.

This procedure is particularly advantageous in the following therapeutic perspectives:

Taking advantage of the good transfection ability generally expressed by viral vectors over non-viral vectors,

When a small amount of viral vector is used, it greatly reduces the risk of viral contamination, local inflammatory and anti-viral immune responses. Viral vectors are introduced only in proportions necessary to produce therapeutic DNA molecules,

Expandable application of some viral vectors: Adenovirus vectors are therefore limited in their application to proliferative cells, such as hematopoietic stem cells. The present invention allows the use of infectivity to produce stable replicable circular molecules in proliferating cells.

Thus, claimed DNA molecules have the ability to efficiently ensure the delivery of the containing therapeutic gene (s) into the cell of interest.

To accomplish this, it contains a replication source characterized by the fact that it functions in mammalian cells and human cells in particular. Advantageously, the replication source used is a conditional replication source, ie whose activity can be regulated. More preferably, the replication source is inactive in the viral vector and is arranged to be active after site-specific recombination.

Advantageously, the replication source used is of eukaryotic, viral or mammalian origin.

Preferred examples of replication sources that can be used within the framework of the invention are in particular Epstein-Barr (EBV) virus replication sources. EBV viruses belong to the Herpesviridae family. Its replication origin comprises two elements: the oriP sequence (1.7 kb), which is involved in replication and whose activity is driven by a protein encoded by the EBNA1 gene. Such sequences can be carried in trans. This element, by itself, allows episomal maintenance at the same time as replication and up to 5-20 copies per cell of plasmid vector. The oriP sequence consists of 20 repeats of 30 bp, formed by 65 bp reverse repeating units and separated by 960 bp from a replication origin comprising 4 incomplete copies of 30 bp. The EBNA1 protein is attached to 30 bp units at the replication origin level and allows for simultaneous replication of cell division of the plasmid with the requirement of cellular factor in the S phase and cis having the oriP sequence. Moreover, EBNA1 permits separation of episomes during endothelial maintenance and cell division, perhaps through the same attachment at the level of repeat units and chromosome structure. Plasmids containing the replication source oriP of the EBV genome and allowing expression of the EBNA1 virus protein (641 amino acids) are maintained in a stable episomal manner in transfected human cells and their replication is simultaneous with cell division (Lupton and Levine, 1985). .

The origin of replication may also be derived from papilloma virus. Papilloma virus utilizes a viral incubation system in which genomes similar to those of Epstein-Barr virus (EBV) remain episomal. This system has been specifically studied for bovine papilloma virus type 1 (BPV-1). The replication source of BPV is active in the presence of proteins E1 and E2. As in the case of EBV, episomal maintenance is independent of replication and is ensured by the attachment of E2 at the level of the MME (minichromosomal maintenance element) sequence, but also requires the presence of E1. On the other hand, in contrast to the EBV oriP / EBNA1 system, episomal replication is not synchronized with cell division (Piirsoo et al., 1996).

The origin of replication may also consist of a sequence capable of self replication or ARS (self replicating sequence). ARS has been isolated from the chromosomes of mammals, particularly humans and mice. Preferably, for humans, the ARS sequence upstream of the c-myc locus (Ariga et al., 1988) and the 4 kb fragment of the mouse adenosine deaminase locus (Virta-Pearlman et al., 1993) are mentioned. have.

In relation to the gene of interest, such gene may be a therapeutic, vaccine, agricultural or veterinary gene. It also contains a transcriptional promoter region that is functional in the target cell or organism and a region located 3 ′ that defines the transcriptional and polyadenylation termination signals. With respect to the promoter region, it is involved in the expression of the gene to be considered when the promoter region can act in a suitable cell or organism. It may also be a region of different origin (involved in the expression, or even synthesis) of other proteins. In particular, it may be a promoter sequence of a eukaryotic or viral gene. For example, it may be a promoter sequence derived from the genome of the target cell. Among eukaryotic promoters, any promoter or derived sequence that specifically or non-specifically, inductively or non-inducedly or strongly or weakly promotes or inhibits the transcription of a gene can be used. These include, in particular, ubiquitous promoters (promoters such as HPRT, PGK, α-actin and tubulin and tubulin genes), promoters of intermediate filaments (GFAP, desmin, bimentin, neurofilament and keratin genes), promoters of therapeutic genes ( For example, promoters such as MDR, CFTR, Factor VIII and ApoAI genes), tissue-specific promoters (promoters such as pyruvate kinase, borrowers, fatty acid binding enteric proteins and smooth muscle α-actin genes) or promoters that respond to stimulation ( Receptors for steroid hormones, retinoic acid receptors, and the like. Likewise, they may be promoter sequences derived from the genome of the virus, such as, for example, promoters of adenovirus E1A and MLP genes, early CMV promoters or promoters of RSV LTRs, and the like. In addition, these promoter regions can be modified by addition of an activating sequence, regulatory sequence or sequence that allows for tissue-specific or dominant expression. Moreover, the gene of interest may also contain signal sequences that direct the synthesized product in the secretory pathway of the target cell. Such signal sequences may be natural signal sequences of synthetic products, but may be other functional signal sequences or artificial signal sequences.

Promoter or mammalian promoters of viral origin selected from the early CMV promoter or LTR of retroviruses are advantageously used.

In addition to the origin of replication and at least one gene of interest, the DNA molecules of the invention comprise regions resulting from specific recombination between two sequences. Such site-specific recombination can be obtained from a variety of systems that bring about site-specific recombination between sequences.

Specific recombination systems for the in situ generation of claimed DNA molecules used within the framework of the present invention may be of various origins. In particular, the specific sequences and recombinases used may belong to different structural classes, in particular the bacteriophage P1 recombinase line.

More preferably, site-specific recombination used in accordance with the process of the present invention can be obtained by two specific sequences that can be recombined with each other in the presence of a specific protein, generally referred to as recombinase. For this reason, the cyclic DNA molecules according to the invention may further comprise sequences resulting from such site-specific recombination. Recombination sequences used within the framework of the present invention generally comprise from 5 to 100 base pairs, more preferably up to 50 base pairs.

Recombinases belonging to the bacteriophage 1 integrase family include phage λ (Landy et al., Science 197 (1977) 1147), P22 and F80 (Leong et al., J. Biol. Chem. 260 (1985) 4468], HP1 of Haemophilus influenza (Hauser et al., J. Biol. Chem. 267 (1992) 6859], integrase Cre of P1 phage, integrase of pSAM2 plasmid (EP 350 341) or FLP recombinase of plasmid 2 m of yeast Saccharomyces cerevisiae. When a DNA molecule according to the invention is recombinantly produced by a site-specific system of the λ bacteriophage integrase family, the DNA molecule according to the invention is generally recombined between two sequences for attachment att of the corresponding bacteriophage or plasmid. It may further comprise a sequence resulting from.

Recombinases belonging to the transposon Tn3 family include, in particular, the resolbases of transposon Tn3 or transposon gd, Tn21 and Tn522 (Stark et al., 1992); Resolbases of plasmids such as those of fragment par of RP4 or invertase Gin of bacteriophage mu. See Abert et al., Mol. Microbiol. 12 (1994) 131]. When the DNA molecule according to the invention is recombinantly produced by a site-specific system of the transposon Tn3 family, the DNA molecule according to the invention is generally produced by recombination between two sequences for recognition of the resolver of the transposon under consideration. Further contains a sequence.

According to a preferred embodiment, in the genetic construct of the present invention, the sequence allowing site-specific recombination is derived from bacteriophage. More preferably, they are sequences for attachment of bacteriophages (attP and attB sequences) or derived sequences. These sequences can be specifically recombined with each other in the presence of a recombinase called integrase. The term derived sequence includes sequences obtained by modification (s) of the sequence for attachment of bacteriophages that retain the ability to specifically recombine in the presence of a suitable recombinase. Thus, they may be reduced fragments of these sequences or vice versa extended by addition of other sequences (such as restriction sites). These may be variants obtained by mutation (s), in particular point mutation (s). According to the invention, the attP and attB sequences of a bacteriophage or plasmid refer to the sequence of a recombinant system specific for the bacteriophage or plasmid, ie the attP sequence present in the phage or plasmid and the corresponding chromosomal attB sequence.

Preferred examples include sequences for attachment of λ phage P22, F80, P1, HP1 or plasmid pSAM2 of Haemophilus influenza, or 2 m.

According to a preferred embodiment of the invention, the sequences that allow site-specific recombination are derived from the recombination system of P1 phage. This P1 phage carries a recombinase called Cre, which specifically recognizes a 34 base pair nucleotide sequence called the lox P region. This sequence consists of two 13 bp two palindromic sequences separated by an 8 bp conserved sequence.

For specific variants, the present invention thus functions in sequence (a) derived from site specific recombination between two loxP regions of a P1 bacteriophage, at least one of its sequences and in mammalian and human cells, and is conditionally functional according to a preferred mode. Relates to a cyclic replicating DNA molecule comprising one replication source that bears one replication source.

In this regard, the present invention also provides specific gene constructs suitable for the production of therapeutic DNA molecules as defined above. Such gene constructs, or recombinant DNAs according to the invention, in particular, comprise an EBNA1 protein gene surrounded by two sequences allowing for site-specific recombination of the gene (s), replication origin and direct orientation. These sequences can be cloned into bacterial plasmids in cassette form, and in the first case plasmid DNA can be transfected into human cells to test the functionality of these sequences. These cassettes are then used in the construction of viral vectors bearing these same sequences integrated into their genomes.

As mentioned above, another aspect of the present invention is a method for in situ production of the aforementioned therapeutic DNA molecules from a viral vector by site-specific recombination. The use of such vectors may advantageously optimize the administration of the claimed DNA molecules in the cells to be treated.

In this regard, the subject matter of the present invention is also a viral vector comprising at least one DNA region surrounded by two sequences that allow for site-specific recombination and placed in a direct orientation, inserted into its genome, wherein the DNA region is at least It contains one replication source and one corresponding gene.

According to a preferred embodiment of the invention, the gene of interest to be incorporated into the replication origin and viral vector is present in an inactivated form and the promoter is cloned in a direct orientation at one end of the expression cassette (FIG. 1). After recombination between two LoxP sites, the promoter seeks its course in front of the gene and is separated therefrom by the LoxP site, the first ATG of the transcript corresponding to the transgene's initiation codon. The oriP sequence is active only in the presence of an EBNA1 protein expressed in the form of an isctronic messenger under the control of the same promoter as a transgene. The translation of EBNA1 is initiated by an internal initiation mechanism at the level of IRES sequences derived from the piconavirus family of brain myocarditis virus (ECMV). Expression of the transgene and EBNA1 protein and replication of the plasmid are thus directly determined by the recombination event between the two LoxP sites.

According to an alternative of the invention, the encapsidated region of the virus is contained in a replicon (a DNA region surrounded by a sequence allowing site-specific recombination). This aspect provides additional safety to the system as described below.

The viral vector used may be of various origins as long as it can transduce animal cells, preferably human cells. As a preferred embodiment of the present invention, vectors derived from adenoviruses, adeno-associated viruses (AAV), herpes virus (HSV) or retroviruses can be used. Particular preference is given to using adenoviruses for ex vivo modification of cells to be administered or transplanted, or retroviruses for transplantation of production cells.

The viruses according to the invention are defective, ie they cannot autonomously replicate in target cells. In general, the genome of a defective virus used within the framework of the present invention thus lacks the minimum sequence necessary for viral replication in infected cells. Such regions may be removed (completely or partially), rendered non-functional, or replaced by other sequences, particularly heterologous nucleic acid sequences in question. Preferably, the defective virus nevertheless retains its genome sequence essential for the encapsidation of the viral particles.

Especially for adenoviruses, type 2 or 5 human adenoviruses (Ad2 or Ad5) or adenoviruses of animal origin (see patent application WO94 / 26914) are preferably used within the framework of the invention. Animal-derived adenoviruses that can be used within the framework of the present invention include dogs, cattle, mice (e.g. Mav1, Beard et al., Virology 75 (1990) 81), sheep, pigs, birds or monkeys. Adenovirus of origin (for example, SAV) is mentioned.

Preferably, the viral vector of the present invention is a defective adenovirus comprising at least one origin of replication and one gene of interest and surrounded by two sequences located in a direct orientation with a defective adenovirus inserted into the genome. They can induce the conditional activity of replication origin and transgene by site-specific recombination, depending on the presence of recombinase.

Advantageously, at least the El region in the genome of this adenovirus of the invention is made non-functional. More preferably, at least one of the E1 gene and the E2, E4, L1-L5 genes is non-functional. Other regions, in particular E3 (WO90 / 02697), E2 (WO94 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697) can also be modified.

According to a preferred embodiment, the adenovirus according to the invention is inserted at the level of an inactivated E1 region in which a DNA region comprising deletions in the E1 and E4 regions and surrounded by two sequences allowing site-specific recombination. According to another preferred embodiment, a DNA region comprising a deletion in the E1 and E4 regions and surrounded by two sequences allowing site-specific recombination is inserted at the level of the inactivated E4 region. As described below, according to certain embodiments of the present invention, the adenovirus also includes a cassette for the expression of the recombinase gene.

The claimed vectors are obtained by recombination with plasmids as described above, i.e. characterized by the fact that they comprise at least one replication source and one corresponding gene having conditional activity between two site-specific recombination regions. .

More specifically, with respect to the definition of the origin of replication of the genes present in the two sequences allowing site-specific recombination and the region of DNA integrated into the claimed viral vector, reference is made to the above definitions.

Defective recombinant adenoviruses according to the present invention may be prepared by techniques known to those skilled in the art. See Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917]. In particular, they are produced by homologous recombination between adenoviruses and, in particular, plasmids carrying the DNA sequences of the invention or by E. By genomic construction in E. coli. Homologous recombination occurs after simultaneous transfection of adenovirus and plasmid with a suitable cell line. The cell line used should preferably contain sequences that (i) are transformed by said element and (ii) complement the defective adenovirus genome, preferably in an integrated form to avoid the risk of recombination. As an example of a cell line, in particular, the left part of the cell line genome, which can be integrated into the genome and complement the E1 and E4 fractions as described in Ad5 adenovirus (12%) or in particular in WO 94/26914 and WO95 / 02697. Human embryonic kidney cell line 293 comprising Graham et al., J. Gen. Virol. 36 (1977) 59 may be mentioned.

The amplified adenovirus is then recovered and purified by conventional molecular biology techniques described in the Examples.

With respect to adeno-associated viruses (AAV), they are relatively small sized DNA viruses that integrate in a stable and site-specific manner into the genome of the cells they infect. They can infect a broad spectrum of cells without inducing any effect on cell growth, morphology or differentiation. In addition, they do not appear to be involved in human pathology. The AAV genome was cloned, sequenced and characterized. It contains about 4700 bases and contains a reverse repeat region (ITR) of about 145 bases at each end and serves as a replication region for the virus. The rest of the genome consists of two essential regions that perform the encapsulation function: the left side of the genome containing the rep gene involved in viral replication and expression of viral genes; Divide into the right part of the genome containing the cap gene encoding the viral capsid protein.

AAV-derived vectors for in vitro and in vivo delivery of genes are described in the literature (WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528). These applications include various AAV-induced constructs in which the rep and / or cap genes are deleted and replaced by the genes, and their use for delivery of the genes in vitro (cells in culture) or in vivo (directly to the organism). It describes. The defective recombinant AAV according to the present invention is a human helper virus of a plasmid containing the nucleic acid sequence of the present invention bounded by two AAV reverse repeat regions (ITRs), and a plasmid carrying AAV capsidation genes (rep and cap genes). It can be prepared by co-transfection with a cell line infected with (eg adenovirus). The resulting recombinant AAV is then purified by conventional techniques.

Regarding herpes viruses and retroviruses, the construction of recombinant vectors is described in Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al., Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like.

In particular, retroviruses are integrative viruses that selectively infect dividing cells. Therefore, they constitute the corresponding vector for cancer application. The genome of retroviruses essentially comprises two LTRs, capsidizing sequences and three coding regions (gag, pol and env). In recombinant vectors derived from retroviruses, the gag, pol and env genes are generally completely or partially deleted and substituted with the corresponding heterologous nucleic acid sequence. Such vectors can be used in various types of retroviruses, for example, MoMuLV ("Rat Molecular Leukemia Virus", also called MoMLV), MSV ("Rat Molecular Sarcoma Virus"), HaSV ("Harvey Sarcoma Virus") ); SNV ("splenic necrotic virus"); It can be prepared from RSV (“Rous Sarcoma Virus”) or Friends virus.

In order to construct a recombinant retrovirus of the present invention, in general, a plasmid comprising the LTR, the capsid sequence and the sequence of the present invention is generally constructed and then transduced a so-called encapsidated cell line which can provide transplasms lacking the plasmid lacking retroviral function. Used to infect Therefore, in general, encapsidated cell lines can express gag, pol and env genes. Such capsidated cell lines, in particular PA317 cell line (US 4,861,719); PsiCRIP cell lines (WO90 / 02806) and GP + envAm-12 cell lines (WO89 / 07150) are described in the prior art. In addition, recombinant retroviruses may include extended capsidation sequences that include portions of the gag gene and modifications at the level of LTR to inhibit transcriptional activity. See Bender et al., J. Virol. 61 (1987) 1639. Recombinant retroviruses are then purified by conventional techniques.

With the preferred vectors according to the invention, more specifically, adenoviruses which are bounded by the reverse repeat sequence of the P1 bacteriophage (loxP region) comprising the DNA region according to the invention in the genome and arranged in direct orientation can be proposed. .

By the description of this type of vector, the following construct with the β-galactosidase (LacZ) gene or the thymidine kinase gene (TK) of the herpes virus can be mentioned (Fig. 1). Such genes may be RNA or therapeutic or vaccine proteins such as enzymes, blood derivatives, hormones, lymphokines: interleukin, interferon, TNF, etc. (FR 9,203, 120), growth factors, neurotransmitters or precursors or synthetic enzymes thereof, Nutritional factors: BDNF, CNTF, NGF, IGF, GMF, αFGF, βFGF, NT3, NT5 and the like; Apolioproteins: ApoAI, ApoAIV, ApoE, etc. (FR 93 05125), genes encoding dystrophin or minidistroffin (FR 9111947) (cDNA, gDNA, RNA, synthetic or semi-synthetic nucleic acid), tumor suppressor genes: p53 , Rb, Rap1A, DCCm, k-rev et al. (FR 93 04745), Factors involved in coagulation: factors VII, VIII, IX, etc., or all or part of natural or artificial immunoglobulins (Fab, ScFv, etc.) Gene encoding, ligand RNA (WO91 / 19813) and the like.

The gene of interest may also be an antisense sequence that allows expression in a target cell to regulate expression of the gene or transcription of cellular mRNA. Such sequences can, for example, be transcribed into RNA complementary to cellular mRNAs in target cells according to the techniques described in EP 140 308 and thereby inhibit their transcription to proteins.

Vectors of the invention are particularly suitable for the expression of sequences encoding virulence factors. These include cell toxins (diphtheria toxin, Pseudomonas toxin, lysine A, etc.), products that induce susceptibility to external bodies (suicide genes: thymidine kinase, cytosine deaminase, etc.) or killer genes that can induce cell death ( Grb3-3) (PCT / FR94 / 00542), anti-ras ScFv (WO 94/29446) and the like). The system of the invention makes it possible in fact to generate viral vectors containing such sequences without toxicity to vectors, in particular to producing cells, and to selectively induce the expression of such toxic molecules in target cells after site-specific recombination. Therefore, this type of construct is particularly suitable for anticancer treatment strategies, for example, where the purpose is to selectively destroy diseased cells. Such systems are also particularly advantageous for the expression of cytokines, interferons, TNFs or TGFs, for example, where non-controlled production can have very significant side effects.

The invention also aims at eukaryotic cells transfected with at least one viral vector or DNA molecule according to the invention as described above.

Another object of the present invention is a method of producing a DNA molecule as defined above, wherein a culture of a host cell containing a viral vector according to the invention is contacted with a recombinase to induce site-specific recombination.

More briefly, the present invention generally comprises at least one replication source and one arranged in a direct orientation, surrounded by two sequences allowing site-specific recombination to produce circularly replicable DNA molecules (i) Contacting a modified host cell containing at least one viral vector comprising in said genome at least one DNA region comprising said gene of interest and (ii) a recombinase that causes site-specific recombination to be induced in situ. A method for producing other annular replicative DNA molecules is characterized.

Various procedures can be proposed for contacting viral vectors with specific recombinases within the framework of the present invention. This is especially true at the level of the host cell either by cotransfection with the plasmid or by co-infection with a viral vector containing the gene for recombinase or by expression of the gene encoding the recombinase present directly in the host cell genome. By induction. Thus, the gene encoding the recombinase may be present in the host cell in a form integrated into the genome, for example, in a plasmid or otherwise in the accompanying viral vector of the adenovirus type. In this case, the viral vector used to generate the DNA molecule according to the invention is as defined above.

According to another method, a cassette for expressing a gene is also carried on a viral vector involved in the expression of that gene.

In this particular case, the method according to the invention is limited by two sequences encoding specific recombination sites and expresses the recombinase gene in its genome in addition to a DNA region comprising at least one origin of replication and one gene. Viral vectors containing cassettes are used.

Such vectors constitute another object of the present invention.

In this regard, according to certain alternatives, the present invention provides a cassette for expressing a first deletion and recombinase protein in an E1 region into which a replicon (a DNA region surrounded by two site-specific recombinant sequences) is inserted. To adenoviruses that have been deleted in the E4 and / or E3 region at a defined level.

In contrast to the embodiments described above, the replicon can be inserted into the deleted portion corresponding to E3 or E4 while the cassette for expressing the recombinase is inserted at the level of the deleted El region.

According to another alternative, a cassette for expressing replicon and recombinase is inserted at the level of the defective El region.

More specifically, as described above, the sequence allowing site-specific recombination is a LoxP sequence and the recombinase is a Cre protein, as described above for its shorthand for the recombinant sequence.

According to a preferred embodiment of the invention, it is practically desirable to be able to modulate and in particular express the expression of such recombinases in host cells. For this reason, regulating the expression of genes encoding recombinases is advantageously proposed within the framework of the present invention. To do this, it is proposed to carry out the expression of genes under the control of regulatory elements. This may in particular be a promoter regulated by an inducible promoter, for example MMTV LTR promoter (Pharmacia) or tetracycline induced by dexamethasone, which makes it possible to regulate the level and / or duration of such gene expression (WO94 / 29442; WO 94/04672). It is understood that MMTV LTR variants can be used that carry other promoters, particularly heterologous regulatory regions (particularly “enhancer” regions).

In another embodiment, expression of the gene encoding recombinase is under the control of a promoter that is regulated to avoid structural accumulation of proteins in the host cell or to minimize "leakage" and cytotoxicity towards the nuclear compartment. Elements that function as transcriptional transactivation domains can thus be associated with them. As representative of this type of element, mention may be made in particular of hormonal receptors, including steroids, retinoid acid and thyroid receptors, more particularly for glucocorticoids, mineralocorticoids, thyroids, estrogens, aldosterones and retinoic acid. These may be mentioned. Constructs of this type between the recombinase Cre and the DNA-binding domain of glucocorticoids are mentioned, for example, in PNAS 93 (1996) 10887.

The possibility of modulating the expression of recombinase is particularly important in the case of the vector of the invention carrying both the replicon and the recombinase expression cassette. Indeed, in this embodiment, if the recombinase is expressed during the generation of a viral vector, for example, the replicon will be cleaved from the viral genome prior to capsidation. For this reason, it is useful that the expression of the recombinase protein may have a system that is inhibited in virus producing cells. This is obtained using a regulatory promoter (tetracycline or MMTV type), as described above, and / or a hormone receptor type element that maintains it in the extranuclear compartment in the presence of hormones in conjunction with recombinase (FIGS. 6 and 7). As a result, recombinases do not work in the absence of hormones and are transported to the nuclear compartment in which recombinases are active in the presence of hormones.

An advantageous approach for regulating recombinase expression is to use recombinase fused with hormone receptors (DNA-binding domains) and thus inactive in the absence of hormones, and then the gene is virus so that the gene is under the control of the replicon promoter. In a vector. This aspect is shown, for example, in FIG. 6B.

In addition, to increase the stability of the system, it is also possible to include a region for capsidation of the viral vector in the replicon, as described above (FIG. 7). This allows the virus stock to be produced to be avoided from being contaminated by vectors that have lost their replicon. Indeed, in spite of the regulatory system mentioned above, if active recombinase expression occurs during virus production, this will result in cleavage of the replicon from the viral genome, thus producing a viral genome containing no replicon. If the region for encapsidation of the virus is carried by the replicon, the viral genome thus produced will not be encapsidated. Thus, only viral genomes carrying replicon and recombinase expression cassettes can be encapsidated.

The subject of the invention is also a pharmaceutical composition comprising at least one viral vector according to the invention or transfected cells according to the invention. Such compositions may be formulated for administration by topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular routes. Preferably, the composition according to the invention contains a pharmaceutically acceptable vehicle for injection formulation. They are particularly suited to the composition of the injection solution upon addition of isotonic sterile physiological saline solutions (mono or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of these salts), or optionally sterile water or physiological saline solution. Acceptable drying, in particular freeze-drying compositions. With respect to retroviruses, they may advantageously use ex vivo infected cells, optionally in the form of renal organs, directly to the capsidized cells or for the purpose of their in vivo replanting (WO 94/24298).

The dose of the vector used for injection can be adjusted according to various parameters, in particular depending on the dosage form used, the pathology involved or the desired duration of treatment. In general, recombinant viruses according to the invention are formulated and administered in the form of 10 ⁴ to 10 ¹⁴ pfu / ml dose. For AAV and adenovirus, doses of 10 ⁶ to 10 ¹⁰ pfu \ ml may also be used. The term pfu (“plaque forming unit”) corresponds to the infectivity of a virion suspension and is determined by infecting a suitable cell culture and generally measuring the number of plates of infected cells after 48 hours. Pfu titer measurements of viral solutions are well documented in the literature.

Depending on the genes present in the region integrated into the genome of the DNA molecules or viral vectors of the present invention, they are hereditary diseases (muscular dystrophy, cystic fibrosis, etc.), neurodegenerative diseases (Alzheimer's, Parkinson's, ALS, etc.), cancer, blood coagulation disorders And for the treatment or prophylaxis of many pathologies, including those associated with Dis lipoproteinini, pathologies associated with viral infections (hepatitis, AIDS, etc.) or in the agricultural and veterinary fields.

The invention will be described more fully with the aid of the following examples which should be considered as illustrative and non-limiting.

Example 1 Description of Vectors According to the Invention (FIGS. 1-3)

1 illustrates the general structure of a vector according to the present invention, more particularly the region between sequences that allows site-specific recombination. A more detailed construction is shown in FIG.

The orientation of the cassette for expressing promoter (P) and transgene (TG) and EBNA1 protein is indicated by the arrow and thus allows expression of these two genes only after recombination in the presence of recombinase Cre. Details of the intergene sequence between promoter (P) and post-recombination are shown in FIG. 3. In this figure, the promoter is RSV virus LTR (P-RSV) and the gene is the thymidine kinase TK gene. The first ATG of messenger RNA corresponding to the messenger RNA of the TK gene is underlined. As shown in FIG. 1, expression of EBNA-1 is mediated by internal initiation of translation using IRES (Internal Ribosome Entry Site), also called CITE (“Cap Independent Translation Entry”) sequence of Brain Myocarditis Virus (ECMV). Obtained from polycistronic messenger. Signal sequences for transcription termination and polyadenylation of the SV40 virus (pA) are introduced at the 3 'end of the coding sequence of EBNA1. The oriP sequence is located between the expression cassette TK-EBNA-1 and the RSV promoter. This sequence is active for replication only in the presence of EBNA-1; Thus, it functions only after recombination and the formation of episomes.

Example 2 Construction of Plasmids LoxP-oriP-TK-EBNA1 and LoxP-ori-LacZ-EBNA1

Plasmid construction steps are shown in FIGS. 4 and 5 and the origin of the sequences used is summarized in Table 1.

2.1. Construction of the plasmid pLoxP-oriP-TK-EBNA1 (FIG. 4)

1. Plasmid pSLori is cleaved with restriction enzymes HpaI and EcoRI and the 1817 bp fragment obtained, corresponding to the oriP sequence, is cloned between the SmaI and EcoRI sites of the previously dephosphorylated plasmid pIC19H to generate plasmid pIC-ori (FIG. 4A).

2. The plasmid pIC-ori was cleaved at the HindIII and BglII sites, dephosphorylated, and the 1900 bp fragment obtained was cloned between the HindIII (174) and BamHI (208) sites of the vector pBS246 to generate the vector pLox-ori. (FIG. 4A).

3. The 399 bp BamHI-SalI fragment of vector pCEP4, corresponding to the transcription and polyadenylation termination signal sequence of SV40 virus, was repaired with Klenow DNA polymerase and then the SmaI and SalI sites of the dephosphorylated vector pIC20R in advance ( Cloned between Clenau DNA polymerase) to generate the vector pICpA (FIG. 4A).

4. Synthetic oligonucleotide 1 5'-GGCC TCTAGACAGCTG GTTATTTTCC-3 comprising the ECMV IRES sequences between nucleotides 16 and 518 of vector pCITE-2 comprising XbaI and PvuII (oligo 1) and BamHI (oligo 2) sites at the 5'-end. is amplified by PCR by '(SEQ ID NO: 1) and 2 5'-GGCC GGATCC CATATTATCATCG-3 ' ( SEQ ID NO: 2). The product obtained is cleaved with restriction enzymes XbaI and BamHI and cloned between the XbaI and PstI sites of the previously dephosphorylated vector pCMV-EBNA1 to produce the vector pCITE-EBNA1. The sequence between the ATG 12 of IRES corresponding to the translational initiation codon, and the methionine codon of the EBNA1 protein followed by the serine codon is removed by site-directed mutation by PCR technique (ex-PCR site). The entire sequence of IRES and EBNA1 obtained after PCR is fully sequenced (FIG. 4A).

5. The XbaI-PstI fragment (2546 bp) of the vector pCITE-EBNA1 was cloned between the XbaI and PstI sites of the previously dephosphorylated plasmid pICpA to generate the vector pCITE-EBNA1-pA (FIG. 4A).

6. CloI-EcoRI fragment (2945 bp) of vector pCITE-EBNA1-pA was cloned into vector pLox-ori, partially cleaved EcoRI site located at the end of ori, cleaved with ClaI and dephosphorylated The vector pLox-ori-CITE-EBNA1-pA is generated (FIG. 4B).

7. ClaI-BsaW1 fragment (1270 bp) of vector pCMV-TK, obtained by partial cleavage with BsaW1 and repair of BsaW1 site using Klenow DNA polymerase, was subjected to ClaI of vector pLox-ori-CITE-EBNA1-pA. Cloning between and PvuII yields the vector pLox-ori-CITE-TK-EBNA1-pA (FIG. 4B).

8. The BamHI-SalI fragment (600 bp) of plasmid pRSV-TK corresponding to the RSV LTR promoter sequence was cloned between the BamHI and SalI sites of the previously dephosphorylated vector pLox-ori-CITE-TK-EBNA1-pA. To generate plasmid pLox-ori-pRSV-TK-CITE-EBNA1-pA (FIG. 4B).

2.2. Construction of the plasmid pLoxP-oriP-LacZ-EBNA1 (FIG. 5)

Cloning 1-6 described in Example 2.1 above and described in FIG. 4A is common for the construction of the vectors pLox-ori-pRSV-TK-CTIE-EBNA1-pA and Lox-ori-pRSV-LacZ-CITE-EBNA1-pA. do.

7. Cloning of the RSV LTR promoter is performed according to step 7 of Example 2.1 (FIG. 5).

8. Nucleic Localization Signal (NLS) The BamHI-StuI fragment (3205 bp) of the vector pRSV-GalIX, corresponding to the LacZ gene, was cloned between the SmaI and BglII sites of the previously dephosphorylated vector pIC20H to generate the plasmid pICLacZ. To generate (FIG. 5).

9. The XbaI-NruI fragment (3205 bp) of vector pICLacZ was cloned between the XbaI and PvuII sites of vector pLox-ori-pRSV-CITE-EBNA1-pA to generate vector pLox-ori-pRSV-LacZ-CITE-EBNA1-pA. To generate (FIG. 5).

Example 3 Concurrent Transfection of Human Cells (Hela-EBNA1; 143B-TK-) Under the Support of Plasmid CMV-Cre (pBS185) and LoxP-oriP-TK-EBNA.1 or LoxP-oriP-LacZ-EBNA1 System verification

3.1. In vitro Verification

The Hela cell line stably expressing the EBNA1 gene is transfected with a plasmid expressing the gene corresponding to the recombinant enzyme Cre under the control of the plasmids pLoxP-oriP-LacZ-EBNA1 and cytomegalovirus promoter (pBS185). The recombination efficiency and function of the cassette expressing the LacZ gene is assessed by β-galactosidase activity, observed only in the presence of the recombinant enzyme Cre in cells. The obtained results demonstrate that the production efficiency of replicons under the action of recombinant enzyme Cre is revealed by activation of LacZ gene expression and episomal formation after recombination between LoxP sites. In addition, maintenance of the proportion of cells expressing the LacZ gene was observed after incubation of co-infected cells for three weeks (once per week (1:10 dilution)), but the expression of LacZ was a control without replication origin oriP. It does not appear in cells transfected with plasmids, demonstrating the functional nature of oriP in the constructs.

In addition, TK ⁻ cell line (143TK ⁻ ) is co-transfected with plasmids LoxP-ori-TK-EBNA1 and pBS185. Transfected cells expressing the TK gene are selected in HAT medium. The stability of TK gene expression during cell division and thus the activity of the oriP-EBNA1 system is confirmed by immunofluorescence by monoclonal antibodies specific for the TK protein of Herpes virus. The presence of episomes and their replication during cell division can be seen by amplification of episomal DNA by Hirt technique, followed by PCR techniques, with specific primers.

3.2. In vivo Verification

The activity of these constructs in vivo was characterized by intratumoral injection of plasmids pLoxP-ori-TK-EBNA1 or pLox-ori-LacZ-EBNA1 and pBS185 DNA induced by subcutaneous injection of Hela or Hela-EBNA1 cells into nude mice (electroporation). Is tested. Injection of cells previously transfected with this same plasmid is performed in parallel. The maintenance of transgenes (TK or LacZ) in tumors transduced by this construct is analyzed by immunohistochemistry, compared to control plasmids that do not have a replication origin.

Example 4. Analysis of Recombinant and Episomal Retention System

4.1. Construction of Plasmid pCre

Plasmid p-Cre contains the gene for the recombinant enzyme Cre, fused at 5 ′ with the nuclear localization signal of the SV40 virus T antigen and expressed using the thymidine kinase (TK) promoter of the herpes virus. Instead of the TK promoter, similar constructs are carried that carry a promoter regulated by a tetracycline or MMTV promoter. In addition, cassettes carrying Cre-ER fusions are also prepared.

4.2. Construction of Plasmid pRep

Without the EBNA1 protein, a replicon plasmid (pRep) containing the fusion of the β-galactosidase gene (Zeo-LacZ), oriP and cytomegalovirus early main promoter (P.CMV) and pleomycin resistance gene is shown below. Construct as follows:

StuI-ClaI fragment (1149-4553) of plasmid pRSV-Gal-IX containing LacZ gene and SV40 virus polyadenylation signal was cloned between ClaI and EcoRV sites (2029-2032) of pLox-ori (Example 2 And FIG. 4) plasmid pLox-ori-LacZ.

The BglII-BglII fragment (473-1226) of pCEP4 containing the CMV early promoter was cloned into the BamH1 site of pLox-ori-LacZ to generate pLX2.

The 5 'terminus (Xba1-ClaI fragment) of the LacZ gene in pLX2 is replaced with the NcoI-ClaI fragment of plasmid pUT651 (CAYLA, Table 1). To facilitate cloning, this fragment is pre-cloned between the EcoRV and ClaI sites of plasmid pIC20RpA and then cleaved with XbaI and ClaI and cloned between the XbaI and ClaI sites of pLX2.

The control plasmid contains the same structure as pRep lacking replication origin (OriP).

4.3.-Construction of the plasmid pRep-EBNA1

Cassettes expressing EBNA1 from ECMV IRES were constructed by site-directed mutations from plasmid pCITE-EBNA1-pA and cloned into adenovirus shuttle vector pAdLX2 (5-2-2) to produce pAdLX2-EBNA1 or pRep-EBNA1. IRES and early EBNA1 are fully sequenced. Expression of EBNA1 using IRES is analyzed by Western blot in Hela cells transfected with pRep-EBNA1. Plasmid pCMV-EBNA1 provided for the construction of pCITE-EBNA1pA serves as a control.

4.4.- in vitro verification

4.4.1. Analysis of Recombination and Episome Maintenance in Hela-EBNA1 Cells

In the first step, transfection experiments of Hela cells (Hela-EBNA1), which essentially express pRep alone or pCre and EBNA1 protein, indicate that simultaneous transfection of two plasmids results in cleavage of the replicon of β-galactosidase gene. And allow expression of its expression. Control cells transfected with the replicon plasmid alone show a constant but substantially negligible β-galactosidase activity background noise. Expression of the transgene is monitored during successive passages of cells at a rate of 2 subcultures / week (consistent with 24 cell divisions) for 1 month. In cells transfected with pRep + pCre, expression of β-galactosidase is maintained for the entire experimental period, but it is rapidly lost in cells cotransfected with a control plasmid that does not carry a replication origin oriP (after several cell divisions). .

In the second step, cells co-transfected with (pRep + pCre) are selected for the presence of pleomycin. Episomal DNA is isolated by Hirt technology or otherwise cleaved and linearized at the level of the unique XboI site and then optionally cleaved with MboI to verify that DNA actually replicated in eukaryotic cells. Southern blot analysis of the sample thus obtained can confirm the presence of the replicon of the predicted size. The estimated copy number per cell is 1-10. Selected cells are maintained in the presence or absence of selection pressure. Under these two conditions, similar maintenance of β-galactosidase expression during cell division is observed with a slow, constant decrease. After 27 divisions, β-galactosidase activity can still be detected in 25% of cells by direct staining in vitro. This result is consistent with 97% separation stability of episomes at each cleavage. This is consistent with the published results; Indeed, rates of loss of 1 to 5% / division have been reported (Simpson et al., 1996).

4.4.2. Functional Analysis of EBNA1 in Hela Cells

Simultaneous transfection experiments of Hela cells with pRep-EBNA1 and pCre can confirm that EBNA1 is correctly expressed using IRES and ensures maintenance of expression of β-galactosidase during serial passage. Hela-EBNA1 cells transfected with the same plasmid served as controls.

4.5. Validation of Tumor Model Induced in Nude Mice in Vivo

Hela and Hela-EBNA1 cells are carcinogenic in nude mice and subcutaneous injection of 10 ⁶ cells leads to the formation of very fast growing tumors that can be detected in 8 days and can be monitored for 1 month. In the first experiment, Hela-EBNA1 cells transfected with pRep and pCre and then selected in the presence of pleomycin are injected in vitro. Analysis of tumors (3 cm in diameter) 21 days after staining with X-gal demonstrates the maintenance of in vivo replicon in this model.

In a second experiment, Hela-EBNA1 cells (comparative: Example 5.2) transfected with pRep and then infected with AdCre are injected. Cells expressing LacZ in the absence of recombination and transfected with two plasmids derived from pRep that carry oriP or otherwise injected simultaneously serve as controls. Tumor analysis after 21 days demonstrates that the third generation Cre adenovirus allows for incision of the replicon and its presence does not modify the first stage of cellular activity or episomal establishment. It should be noted that no β-galactosidase activity could be detected in cells transfected with control plasmids carrying no oriP. These results indicate that the constructs according to the invention function in tumor cells in vivo; Episomes also show stabilization after 21 days.

Example 5 Construction of First and Third Generation Recombinant Adenoviruses

This example describes the construction of a viral vector according to the invention comprising a region capable of producing cyclic replicating molecules in vivo by site-specific recombination. These viral vectors further comprise a sequence encoding a recombinant enzyme that allows recombination.

5.1. Preparation of first generation adenovirus (from plasmid pBS185, pLox-oriP-TK-EBNA1 and pLox-oriP-LacZ-EBNA1)

The complete sequence of the replicon comprising the cassette and the LoxP site expressing the recombinant enzyme Cre can be expressed in whole or in part of the E1 region from the vectors pBS185 and pLox-oriP-TK-EBNA1 or pLox-oriP-LacZ-EBNA1 (Add1327; ΔE1). -E3) is cloned into the E1 region of the excluded adenovirus vector. Recombinant adenovirus is isolated in cell 293 by conventional homologous recombination techniques. The viral vector was also expressed by a plasmid containing the genome of adeno 5 ΔE1ΔE3. It can be prepared by double recombination in E. coli, and then the viral genome is encapsulated with adenovirus particles in the appropriate cell line. In view of cloning ability, the LacZ gene can be advantageously replaced with smaller marker genes.

5.2. Preparation of Third Generation Adenovirus

The use of third generation adenoviruses has certain advantages over the first generation, in terms of stability as well as in terms of reducing inflammatory responses and increasing stability of transgene expression. Such vectors also have increased cloning capacity and the absence of direct cytopathic effects in vitro and in vivo.

Third generation vectors (Adl1007; ΔE1ΔE3ΔE4) are also known as homologous recombination techniques (WO 96/22378) or E. coli in appropriate packaging cells. It can be prepared by double recombination in E. coli followed by packaging.

Both adenoviruses are the third generation adenovirus genomes pXL2811 (pRSV-bGal-ΔE1, ΔE3, ΔE4-dl1007-SspI) and pXL2789 (pΔE1, Δ, intended to modify the E1 region according to the method described above. E3, ΔE4-dl1007-SsaI) containing E. E. coli cells are constructed by double recombination with suicide plasmid (Kana-SacB) pMA37 or pXL3048 (Crouzet et al., 1997 PNAS, 94: 1414; WO96 / 25506).

5.2.1. Preparation of Cre Adenovirus (Cre-Ad)

XhoI-BamHI (451-2057) (Table 1) of plasmid pMC-Cre is repaired with Klenow and cloned into the EcoRV site of suicide plasmid pMA37.

The adenovirus genome obtained by double recombination with plasmid pXL2811 is cleaved with PacI and linearized and transfected into IGRP2 cells (WO96 / 22378) using lipofectamine (GIBCO). The 3.0 Cre-Ads thus generated are amplified in the same cells and then purified according to conventional techniques (cesium chloride) or by chromatography (FR96 / 08164).

The structure of the Cre adenovirus genome can be known by enzyme cleavage.

5.2.2. Preparation of Rep Adenovirus (Rep-Ad)

Plasmid pLX2 was cleaved with BamHI and then recyclized to remove the 5 'SV40 polyadenylation signal of the LacZ gene. The NotI-NotI fragment (1-6145) of the plasmid thus obtained was repaired with Klenow and then cloned into the EcoRV site of the suicide vector pXL3048 previously cleaved with BamHI and SalI, and then repaired with Klenow to destroy these two sites. Recyclization yields plasmid pAdLX2. The XhoI-SalI fragment (441-3457) of the mutated plasmid pCITE-EBNA1pA was cloned into the Xho1 site of plasmid pAdLX2 obtained beforehand to generate plasmid pAdLX2-EBNA1 (pRep-EBNA1).

According to the techniques described above for Cre-Ad, Rep-Ad is obtained by recombination with plasmids pAdLA2-EBNA1 and pXL2789.

5.2.3. Preparation of Rep / Cre Adenovirus (FIG. 6)

Rep-Cre adenoviruses carrying both replicon and genes for the recombinant enzyme Cre are constructed. The method can in particular increase the delivery efficiency of the replicon within. The Cre expression cassette is inserted into the El, E3 or E4 region of the adenovirus genome. During proliferation in IGRP2 cells, expression of fully regulated recombinant enzymes is required to prevent cleavage of the replicon from the adenovirus genome.

Regulation of the expression of recombinant enzymes is observed at the level of transcription (tissue-specific promoter or inducible promoter activated in vivo) or post-transcriptional level (fusion of Cre with the attachment domain of the steroid hormone receptor Cre-ER).

To construct such adenoviruses, replicon is introduced into plasmid pXL2789 as described in the Examples above. Expression cassettes of Cre, including promoter tet or MMTV, or Cre-ER fusion, were introduced into the plasmid by double homologous recombination in E. coli to form plasmids pRep-Cre1 (Rep and Cre in E1) and pRep-Cre2 ( Rep of E1 and Cre) of E4 are generated. These plasmids are treated with PacI to extract the recombinant viral genome introduced into IGRP2 cells to produce the corresponding virus.

5.2.4. Preparation of Rep / Psi Adenovirus (FIG. 7)

This construct can advantageously avoid contamination of Rep-Cre-Ad due to adenovirus lacking replicon in case complete control of Cre activity cannot be achieved. The method is based on the insertion of the encapsulation signal Psi into the replicon. Vector pXL3048 is modified with site-directed mutations at the level of the ITR region to eliminate encapsulation signals and introduce LoxP sites, resulting in plasmid pXL3048-ΔPsi-LoxP. The replicon sequence from which "left LoxP" has been removed is separated from plasmid pAdLX2 or pAdLX2-EBNA1 by enzymatic cleavage and cloned into the EcoRV site of plasmid pXL3048-ΔPsi-LoxP.

Example 6 System Validation by Co-Infection with Two Recombinant Adenoviruses

The function of the viral vectors of the invention is regulated in vitro and in vivo:

6.1. Validation by infection of various cell lines in vitro

Recombinant enzyme activity of Cre adenovirus is demonstrated in in vitro Hela-EBNA1 cells and mouse embryonic cell line (LoxP-βgal) transfected with pRep, where expression of LacZ can be activated by recombination between two LoxP sites.

The cleavage effect of adenovirus genome origin replicon is studied in IGRP2 cells co-infected with Rep-Ad and Cre-Ad.

Direct analysis of viral DNA isolated by Hirt technology and cleaved with XhoI shows complete loss of fragments corresponding to unresected replicons from the Rep-Ad genome, demonstrating recombination efficiency between the two LoxP sites. Southern analysis of the same sample can show a 9.2 kb fragment corresponding to the replicon.

In Hela cells co-infected with Rep-Ad and Cre-Ad, β-galactosidase activity was observed in 50% of cells after 48 hours, but β-galactosidase activity was not detected in cells infected with Rep-adeno only. Do not. After 96 hours, the number of cells expressing LacZ increases with cell division. After cell passage, LacZ expression is maintained for an additional 20 days after co-infection. These results indicate that (i) replicon can be efficiently delivered into cells by co-infection of these two adenoviruses, and (ii) recombination releases the replicon to activate expression of the transgene and (iii) It demonstrates that cones can ensure stable expression of transgenes during cell division.

6.2. In vivo Verification

In vivo validation was performed in nude mice, either normal human cells (keratin producing cells, hematopoietic stem cells (CD34 +), bronchial epithelial cells, myoblasts, etc.) previously co-infected with Rep-Ad and Cre-Ad or carcinogenic human cells (MDA). , HT29, etc.).

The expression of the transgene and the stability of the episomes during cell division are confirmed by the techniques described above.

Sequence Listing

Claims (21)

(1) a DNA region comprising: (a) the origin of replication oriP of the Epstein-Barr Virus (EBV); (b) the gene of interest; (c) promoters that function in mammalian cells; (d) a nucleic acid sequence encoding the Epstein-Barr virus nuclear antigen 1 (EBNA1) protein; And (e) Internal Ribosome Entry Site (IRES) sequence of Encephalomyocarditis virus (EMV); And (2) a replication-deficient adenovirus vector located in direct orientation and comprising two recombinant nucleic acid sequences capable of site-specific recombination, In the above, the nucleic acid sequence encoding the IRES sequence EBNA1 protein and the corresponding gene, the DNA region is located between the two recombinant nucleic acid sequences, nucleic acid encoding the EBNA1 protein by site-specific recombination between the recombinant nucleic acid sequence An adenovirus vector whose sequence and corresponding genes are operatively linked to a promoter. The adenovirus vector according to claim 1, wherein the two recombinant nucleic acid sequences perform site-specific recombination in the presence of recombinase. 3. The adenovirus vector according to claim 2, wherein the sequence allowing two site-specific recombination is derived from bacteriophages. 4. The adenovirus vector according to claim 3, wherein the bacteriophage is a P1 bacteriophage. 5. The adenovirus vector according to claim 4, wherein the two recombinant nucleic acid sequences are reverse repeat sequences of the loxP region of the P1 bacteriophage. 3. The adenovirus vector according to claim 2, further comprising a gene encoding a corresponding recombinase outside the region bounded by two recombinant nucleic acid sequences. 7. The adenovirus vector according to claim 6, wherein the gene encoding the recombinase is placed under the control of an inducible promoter. 8. The adenovirus vector according to claim 7, wherein the promoter is selected from a mouse mammary tumour virus (MMTV) promoter derivable by dexamethasone and a promoter derivable by tetracycline. 8. The adenovirus vector of claim 7, wherein the gene for recombinase further comprises a regulatory element encoding a hormone receptor binding domain. 10. The adenovirus vector according to claim 9, wherein the adenovirus vector is a receptor selected from among glucocorticoid, mineralocorticoid, thyroid, estogen, aldosterone and retinoic acid receptors. The viral vector of claim 1, wherein the viral capsidation region is comprised in a DNA region surrounded by two sequences that allow site-specific recombination. 12. The adenovirus vector according to claim 11, which is an adenovirus vector comprising a deletion of all or part of an El region. 13. The adenovirus vector according to claim 12, which is an adenovirus vector further comprising a deletion of all or part of the E4 region. A cell comprising the adenovirus vector according to any one of claims 1 to 13. 15. The cell of claim 14 which is a eukaryotic cell. A pharmaceutical composition for treating cancer, comprising the adenovirus vector according to any one of claims 1 to 13 or a cell comprising the adenovirus vector. A cell population containing an adenovirus vector according to any one of claims 1 to 13 is contacted with a recombinase to allow site-specific recombination of two recombinant nucleic acid sequences of the adenovirus vector in situ. In situ preparation of a cyclic replicating DNA molecule. 18. The method of claim 17, wherein the contact with recombinase is performed by transfecting or infecting said cells with a plasmid or viral vector containing the gene for said recombinase. (i) transforming the cell by the adenovirus vector according to claim 7; And (ii) inducing the expression of a recombinase allowing recombination between two recombinant nucleic acid sequences. (i) transforming the cells with the adenovirus vector according to claim 7 or 8; And (ii) inducing the expression of a recombinase that allows recombination between two recombinant nucleic acid sequences. (i) the gene containing the sequence required for its expression, (ii) the origin of replication oriP of the EBV virus, (iii) a region formed by specific recombination between two sequences, (iv) a nucleic acid sequence encoding EBNA1, and (v) contains at least the IRES sequence of EMCV, wherein the IRES sequence is located between the gene of interest and the nucleic acid sequence encoding the EBNA1 protein, wherein both the gene and the nucleic acid sequence encoding EBNA1 function in mammalian cells. Is operatively connected to the same promoter. 14. A replicable circular DNA molecule formed by site-specific recombination between two recombinant nucleic acid sequences of an adenovirus vector according to any one of claims 1 to 13.