MXPA98010328A - Terapeut dna production procedure - Google Patents

Abstract

The present invention relates to the preparation of DNA, particularly plasmid. It relates more particularly to the production of bacterial plasmid DNA that is useful in gene therapy, in the form of a plasmid, of a subcoiled, relaxed or line minicircle.

Description

PROCEDURE FOR THE PRODUCTION OF THERAPEUTIC DNA The present invention relates to the preparation of DNA, in particular plasmid. It relates more particularly to the production of bacterial plasmid DNA which is used in gene therapy, in the form of a plasmid, of a subcoiled, relaxed or linear minicircle, and in which the immunogenic properties are reduced until they are suppressed. The invention also relates to microorganisms useful for the production of DNA, as well as to pharmaceutical compositions.

Gene therapy consists in correcting a deficiency or an anomaly by introducing one. genetic information in the affected cell or organ. This information can be entered either in. in vitro in a cell extracted from the organ and then reinjected into the organism, either in vivo, directly in the targeted tissue. It is a molecule of high molecular weight and negative charge, DNA has difficulties to spontaneously cross the phospholipid cell membranes. Thus the different vectors are used in order to allow the transfer of the gene: the viral vectors of a part, the chemical and / or biochemical, natural or synthetic vectors, on the other hand. Viral vectors (retroviruses, adenoviruses, REF virus: 28909 adeno-associated, ...) are very effective, in particular for the passage of membranes, but present a number of risks such as pathogenicity, recombination, replication, immunogenicity, ... Chemical and / or biochemical vectors make it possible to avoid these risks (for reviews, see Behr, 1993, Cotten and Wagner, 1993). These are for example the cations (calcium phosphate, DEAE-dextran, ...) that are treated in the form of precipitates with DNA, which can be "phagocytosed" by the cells. It can also be liposomes in which DNA is incorporated and which fuse with the plasmid membrane. Synthetic gene transfer vectors in general are lipids or cationic polymers that complex DNA and form a particle that carries negative charges on the surface. These particles are able to interact with the negative charges of the cell membrane, after crossing it. Examples of such vectors are dioctadecylamidoglycylspermine (DOGS, Transfectam ™) or N- [1- (2, 3-dioleyloxy) propyl] -N, N, N-trimethylamino chloride (DOTMA, Lipofectin ™). Chimeric proteins were also developed; they are made up of a polycationic part that condenses the DNA, linked to a ligand that is fixed on a membrane receptor and drags the complex inside the cells by endocytosis. Thus it is theoretically possible to "direct" a tissue or certain cell populations, in order to improve the in vivo bioavailability of the transferred gene.

The plasmids currently used in gene therapy generally carry (i) an origin of replication, (ii) a marker gene such as a gene for resistance to an antibiotic (kanamycin, ampicillin ...) and (iii) one or more transgenes with the sequences necessary for its expression (enhancer (s), promoter (s), polyadenylation sequences ...). This type of plasmids is currently used, for example, in gene therapy at the level of clinical trials such as the treatment of melanomas, Nabel et al., 1992, or at the level of experimental studies.

The use of plasmid DNA in gene therapy, however, poses a number of problems.

In particular, this implies the possibility of producing significant amounts of DNA of pharmacological purity. Indeed, in these gene therapy techniques, the medicine is constituted by the same DNA and it is essential to be able to manufacture, in the adapted amounts, the DNAs that have the appropriate properties for a therapeutic use in man. In this regard, different methods of production and / or purification have been described in the prior art, allowing to improve the quality of the plasmid DNA (PCT / FR95 / 01468; FR9603519).

On the other hand, the use of DNA carrying antibiotic resistance genes or of functional origins of replication can also present certain drawbacks, linked in particular to their dissemination in the organism. Different approaches have also been developed to limit these drawbacks (PCT / FR96 / 00274, FR95 10825).

Another drawback of the plasmid DNAs used up to the present lies in their origin. In fact, they are molecules produced essentially in prokaryotic organisms (bacteria) or lower eukaryotes (yeasts), which potentially possess the immunogenic radicals in man. The immunological properties of DNA are still very little known. The bacterial DNA in mice led i) to the synthesis of antibodies that recognize double-stranded, single-stranded bacterial DNA that has allowed immunization but did not react with mammalian double-stranded DNA, ii) stimulation of macrophages and cytokines (D. Pisetsky "the Immunologic Properties od DNA" J. Immunol. 156 (1996) 1). The DNA macromolecule is thus said to be immunogenic. On the other hand, a molecule can also lead to a stimulation of the immune system without being immunogenic (for example foreign bodies that lead to an immune response through cellular mediation). The first evidence suggesting that bacterial DNA leads to an immune response was described by Pisetsky et al. (1991 J. Immunol., 147, p.759). It has been shown that the DNA of three bacterial species can stimulate the proliferation of lymphocytes of mice while the DNA extracted from three animal species does not lead to this stimulation. After, Yamamoto et al. (1992 Microbiol, Immunol, 36 p983) observed that the bacterial DNA of six species leads in BALB / c mouse spleen cells to an increase in NK activity "natural killer" and to the induction of interferon production. But the DNA extracted from ten species of vertebrates did not lead to any of these responses. In addition, Krieg et al. described in 1995 (Nature vol374 p546) that a genomic DNA fragment of E. coli induced in vitro the proliferation of murine B cells and the secretion of IgM immunoglobulins, whereas this same bacterial DNA, treated in vitro with a CpG methylase, did not He induced such a response. Krieg et al. they also indicated that in the presence of unmethylated DNA, interferon g is produced, and reacts as a co-stimulatory factor of B cells by modulating the production of IL-6 by B cells (Krieg et al., 1996 J. Immunol 156 p558). In addition, an oligonucleotide that has a non-methylated CpG radical and framed in 5 'by 2 purines and in 3' by 2 pyrimidines led in vivo a coordinated secretion by interleukins IL-6 and IL-12, and interferons g by cells NK (INF-g), B cells (IL-6 and IL-12) and CD4 + T lymphocytes (IL-6 and IFN-g) (Krieg et al., 1996 Proc. Nati, Acad. Sci. USA 93 p2879 ).

The plasmid DNA used hitherto in gene therapy is produced essentially in prokaryotic cells, and consequently has a methylation profile comparable to that of bacterial genomic DNA. It has also been shown that plasmid DNA that was injected into muscle or liver retained the prokaryotic methylation profile (Wolf et al., 1992 Hum. Mol.Genet.1 p363; Malone et al., 1995 J. Biol. Chem. .269 p29903). In fact, the plasmid DNA used has a potential for stimulation of the important immune system.

Thus, it would be particularly advantageous to be able to Plasmid DNA that has reduced immunological properties, until suppressed. It would also be particularly advantageous to be able to have a method that allows producing, a Scale compatible with an industrial use, plasmid DNA of this type.

The present solution provides a solution to these problems. The applicant is interested in the immunogenic properties of bacterial DNA. The applicant has now indicated a method for producing pharmaceutical grade plasmid DNA, potentially devoid of undesirable immunogenic effects. The firm has also shown that methylation of certain DNA residues would reduce the immunogenic potential of plasmid DNA, without affecting its ability to transfect cells and express a nucleic acid of interest.

One aspect of the invention is to prepare DNA, particularly plasmids, of therapeutic quality. According to another aspect, the present invention relates to the use in gene therapy of methylated plasmid DNA in the cytosines of the 5'-GC-3 'dinucleotides. A third aspect of the invention relates to pharmaceutical compositions containing the methylated plasmid DNA. The invention also relates to a method of in vivo methylation of DNA by the expression of a methylase. The invention in other aspects, relates to the expression cassettes, the host microorganisms usable for methylation, the preparation of therapeutic compositions, and the methods of transferring genes.

A first objective of the invention therefore relates to a method of producing DNA useful in gene therapy, characterized in that said DNA is produced in a cell that contains a cassette for the expression of a DNA methyltransferase that allows methylation of the cytokine residues of the dinucleotides. '-CG-3' The present invention thus relates to the production of DNA, particularly plasmid, methylated in the cytokine residues of the 5'-CG-3 'dinucleotides.

The methylation of plasmid DNA in vitro is documented in the literature (Adams et al., 1992 FEBS Letters 309 p97; Doerfler 1994 FEBS Letters 344 p251; Komura et al. 1995 Biochim. Biophys. Acta 1260 p73).

Meanwhile, this form of methylation is not visualized for the industrial production of the plasmid that would be used in gene therapy. A process for the production of plasmid DNA must in fact make it possible to reproducibly produce significant quantities of plasmids and homogenates and to purify this DNA by methods acceptable for pharmaceutical use. It is quite clear that a methylated DNA in vitro can be more or less related from batch to batch (Doerfler 1994 FEBS Letters 344 p251) and that the quantities produced are limited.

The present invention now demonstrates that it is possible to methylate a plasmid of interest directly in the course of production, coexpressing in the host cell the gene encoding a methylase. The present invention also shows that, according to this method, important amounts and homogenates of the methylated plasmid can be produced and the methylated plasmid DNA can be purified according to the methods already described. The applicant also has shown, advantageously, that the plasmid DNA thus methylated retains the ability to transfect the target cells and, if appropriate, to replicate them. Of particular note, the applicant also demonstrated that the plasmid DNA thus methylated can, in vivo, express the nucleic acids of interest.

Numerous studies reinforce the idea that hypermethylation correlates with inhibition or inactivation and that actively transcribed promoters are often hypo or non-methylated. Thus, they are viral promoters, if the late E2A promoter of the genome of adenovirus type 2 is completely methylated in the 5'-CG-3 'dinucleotides, in the hamster transformed cells HE1, and not methylated in the transformed cells of hamster HE2; the E2A gene is silent in HE1 and is transcribed in HE2 (W. Doerfler 1995 Curr. Top, Microbiol.Immunol.197 p207). Another example is described by Kohn et al. (1994 Proc. Nati, Acad. Sci. USA 91 p2567) demonstrating the absence of expression from the translated retroviral vector LTR in the cells of hematopoietic strains is associated with methylation in vivo. The inhibition of the expression of the transporter gene, under the control of a viral promoter, when it was also demonstrated that this gene is introduced in transient transfection by a methylated plasmid in vitro (Adams et al., 1992 FEBS Letters 309 p97; Doerfler 1994 FEBS Letters 344 p251; Komura et al., 1995 Biochim Biophys, Acta 1260 p73). On the other hand, Razin et al. (cited) demonstrated that the promoter of the gene encoding the herpes simplex type I thymidine kinase and the gene coding for mouse metallothionein are inactive when the transient expression in mouse L cells and murine teratocarcinoma cells F9 if these promoters are methylated in the 5'-CG-3 'dinucleotides.

The present invention thus describes for the first time a method that allows the production of methylated plasmid DNA, homogeneous and compatible with industrial use, and demonstrates the possibility of using this type of plasmid for the expression of genes in vitro, ex vivo or in vivo , particularly in gene therapy applications.

The method according to the invention can be used in different types of cellular hosts. It is particularly concerned with any non-human cell, essentially devoid of a cytokine methylation system of the 5'-CG-3 * dinucleotides. The absence of methylation can be the result of the absence of appropriate enzymatic activity, due either to insufficient expression of a corresponding gene, or to the absence of said gene. It is preferably simple prokaryotic or eukaryotic cells.

Advantageously, the cellular host is a bacterium. Among the bacteria, mention may be more preferably made of E. coli, B. subtilis, Streptomvces, Pseudomonas (P. putida, P. aerusinosa), Rhizobium meliloti, Asrobacterium tumefaciens, Staohylococcus aureus, Streptomvces pristinaespiralis, Enterococcus faecium or Clostridium. Enterobacteria such as Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerocenes, Erwinia carotovora or Serratia marcescens can also be used. Preferably, the cell host used is a non-pathogenic organism and can produce important amounts of plasmid DNA and homogenes. By way of a particularly preferred example, E. coli is used.

The method of the invention allows the production of therapeutic quality DNA.

The DNA can be in the entire DNA molecule, single strand or double strand, linear or circular, replicating or not, integrative or not, in the form of a plasmid, of a subcoiled, relaxed or linear minicircle. In the text below, the DNA will also be referred to as plasmid DNA or TG plasmid (for the plasmid useful in gene therapy).

The TG plasmids generally used in gene therapy carry essentially (i) an origin of replication, (ii) one or more nucleic acids of interest (therapeutic gene) with the sequences necessary for their expression (enhancer (s), promoter (s) , polyadenylation sequences ...) and optionally (iii) a marker gene.

The choice of the replication gene is determined primarily by the cell host used for production. It may be an origin of replication from a plasmid of the incompatibility group P (example = pRK290) that allows replication in strains of E. coli pol A. In general, it can be any origin of replication from a plasmid which replicates in prokaryotic or lower eukaryotic cells. This plasmid can be a derivative of pBR322 (Bolivar et al., Gene 2 (1977) 95), a derivative of pUC (Viera et Messing, Gene 19 (1982) 259), or of other plasmids that are derived from the same group of incompatibility, that is, ColEl or pMBl for example. These plasmids can be chosen on the other hand in other incompatibility groups that replicate in Escherichia coli. They may be plasmids derived from plasmids belonging to the incompatibility groups A, B, Fl, FU, FU, FIV, Hl, Hll, II, 12, J, K, L, N, OF, P, Q, T, U , W, X, Y, Z or 9 for example. Other plasmids can also be used, among which the plasmids do not replicate in E. coli but in other hosts such as B¿_ subtilis, Streptomyces, P. putida, P. aeruginosa, Rhizobium meliloti, Aqrobacterium tumefaciens, Staphylococcus aureus, Streptomyces pristinaespiralis , Enterococcus faecium or Clostridium. A preferential title, origins of replication derived from plasmids that are replicated in E. coli are used. According to a particular variant, the origin of replication can be a conditional origin, that is, of which the activity depends on factors in trans. The use of this type of replication origin prevents replication of plasmid DNA after administration, for example in man (FR95 10825).

Among the marker genes one can cite a resistance gene, particularly an antibiotic (ampicillin, kanamycin, geneticin, hygromycin, etc.), or any gene that gives the cell a function it does not possess (for example, a gene that was deleted). of the chromosome or became inactive), the gene in the plasmid that restores this function.

According to a particular embodiment, the plasmid DNA contains the sequences that make it possible to eliminate, after the production phase, all essentially non-therapeutic regions (origin of replication, marker gene, etc.). A particularly advantageous approach to generating this type of molecule (minicircles) was described in the application PCT / FR96 / 00274.

The plasmid DNA according to the invention is preferably a double-stranded DNA molecule containing one or more nucleic acids of interest with the sequences necessary for their expression. According to a preferred embodiment, it is a circular, replicating or integrative molecule. Advantageously, the plasmid DNA contains essentially one or several nucleic acids of interest with the sequences necessary for their expression (miniplasmid).

The nucleic acid of interest can be all nucleic acid (cDNA, gDNA, synthetic or semi-synthetic DNA, etc.) of which transcription and optionally translation into a cell generate products having a therapeutic, vaccine, agronomic or veterinary interest.

Among the nucleic acids that have the therapeutic properties, the genes encoding the enzymes, the blood derivatives, the hormones, the lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), the growth factors, the neurotransmitters or their precursors or synthetic enzymes, the 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), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), the genes that code for the factors involved in coagulation: Factors VII, VIII, IX, etc, suicide genes: Thymidine kinase, cytosine deaminase, etc; or even all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.), an RNA ligand (W091 / 19813) etc. The therapeutic gene can also be a gene or an antisense sequence, from which the expression in the target cell allows to control the expression of genes or the transcription of cellular mRNAs. Such sequences can for example be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in EP 140 308.

The nucleic acid of interest can also be a vaccine gene, ie a gene that codes for an antigenic peptide, capable of generating an immune response in man or animal, from the point of view of carrying out vaccines. They can be particularly antigenic peptides specific to epstein-barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo-rabies virus, or even tumor-specific (EP 259 212).

In general, in plasmids, the nucleic acid of therapeutic, vaccine, agronomic or veterinary interest also contains a promoter region of functional transcription in the target cell or organism (eg, mammals, particularly man), as well as a region located at 3 ', and which specifies a transcriptional end signal and a polyadenylation site. Concerning the region: 'promoter, it may be a promoter region naturally responsible for the expression of the gene considered when it is capable of functioning in the referred cell or organism. They may also be regions of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, may be promoter sequences exiting the genome of the target cell. Among the eukaryotic promoters, any promoter or derived sequence that stimulates or represses the transcription of a gene in a specific manner or not, inducible or not, strong or weak can be used. It can be in particular of 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 the therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAI, etc. genes), of tissue-specific promoters (pyruvate kinase gene promoter, villin, intestinal fatty acid binding protein, α-actin smooth muscle, etc.) or even promoters that respond to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.). Also, they may 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 LTR promoter of RSV or MMTV, 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 contain 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 another functional signal sequence, or an artificial signal sequence.

According to the nucleic acid of interest, the methylated plasmid DNAs of the invention can be used for the treatment or prevention of numerous pathologies, including genetic diseases (dystrophy, cystic fibrosis, etc.), neurodegenerative diseases (Alzheimer's, Parkinson's, ALS, etc.) , cancers, pathologies linked to coagulation disorders or dislipoproteinemias, pathologies linked to viral infections (hepatitis, AIDS, etc.), or in the agronomic and veterinary domains, etc. They are particularly advantageous for the treatment of pathologies in which a durable expression without immunological reaction is desired, particularly in the domain of genetic, neurodegenerative and cardiovascular diseases.

As indicated above, the method according to the invention utilizes a host cell that contains a cassette for the expression of a DNA methyltransferase that allows methylation of the cytosine residues of the 5'-CG-3 'dinucleotides.

After DNA synthesis, certain purines and pyrimidines are chemically modified, for example by methylation. Thus, 5-methylcytosine or N6-methyladenine enter into the composition of certain DNA. These modifications take place with the help of DNA methyltransferases, maintenance or de. novo, which transfer a methyl group of S-adenosyl-L-methionine to the adenine or cytosine residues that can be located at specific positions in the sequences. For example, in E. coli two DNA methyltransferases are well known, DNA methyltransferase dam, which methylates the adenosine residues within the 5'-GATC-3 'sequences, and the DNA methyltransferases dem, which methylates the second cytidine residue of the sequences 5 '-CCA / TGG-3'. Other DNA methylases have been studied in bacteria, which methylate a residue contained in a recognition site of a restriction enzyme. For example, the enzyme M.HpalI methylates the second cytosine residue in the sequence 5'-CCGG-3 '.

Most simple eukaryotes and invertebrates contain relatively little 5-methylcytosine and N6-methyladenine. However, methylation of bases in vertebrates is more important and in this case 5-methylcytosine is the most frequent of the methylated bases. In effect, more than 95 percent of the groups that metilate vertebrate DNA are found in the C residues of few 5'-CG-3 'dinucleotides (the 0.8% frequency of 5'-CG-3 * in the Mammalian sequences are very weak although the percentage in GC is on average 40% and that an unbiased array will lead to a frequency of 4% of 5'-CG-3 '). And, more than 50 percent of the set of dinucleotides can be methylated. Different evidences suggest that the degree of methylation of certain sequences containing the 5'-CG-3 'dinucleotide may be a determining factor in mammals in the regulation of the expression of particular genes, the inactivation of the X chromosome, oncogenesis (1993). eri DNA methylation: Molecular Biology and Biological Significance, Eds Jost et Saluz), and even hereditary diseases (Bates et al 1994 BioEssays 16 p277).

The present invention uses a cassette for the expression of a DNA methyltransferase that allows methylation of the cytosine residues in the 5'-CG-3 'dinucleotides. In fact, in the sense of the present invention, methylated DNA means more particularly methylated DNA in the cytosine residues of the 5-CG-3 'dinucleotides. Advantageously, the DNA methyltransferase used preferably methylates the cytosine residues of the 5'-CG-3 'dinucleotides, ie it does not affect almost the adenine residues, nor the cytosine residues that occur in a context different from the 5'-CG- dinucleotides. 3'. Advantageously, methylated plasmid DNA is understood to mean a plasmid DNA in which at least 50% of the cytosine residues of the 5'-CG-3 'dinucleotides are methylated. More preferably, at least 80%, advantageously 90% of said residues are methylated.

The methylation of plasmid DNA can be verified in different ways. In particular, it can be controlled by directing the plasmid preparations by restriction enzymes in which cleavage is not possible if the cytosine residue of the 5'-CG-3 'dinucleotide, contained in the cutting site, is methylated. Mention may be made, for example, of the restriction enzymes Hpa.II, AatII, BstBI. The methylation can also be determined by chromatography. Thus, the amount of non-methylated plasmid present in the methylated plasmid preparation was quantified in the following way: 1% or 5% of the non-methylated plasmid and fully digested with Hpall was added to the undigested unmethylated plasmid. These samples, as well as the methylated plasmid digested by Hpall, were analyzed by liquid anion exchange chromatography and detected at 260 nm, which allows to separate and quantify the undigested DNA from the digested DNA. It is verified that the methylated plasmid contains less than 5% unmethylated plasmid DNA, otherwise it is said that more than 95% of the plasmid DNA is methylated.

Advantageously, the process of the invention is characterized in that the DNA methyltransferase methylated preferably the cytosine residues of the 5'-CG-3 'dinucleotides.

Advantageously, in the process according to the invention, more than 50% of the cytosine residues of the 5-CG-3 'dinucleotides of the plasmid DNA are methylated. Even more preferably, more than 80%, particularly more than 90% of the cytosine residues are methylated from the 5 '-CG-3' dinucleotides of the plasmid DNA.

Several mammalian DNA methyltranferases were characterized that allow the cytosine residues to be methylated in the sequences containing all the 5'-CG-3 'dinucleotide and the corresponding genes, for example the mouse one, were cloned.

(Bestor et al 1988, J. Biol. 203 p971) or the man's (Yen et al., 1992 Nucí Acids Res. 20 p2287). These enzymes have a molecular weight between 135 and 175 kD.

They mock the semi-stained DNA much faster than the unmethylated one, suggesting that these are maintenance methylases (Smith 1994 Progress in Nuclear Acid Research and Molecular Biology 49 p65). The homologous enzyme in E. coli does not exist. On the other hand, the M. sssl from Spiropiasma methylase exclusively and complements the cytosine residues of all 5'-CG-3 'dinucleotides with a comparable speed, either the semi-stained or unmethylated substrate (Razin et al., 1992 FEBS letters 313 p243 Baker et al 1993 Biochim Biophys, Acta 196-864). This enzyme was isolated from the strain Spiroplasma sp MQ1. Its molecular weight is 42 kD and the gene was cloned and subexpressed in E. coli (Razin et al., 1990 Nucí Acids, Res. 18 pll45 and EP0412676A1 derwent 91045812).

Preferably, the DNA methyltransferase is chosen from the M.SssI methylase, the mouse methylase and the human methylase. Advantageously, the M.SssI methylase is used.

The expression cassette of DNA methyltransferase generally contains a nucleic acid encoding a DNA methyltransferase that allows methylation of the cytosine residues of the 5'-CG-3 'dinucleotides under the control of a promoter. The promoter used for this purpose can be any functional promoter in the chosen host cell. In this regard, it may be a functional promoter such as defined above. These are prokaryotic cell hosts, the promoters of the lactose operon (Plac) of the tryptophan operon can be more particularly mentioned.

(Ptrp), the Plac / Ptryp hybrid promoters, the PL promoter or PR of the bacteriophage lambda, the promoter of the tetA gene (e_n Vectors 1988 pl79 Rodriguez et Denhardt editeurs), etc.

In a preferred embodiment, a promoter different from that responsible for the expression of the nucleic acid of interest in the plasmid DNA is used. It is particularly advantageous to use an inducible promoter, which makes it possible to control the expression of methylase. The inducible promoter can be for example the bacteriophage T7 promoter or the Plac promoter.

Advantageously, the expression cassette also contains end-of-transcription signals (transcriptional terminators), such as ribosomal terminators.

The cassette of the expression of the DNA methyltransferase can be carried by a replicator vector, or integrated into the genome of the host cell.

In the case of a replicator vector, a vector compatible with the TG plasmid is advantageously used, that is to say able to co-reside in the same cell. Two different plasmids can replicate in the same cell if the control of the replication of each plasmid is different. Thus the compatible plasmids belong to two incompatibility groups. Now there are approximately 30 plasmid incompatibility groups that replicate in Enterobacteriaceae (Maas et al., 1988 Microbiol, Rev. 52 p375). In fact, there are numerous possibilities for replicating two plasmids in the same cell and several examples are described in the literature. One can cite, for example, the replication of ColEl-derived plasmids with the plasmids having R6K or pl5A or RSF1010 or RK2 replicons; one can also cite the replication of plasmids derived from RK2 with the plasmids derived from R6K or RFS1010 or pSa or ColEl (in Vectors 1988 p287 Rodríguez et Denhardt editeurs). This list is not limitative and other examples are also described in Vectors 1988 p287 Rodriguez et Denhardt edíteurs. Advantageously, the replicator vector used has a different number of copies in the host cell such as the TG plasmid. Thus, the vector carrying the gene coding for methylase, in which the expression can be induced, is of low copy number (derived for example from pACYC 184 or RK2), when the plasmid TG is in high copy number (derived from ColEl). A sequence can also be cloned into the TG plasmid which allows a triple helical sequence to be formed with an appropriate oligonucleotide such that the TG plasmid can be separated from another plasmid by an affinity purification.

The expression cassette of DNA methyltransferase can also be integrated into the genome of the host cell. The integration can be carried out by homologous recombination, to the extent that the expression cassette is framed by the adjacent fragments of a non-essential gene of the host genome and cloned into a plasmid that can not replicate in the host considered. This plasmid can be i) a derivative of ColEl in a strain of E. coli polAt? (Gutterson et al., 1983 Proc. Nati, Acad. Sci. USA 80 p4894); ii) a thermosensitive derivative of pSClOl in any strain of E. coli (S. Kushner et al., 1989 J. Bacteriol 171 p4617); iii) a suicide vector such as M13mpl0 in the strains of E. coli sup * (Blum el coll 1989 J. Bacteriol 171 p538) or even iv) a plasmid that does not contain the g origin of R6K in any strain of E. coli devoid of the pir gene (Filutowicz et al., 1994 Prog. In Nucleic Acid Res. and Mol. Biol. 48 p239).

The expression cassette can be introduced into the host cell before, after or at the same time as the plasmid DNA. It is an integrative cassette, it is introduced in general before, and the cells containing said cassette are selected and used for the production of the plasmid DNA.

A particular aspect of the invention is to express the gene encoding M.SssI methylase in bacterial cells (particularly E. coli) containing a TG plasmid. As indicated in the examples, said plasmid is then methylated in the cytosines of the 5'-CG-3 'dinucleotides. More specifically, the TG plasmid is transformed into a strain of E. coli mcrA mcrB D (mcrC-mmr) which already contains a plasmid carrying the gene encoding M.ssssl methylase and is compatible with the TG plasmid. In the course of the growth of the bacterium the two callus plasmids are replicated and methylated (Gotschlich et al., 1991 J. Bacteriol. 173 p5793).

The plasmid DNA or the expression cassette can be introduced into the host cell by any technique known to the person skilled in the art (transformation, transfection, conjugation, electroporation, pulsation, precipitation, etc.). The transformation can be ied out particularly by the transformation technique with CaCl2 (Dagert et Ehrlich, Gene 6 (1979) 23), or that indicated by Hanahan et al. (J. Mol. Biol. 166 (1983) 557) or any technique derived therefrom (Maniatis et al., 1989), as well as by electrotransformation (Wirth et al., Mol. Gen. Genet. 216 (1989) 175 ) or by TSB (Transformation and Storage Buffer; Chung et coll. 1988 Nucleic Acids Res. 16 p3580).

Also see the general techniques of Molecular Biology above.

The methylated plasmid DNA according to the invention can be purified immediately by any technique known to the skilled person (precipitation, chromatography, centrifugation, dialysis, etc.). In the particular case of using an expression vector of the replicating methyltransferase, the TG plasmid must be separated in addition to said vector. Different techniques may be used, based on differences in size or mass of two plasmids, or in the digestion of the vector at the level of restriction sites present only in the vector and not in the TG plasmid. A particularly advantageous purification method is based on the affinity between a specific sequence present in the TG plasmid and an immobilized oligonucleotide. This triple helix purification has been described in detail in the applications FR9603519 and FR94 15162, which are incorporated herein by reference.

A particularly advantageous result of the invention is that the plasmid DNA methylated under the conditions of the invention leads to the expression of the gene under the control of the promoter as good as that obtained with the unmethylated plasmid DNA. This methylated plasmid DNA should not carry the immune stimulation associated with the bacterial DNA and consequently has a certain advantage for being used in non-viral gene therapy.

The methylated plasmid DNAs according to the invention can be used in any application of vaccination or gene and cell therapy, by the transfer of a gene to a given organism, tissue or cell. In particular, they can be used for direct administration in vivo, or for the modification of in. vitro or ex vivo, for the purpose of its implantation in a patient. In this regard, the molecules according to the invention can be used as such (in the form of naked DNA), or in association with different chemical and / or biochemical, synthetic or natural vectors. It can be in particular cations (calcium phosphate, DEAE-dextran, ...) that are treated in the form of precipitates with DNA, which can be "phagocytosed" by the cells. It can also be liposomes in which the DNA molecule is incorporated and which fuse in the plasmic membrane. Synthetic gene transfer vectors are generally lipids or cationic polymers that complex DNA and form a particle that carries positive charges on the surface. These particles are able to interact with the negative charges of the cell membrane, and then pass through it. Examples of such vectors are DOGS (Transfectam ™) or D0TMA (Lipofectin ™). Chimeric proteins were also developed: they are composed of a polycationic part that condenses the DNA, bound to a ligand that binds to a membrane receptor and enters the complex in the cells by endocytosis. DNA molecules according to the invention can also be used to transfer genes into cells by physical transfection techniques such as bombardment, electroporation, etc. Furthermore, prior to their therapeutic use, the molecules of the invention can optionally be linearized, for example, by enzymatic cleavage.

In this regard, another objective of the present invention relates to any pharmaceutical composition containing a plasmid DNA methyl or as defined above. This DNA may be naked or associated with a chemical and / or biochemical transfection vector. The compositions according to the invention can be formulated for the purpose of topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the DNA molecule is used in an injectable or in application form. It can be mixed with any pharmaceutically acceptable carrier for an injectable formulation, particularly for a direct injection at the site to be treated. It can be sterile solutions in particular, isotonic, or dry compositions, particularly lyophilized, which, by addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutes. It can be, in particular, Tris or PBS buffers diluted in glucose or sodium chloride. A direct injection of the nucleic acid in the region of the patient's reach is interesting because it allows to concentrate the therapeutic effect at the level of the affected tissues. The doses of nucleic acids used can be adapted according to different parameters, and particularly depending on the gene, the vector, the administration form used, the aforementioned pathology or even the duration of the investigated treatment.

The present invention will be described more fully with the help of the following examples, which should be considered as illustrative and not limiting.

DESCRIPTION OF THE FIGURES Figure 1. Map of plasmid pXL2784 Figure 2. Restriction map of plasmid pXL2784 Figure 3. Digestion profile of plasmids 1-pXL2784, 2-pX12784 methylated, 3-pXL2784 + pAIT2 methylated and 4-pAIT2 methylated, digested by the enzymes A-AatII, B-BstBI, C-HindIII, D-Hpall, E-EcoRI (M is the molecular weight marker of 1KB of scale).

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, transformation of E. coli, precipitation of nucleic acids etc, are described in the literature (Maniatis et al., 1989, Ausubel et al., 1987). The nucleotide sequences were determined by the chain termination method following the protocol already presented (Ausubel et al., 1987).

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

For ligatures, the DNA fragments were separated according to their size in 0.7% agarose gels or 8% acrylamide, purified by electrophoresis and then electroeluted, extracted with phenol, precipitated with ethanol and then incubated in a buffer 50 mM Tris-HCl pH 7.4, 10 mM MgCl 2, 10 M 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 automated DNA synthesizer Applied Biosystems 394 DNA / RNA Synthesizer using the manufacturer's recommendations.

The LB and 2XTY culture media were used for the bacteriological part (Maniatis et al., 1989).

The plasmid DNAs were also purified following the alkaline lysis technique (Maniatis et al., 1989).

EXAMPLES Example 1. Description of the GT plasmid Numerous eukaryotic expression cassettes carried by the replicating plasmids in the bacterium E___ coli are known to the person skilled in the art. These cassettes can express the reporter genes as the gene that encodes the b-galactosidase of E. coli, or the chloramphenicol acetyltransferase of the transposon Tn9, or the luciferase, or the genes of interest in gene therapy. These cassettes contain a promoter that can be viral or eukaryotic. These expression systems can be tissue-specific and / or inducible or have a ubiquity of expression. The cassette used in this example comprises the gene luc, which codes for the luciferase of Photinus pyralis and the promoter / enhancer pCMV, intermediate human cytomegalovirus enhancer. The luc gene possesses 4.78% of dinucleotides 5'-CG-3 ', and the viral promoter pCMV 5%. These percentages are therefore high with respect to the weak frequency of 0.8% of 5'-CG-3 'in mammalian sequences. In the presence of a methylase (such as M. Sssl methylase or endogenous CpG methylases in mammals), the pCMV promoter and the luc gene can thus be highly methylated.

This expression cassette was cloned into the replicating plasmid in E. coli pXL2784 in which the map is presented in figure 1. The plasmid has a size of 6390 bp and contains 5.8% of dinucleotides 5'-CG-3 '. Plasmid pXL2784 was constructed from vector pXL2675 (2.513 kb), minimum replicon of ColEl from pBluescript (ORÍ) and having as a selection marker the Tn5 transposon gene coding for kanamycin resistance. Plasmid pXL2784 also contains a TH (GAA) 17 sequence that can be ligated to an oligomer (CTT) n where n = 1 to 17, to locally generate a triple helical structure and allow an affinity purification. Plasmid pXL2784 possesses the cer locus (382 bp) of ColEl; the cer locus contains a specific site sequence of the XerC / XerD recombinases and leads to the resolution of plasmid dimers (Summers et al., 1988 EMBO J. 7 p851). The transgene cloned in this plasmid pXL2784 is an expression cassette (3.3 kb) of the luc gene that encodes the luciferase of Photinus pyralis (originating from pioneering pGL2 from Promega) under the control of the human cytomegalovirus pCMV enhancer / promoter (which comes from pcDNA3 of Invitrogen).GnI Example 2: Construction of an expression cassette of a DNA methyltransferase This example describes the structure of a cassette for the expression of M. sssila methylase from Spiroplasma sp. MQ1. It is understood that the same principle can be applied in the construction of the expression cassette of any other enzyme according to the invention.

The expression cassette used comprises the gene encoding the M. sssl methylase from Spiroplasma sp. MQ1 that is expressed under the control of the plac promoter. Thus, in the presence of IPTG (isopropylthio-β-D-galactoside) the methylase is synthesized and activated (Gotschlich et al., 1991 J. Bacteriol, 173 p5793).

This cassette is present in the plasmid pAIT2, which has as replicon the pACYC184 and also carries the transposon gene Tn903 that codes for the resistance to lividomycin, allowing the selection of transformed host cells.

Example 3. Production of plasmid DNA pXL2784 methylated in the cytosine residues of the 5'-CG-3 'dinucleotides.

The plasmid pXL2784 was methyl in the cytosines of all the 5'-CG-3 'dinucleotides with the M. Sssl methylase.

Spiroplasma sp. MQ1. The methylation form according to the invention uses this enzyme and the plasmid is methylated in the course of production in the bacterium.

For this, the strain of E. coli ER 1821 in which the genotype is F "1" endAl thi-1 supE44 mcrA5 D (mrr-hshRMS-mcrB) 1-:: IS10, and which carries the plasmid pAIT2, was transformed by the TSB method (Transformation and Storage Buffer; Chung et al. 1988 Nucleic Acids Res. 16 p3580) by the plasmid pXL2784.

The transformants were selected in LB medium containing kanamycin 50 mg / l and lividomycin 10Omg / in order to select the pXL2784 which carries the transposon Tn5 gene that codes for the kanamycin resistance and the pAIT2 that carries the transposon gene Tn903 for the resistance to lividomycin. When a transformant ER1821, pAIT2, pXL2784 was grown in LB kanamycin medium 50 mg / l + lividomycin 100 mg / l + 2.5 mM IPTG at 37 ° C for 15 hours, the extracted plasmid DNA was methylified.

Methylation was verified by digesting the plasmid preparations with the restriction enzymes Hpall, AatII, BstBl. The integrity and presence of two plasmids were verified by digesting these preparations with the restriction enzymes Hinddll «and EcoRI, see figure 2. The restriction enzymes Hpall, AatII, BstBl are three enzymes in which the cut is not possible if the residue cytosine of nucleotide 5'-CG-3 ', contained in the cutting site, is methylated. In the pCMV promoter four AatII recoion sites were located; in the luc gene, two BstBl recoion sites and eleven Hpall recoion sites were mapped; This last enzyme cut the plasmid pXL2784 into 30 fragments. In the photograph of agarose gel colored with ethidium bromide (figure 2), it is verified that with the plasmid preparation pAIT2 + methylated pXL2784 or with the plasmid preparation pXL2784 methylated and purified by affinity chromatography (see example 4) no digestion takes place with the enzymes Hpall, AatII, BstBl when the digestions by the enzymes HindIII and EcoRI lead to the expected profile after the restriction map. The digestions controlled by the enzymes Hpall, AatII, BstBl of the plasmid pXL2784 present the discounted profile after the restriction map.

These results show that the extracted plasmid DNA was methyl, in the cytosine residues of the 5'-CG-3 'dinucleotide. These results show that methylation refers to more than 90% of the cytosines.

Example 4. Use of methylated plasmids for the transfer of genetic material.

This example demonstrates that the methylated plasmid DNA according to the invention retains its ability to transfect cells, to replicate, and to express a gene of interest.

A protocol for preparing the solutions used for transfection Two batches of plasmids were used for the comparative studies: a) the pXL2784, b) the methylated pXL2784.

Plasmid pXL2784 methylated was obtained in the form of a mixture with plasmid pAIT2 which ensured methylation after bacterial co-transformation. A fractionation by affinity chromatography was performed to purify the plasmid of interest and the technique used is described in the application No. FR 94tl5162. A dialysis step against 0.15M NaCl could be carried out to eliminate the buffer constituting the elution phase of the column.

When the plasmid pXL2784 was used in reference it was purified according to the same protocol as described above.

In this example, DNA vectorization is ensured by a cationic lipid, RPR120535A, which belongs to a series described in patent application No. FR 95 13490. It is understood that any other chemical or biochemical transfer vector can be used.

The transfection solutions were prepared from a volume-to-volume mixture of DNA at 30 μg / ml and aqueous solution of cationic lipid RPR 120535 at 90 μM, the cationic lipid / DNA ratio is therefore 3 nanomoles of cationic lipid / μg DNA After homogenization at the vortex and incubation for at least 15 minutes at room temperature, the DNA / lipofectant solutions are distributed to the 4. 8% (V / V) final in the wells where the cells were washed with medium devoid of proteins (serum) and placed in growth during the transfection time in medium devoid of serum.

B transfection protocol The samples of 1,105 cells [NIH3T3 (mouse fibroblasts) and Hela (human uterine carcinoma)] in exponential growth phase in 2 cm2 (500μl of medium without serum / well) were treated with 25 μl of transfection solution corresponding to the contribution of 0.375 μg of DNA / 1,105 cells. After an incubation of 2 hours at 37 ° C under 5% C02 in a humid atmosphere, the growth medium is supplemented with final 8% calf fetus serum (V / V).

At 40 hours post-transfection the cells were washed with PBS and used with a buffer containing Triton X-100 at 1% and 2mM DTT. The expressed luciferase activity was dosed by light emission [RLU = relative unit of light] in the presence of luciferin, coenzyme A and ATP for 10 seconds and was related to mg of proteins extracted with the lysis buffer.

C results The results obtained according to the conditions described above are shown in the following table.

Enzymatic activity in RLU / 10 seconds / mg protein (coefficient of variation% [3 transfection experiments per result] Taking into account the coefficients of variation obtained for this type of experiments, it can be concluded that there are no significant differences in the expression of the two plasmids used in the same transfection conditions. In addition, the luciferase activity obtained is of the same order of magnitude for the two purification steps considered.

It is noted that in relation to this date, the best method known to 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 (19)

1. A method of producing DNA useful in gene therapy, characterized in that the DNA is produced in a cell that contains a cassette for the expression of a DNA methyltransferase that makes it possible to methylate the cytosine residues of the 5'-CG-3 'dinucleotides.

2. Method according to claim 1, characterized in that the cell is a prokaryotic cell.

3. Method according to claim 2, characterized in that the cell is a bacterium.

4. Process according to claims 1 to 3, characterized in that the expression cassette is carried by a replicating vector.

5. Process according to claims 1 to 3, characterized in that the expression cassette is integrated into the genome of the cell.

6. Process according to one of the preceding claims, characterized in that the expression cassette comprises a nucleic acid encoding a DNA methyltransferase that allows methylating the cytosine residues of the 5'-CG-3 'dinucleotides under the control of a promoter.

7. Process according to claim 6, characterized in that the promoter is an inducible promoter.

8. Process according to claim 1, characterized in that the DNA methyltransferase methylated preferably the cytosine residues of the dinucleotides 5'-CG-3 *.

9. Process according to claim 8, characterized in that the DNA methyltransferase is chosen from the M.SssI methylase, the mouse methylase and the human methylase.

10. Process according to claim 1, characterized in that more than 50% of the cytosine residues of the 5f-CG-3 'dinucleotides of the plasmid DNA are methylated.

11. Process according to claim 1, characterized in that more than 80% of the cytosine residues of the 5'-CG-3 'dinucleotides of the plasmid DNA are methylated.

12. Process according to claim 1, characterized in that more than 90% of the cytosine residues of the 5'-CG-3 'dinucleotides of the plasmid DNA are methylated.

13. Use of a plasmid DNA for the preparation of a pharmaceutical composition intended for the therapeutic or diagnostic treatment of the human or animal body, characterized in that more than 50% of the cytosine residues of the 5'-CG-3 'dinucleotides of the plasmid DNA are methylated.

14. Use of a plasmid DNA for the preparation of a pharmaceutical composition for the therapeutic or diagnostic treatment of the human or animal body, characterized in that more than 80% of the cytosine residues of the 5'-CG-3 'dinucleotides of the plasmid DNA are methylated.

15. Use of a plasmid DNA for the preparation of a pharmaceutical composition for the therapeutic or diagnostic treatment of the human or animal body, characterized in that more than 90% of the cytosine residues of the 5'-CG-3 'dinucleotides of the plasmid DNA are methylated.

16. Process for the preparation of a pharmaceutical composition for the therapeutic or diagnostic treatment of the human or animal body comprising the following steps: - the production of DNA by culture of a cell comprising said DNA and a cassette for the expression of a DNA methyltransferase that allows methylation of the cytosine residues of the 5'-CG-3 'dinucleotides, - the recovery of said DNA, and, - the conditioning of said DNA with a pharmaceutically acceptable vehicle.

17. Process according to claim 16, characterized in that the DNA is a plasmid DNA containing a nucleic acid of therapeutic interest.

18. Process according to claim 16, characterized in that the DNA is a minicircle containing a nucleic acid of therapeutic interest.

19. Composition containing a bacterial DNA in which at least 50% of the cytosine residues of the '-CG-3' dinucleotides are methylated.