EP0914418A1

EP0914418A1 - Method for producing therapeutic dna

Info

Publication number: EP0914418A1
Application number: EP97930569A
Authority: EP
Inventors: Jo[L Crouzet; Béatrice Cameron
Original assignee: Rhone Poulenc Rorer SA
Current assignee: Aventis Pharma SA
Priority date: 1996-07-04
Filing date: 1997-06-24
Publication date: 1999-05-12
Also published as: AU3447597A; FR2750704A1; US20020187527A1; CZ438298A3; SK181498A3; WO1998001540A1; NO986177L; AU722909B2; BR9710173A; US6410273B1; NO986177D0; KR20000022488A; FR2750704B1; ZA975959B; JP2000514293A; CA2258792A1; IL127620A0

Abstract

The invention discloses the preparation of DNA, in particular plasmid DNA. More particularly it concerns the production of bacterial plasmid DNA to be used in gene therapy, in the form of plasmid, minicircle supercoiled, loose or linear.

Description

PROCESS FOR PRODUCING THERAPEUTIC DNA

The present invention relates to the preparation of DNA, in particular plasmid DNA. It relates more particularly to the production of bacterial plasmid DNA which can be used in gene therapy, in the form of plasmid, of supercoiled, relaxed or linear minicircle, and whose immunogenic properties are reduced or even eliminated. The invention also relates to microorganisms which can be used for the production of DNA, as well as pharmaceutical compositions.

Gene therapy consists of correcting a deficiency or an anomaly by introducing genetic information into the affected cell or organ. This information can be introduced either in vitro into a cell extracted from the organ and then reinjected into the body, or in vivo, directly into the targeted tissue. Being a molecule of high molecular weight and negative charge, DNA has difficulties in spontaneously crossing phospholipid cell membranes. Different vectors are therefore used to allow gene transfer: viral vectors on the one hand, chemical and / or biochemical vectors, natural or synthetic, on the other hand. Viral vectors (retroviruses, adenoviruses, adeno-associated viruses, etc.) are very effective, in particular for the passage of membranes, but present a certain 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 cations (calcium phosphate, DEAE-dextran, ...) which act by forming precipitates with DNA, which can be "phagocytosed" by cells. They can also be liposomes in which the DNA is incorporated and which fuse with the plasma membrane. Synthetic gene transfer vectors are generally lipids or cationic polymers which complex DNA and form with it a particle carrying positive charges on the surface. These particles are capable of interacting with the negative charges of the cell membrane, then of crossing it. Examples of such vectors that may be mentioned are dioctadecylamidoglycylspermine (DOGS, TransfectamTM) ₀ or N- chloride [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium (DOTMA, LipofectinTM). Chimeric proteins have also been developed: they consist of a polycationic part which condenses DNA, linked to a ligand which binds to a membrane receptor and drives the complex into cells by endocytosis. It is thus theoretically possible to "target" a tissue or certain cell populations, in order to improve the bioavailability in vivo of the transferred gene.

The plasmids currently used in gene therapy generally carry (i) an origin of replication, (ii) a marker gene such as an antibiotic resistance gene (kanamycin, ampicillin, etc.) and (iii) one or more transgenes with sequences necessary for their expression (enhancer (s), promoter (s), polyadenylation sequences ...). This type of plasmid is for example currently used 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 large quantities of DNA of pharmacological purity. In fact, in these gene therapy techniques, the drug is made up of DNA itself and it is essential to be able to manufacture, in suitable quantities, DNAs having properties suitable for therapeutic use in humans. In this regard, various production and / or purification methods have been described in the prior art, making it possible to improve the quality of the plasmid DNA (PCT / FR95 / 01468; FR96 03519).

Furthermore, the use of DNA carrying genes for resistance to antibiotics or to functional replication origins can also have certain drawbacks, linked in particular to their dissemination in the body.

Different approaches have also been developed to limit these drawbacks

(PCT / FR96 / 00274, FR95 10825). Another drawback of the plasmid DNAs used up to now lies in their origin. These are indeed molecules produced essentially in prokaryotic (bacteria) or lower eukaryotic (yeast) organisms, which potentially have immunogenic motifs in humans. Little is known about the immunological properties of DNA. Bacterial DNA in mice leads to i) the synthesis of antibodies recognizing double-stranded and single-stranded bacterial DNA which have allowed immunization but which does not react with mammalian double-stranded DNA, ii) the stimulation of production of macrophages and cytokines (D. Pisetsky "the Immunologie Properties od DNA" J. Immunol. 156 (1996) 1). The DNA macromolecule is therefore called immunogenic. In addition, a macromolecule can also lead to stimulation of the immune system without being immunogenic (for example a foreign body leading to a cell-mediated immune response). The first evidence suggesting that bacterial DNA leads to an immune response has been described by Pisetsky et al. (1991 J. Immunol. 147 pl759). They have shown that the DNA of three bacterial species can stimulate the proliferation of mouse lymphocytes while the DNA extracted from three animal species does not lead to this stimulation. Then, Yamamoto et al. (1992 Microbiol. Immunol. 36 p983) observed that the bacterial DNA of six species leads in the spleen cells of BALB / c mice to an increase in NK "natural killer" activity and to the induction of production interferons. But the DNA extracted from ten vertebrate species does 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 induces in vitro the proliferation of murine B cells and the secretion of an IgM unoglobulin, whereas this same bacterial DNA treats in vitro with a CpG methylase, does not induce such a response. Krieg et al. also indicate that in the presence of unmethylated DNA, interferon g is produced, and acts as a costimulating factor for the differentiation 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 having an unmethylated CpG motif and framed in 5 'by 2 purines and in 3' by 2 pyrimidines leads in vivo to a coordinated secretion of the interleukins IL-6 and IL-12, and of interferons g by the cells. NK (IFN-g), B cells (IL-6 and IL-12) and CD4 + T cells (IL-6 and IFN-g) (Krieg et al. 1996 Proc. Natl. Acad. Sci. USA 93 p2879).

The plasmid DNA used to date in gene therapy is essentially produced in prokaryotic cells, and therefore has a methylation profile comparable to that of bacterial genomic DNA. It has also been demonstrated that the plasmid DNA which has been injected into the muscle or into the liver, and then extracted, retains the procaroyte methylation profile (Wolf et al. 1992 Hum. Mol. Genet.1 p363; Malone et al 1995 J. Biol. Chem. 269 ρ29903). As a result, the bacterial plasmid DNA used has a significant potential for stimulating the immune system.

It would therefore be particularly advantageous to be able to have plasmid DNA having reduced or even suppressed immunological properties. It would also be particularly advantageous to be able to have a process making it possible to produce, on a scale compatible with industrial use, plasmid DNAs of this type.

The present invention provides a solution to these problems. The Applicant has in fact been interested in the immunogenic properties of bacterial DNA. The Applicants have now developed a process for producing pharmaceutical grade plasmid DNA, potentially devoid of undesirable immunogenic effects. The Applicant has also shown that the methylation of certain DNA residues makes it possible to reduce the immunogenic potential of plasmid DNAs, without affecting their capacity to transfect cells and to express therein a nucleic acid of interest.

One aspect of the invention is to prepare DNA, in particular plasmid DNA, of therapeutic quality. According to another aspect, the present invention relates to the use in gene therapy of methylated plasmid DNA on the cytosines of the 5'-CG-3 'dinucleotides. A third aspect of the invention relates to pharmaceutical compositions comprising methyl plasmid DNAs. The invention further relates to a method for in vivo methylation of DNA by expression of a methylase. The invention in other aspects relates to expression cassettes, host microorganisms usable for methylation, the preparation of therapeutic compositions, and methods of gene transfer.

A first object of the invention therefore relates to a process for the production of DNA usable in gene therapy, characterized in that said DNA is produced in a cell containing a cassette for expression of a DNA methyltransferase making it possible to methylate the cytosine residues of the dinucleotides 5'-CG-3 '.

The present invention therefore relates to the production of DNA, in particular plasmid DNA, methylated on the cytosine residues of the 5'-CG-3 'dinucleotides.

Plasma DNA methylation in vitro is documented in the literature (Adams et al. 1992 FEBS Letters 309 p97; Doerflerl994 FEBS Letters 344 p251; Komura et al. 1995 Biochim. Biophys. Acta 1260 p73). However, this methylation method cannot be envisaged for the industrial production of plasmids which would be used in gene therapy. A process for the production of plasmid DNA must indeed make it possible to reproducibly produce large and homogeneous quantities of plasmids and to purify this DNA by methods acceptable for pharmaceutical use. It is quite clear that DNA methylated in vitro may be more or less released from batch to batch (Doerflerl994 FEBS Letters 344 p251) and that the quantities produced are limited.

The present invention now shows that it is possible to methylate a plasmid of interest directly during production, by coexpressing in the host cell the gene coding for a methylase. The present invention also shows that, according to this method, large and homogeneous quantities of methylated plasmid can be produced and the methylated plasmid DNA can be purified according to methods already described. The Applicant has also demonstrated, advantageously, that the plasmid DNA thus methylated retains the capacity to transfect target cells and, if necessary, to replicate therein. In a particularly remarkable manner, the applicant has further demonstrated that the plasmid DNA thus methylated can, in vivo, express nucleic acids of interest. Numerous studies indeed reinforce the idea that hypermethylation is correlated with inhibition or inactivation of promoters and that actively transcribed promoters are often hypo-or non-methylated. Thus, as regards viral promoters, if the late promoter E2A of the genome of the adenovirus type 2 is fully methylated on the dinucleotides 5'-CG-3 ', in the transfoimated cells of hamster HE1, and not methylated in the transformed hamster HE2 cells; the E2A gene is silent in HE1 and transcribed in HE2 (W. Doerfler 1995 Curr. Top. Microbiol. Immunol. 197 p209). Another example is described by Kohn et al. (1994 Proc. Natl. Acad. Sci. USA 91 p2567) which show that the absence of expression from the retroviral vector LTR transduced in hematopoietic stem cells is associated with methylation in vivo. The inhibition of the expression of a reporter gene, under the control of a viral promoter, when this gene is introduced in transient transfection with a methylated plasmid in vitro has also been demonstrated (Adams et al. 1992 FEBS Letters 309 p97 ; Doerfler 1994 FEBS Letters 344 p251; Komura et al. 1995 Biochim. Biophys. Acta 1260 p73). Furthermore, Razin et al. (cited above) have shown that the promoter of the gene coding for thymidine kinase of herpes simplex type I and that of the gene coding for mouse metallothionein are inactive during transient expression in mouse cells L and murine cells teratocarcinomas F9 if these promoters are methylated on the 5 '-CG-3' dinucleotides.

The present invention therefore describes for the first time a process allowing 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, in particular in gene therapy applications.

The method according to the invention can be implemented in different types of cellular hosts. These include any non-human cell, essentially lacking a methylation system for the cytosines of the 5'-CG-3 'dinucleotides.

The absence of methylation may 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. They are preferably simple prokaryotic or eukaryotic cells.

Advantageously, the cell host is a bacterium. Among the bacteria, there may be mentioned more preferably E, CQli. fi. ___ ύll_, Streptomvces. Pseudomonas (putida. E aeruginosa ⁾ . RhizQbiui melitoti, Agrohacterium tumefaciens. Staphvlococcus _w___, StreptOTTiyceS pristinaespiralts. Enterococcus faecium or Clostridium. Enterobacteria such as Salmonella typhimurium can also be used. Klebsiella ηeurηoniae, Enterobacter aerogenes. Erwinia carotovora or Serratia marcescens. Preferably, the cell host used is a non-pathogenic organism and makes it possible to produce large and homogeneous quantities of plasmid DNA. As a particularly preferred example, E. coli is used.

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

The DNA can be any DNA molecule, single-stranded or double-stranded, linear or circular, replicative or not, integrative or not, in the form of plasmid, supercoiled, loose or linear mini-circle. In the remainder of the text, the DNA will also be referred to as plasmid DNA or TG plasmid (for plasmid usable in gene therapy).

The TG plasmids generally used in gene therapy essentially carry (i) an origin of replication, (ii) one or more nucleic acids of interest (therapeutic gene) with sequences necessary for their expression

(enhancer (s), promoter (s), polyadenylation sequences, etc.), and optionally (iii) a marker gene.

The choice of the origin of replication is mainly determined by the cell host used for production. It may be a source of replication from a plasmid of the incompatibility group P (example = pRK290) which allows replication in E. strains. coli pol A. More generally, it can be any origin of replication resulting from a plasmid replicating in prokaryotic cells or lower eukaryote. This plasmid can be a derivative of pBR322 (Bolivar et al., Gene 2 (1977) 95), a derivative of pUC (Viera and Messing, Gene 19 (1982) 259), or other plasmids derived from the same group of incompatibility, that is to say of ColEl or pMBl for example. These plasmids can also be chosen from other incompatibility groups which replicate in Escherichia coli. They may be plasmids derived from plasmids belonging to the incompatibility groups A, B, FI, Fil, Fin, IVF, 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 plasmids which do not replicate in E. coli but in other hosts such as & s liS. Streptomvces. P. putida. P. aeruginosa. Rhizobium meliloti. Agrobacterium tu efaciens. Staphvlococcus aureus. Streptomvces pristinaespiralis. EnterQgPCÇUS faecium or Clostridium. Preferably, the origins of replication from plasmids replicating in E. coli are used. According to a particular variant, the origin of replication can be a conditional origin, that is to say the activity of which depends on the presence of factors in trans. The use of this type of origin of replication prevents replication of the plasmid DNA after administration, for example in humans (FR95 10825).

Among the marker genes, mention may be made of a resistance gene, in particular to an antibiotic (ampicillin, kamamycin, geneticin, hygromycin, etc.), or any gene conferring on the cell a function which it no longer possesses (for example a gene which has deleted on the chromosome or made inactive), the gene on the plasmid restoring this function.

According to a particular embodiment, the plasmid DNA comprises sequences making it possible to eliminate, after the production phase, all the essentially non-therapeutic regions (origin of replication, marker gene, ete). A particularly advantageous approach to generate this type of molecule

(minicircles) was described in application PCT / FR96 / 00274.

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

The nucleic acid of interest can be any nucleic acid (cDNA, gDNA,

Synthetic or semi-synthetic DNA, ete) whose transcription and possibly translation in a cell generate products having a therapeutic, vaccine, agronomic or veterinary interest.

Among the nucleic acids having therapeutic properties, there may be mentioned more particularly the genes coding for enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, ete (FR 9203120), growth factors, neurotransmitters or their synthetic precursors or enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, ete; apolipoproteins: ApoAI, ApoAIV, ApoE, ete (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, ete (FR 93 04745) , genes coding for factors involved in coagulation: Factors VII, VIII, IX, summer, suicide genes: Thymidine kinase, cytosine deaminase, summer; or all or part of a natural or artificial immunoglobulin (Fab, ScFv, ete), a RNA ligand (WO91 / 19813) etc. The therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can, for example, be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.

The nucleic acid of interest can also be a vaccinating gene, that is to say a gene coding for an antigenic peptide, capable of generating in humans or animals an immune response, with a view to carrying out vaccines. They may in particular be antigenic peptides specific for the epstein barr virus, for the virus HIV, hepatitis B virus (EP 185 573), pseudo-rabies virus, or even specific for tumors (EP 259212).

Generally, in plasmids, the nucleic acid of therapeutic, vaccine, agronomic or veterinary interest also contains a promoter region for functional transcription in the target cell or organism (ie mammals, in particular humans), as well that a region located in 3 ', and which specifies a transcriptional end signal and a polyadenylation site. As regards the promoter region, it may be a promoter region naturally responsible for the expression of the gene considered when it is capable of functioning in the cell or the organism concerned. They can also be regions of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the target cell. Among the eukaryotic promoters, one can use any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific way or not, inducible or not, strong or weak. They may in particular be ubiquitous promoters (promoter of the HPRT, PGK genes, a-actin, tubulin, ete), promoters of intermediate filaments (promoter of the GFAP genes, desmin, vimentin, neurofilaments, keratin, ete), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAI, ete genes), of tissue-specific promoters (promoter of the pyruvate kinase gene, villin, intestinal fatty acid binding protein, muscle a-actin smooth, ete) or promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, ete). Likewise, they may be promoter sequences originating from the genome of a virus, such as for example the promoters of the El A and MLP genes of adenovirus, the CMV early promoter, or even the RSV LTR promoter or MMTV, etc. In addition, these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression. Furthermore, the gene of interest can also include a signal sequence directing the product synthesized in the secretory pathways of the target cell. This signal sequence can be the natural signal sequence of the synthesized product, but it can also be any other functional signal sequence, or an artificial signal sequence.

Depending on the nucleic acid of interest, the methyl 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, parkinson , ALS, ete), cancers, pathologies linked to coagulation disorders or dyslipoproteinemias, pathologies linked to viral infections (hepatitis, AIDS, summer), or in the agronomic and veterinary fields, etc. They are particularly advantageous for the treatment of pathologies in which lasting expression without immunological reaction is desired, in particular in the field of genetic, neurodegenerative and cardiovascular diseases.

As indicated above, the method according to the invention uses a host cell containing an expression cassette for a DNA methyltransferase making it possible to methylate the cytosine residues of the dinucleotide 5'-CG-3 '.

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

Most simple eukaryotes and invertebrates contain relatively little 5-methylcytosine and N ^ -methyladenine. However, the methylation of bases in vertebrates is more important and in this case 5-methylcytosine is the most frequent of the methylated bases. Indeed more than 95 percent of the methylated groups of vertebrate DNA is found on the C residues of the rare 5 '-CG-3 ^* dinucleotides (the frequency of 0.8% of 5' -CG-3 'on mammalian sequences is very low although the percentage in GC is on average 40% and that an unbiased arrangement would lead to a frequency of 4% of 5'-CG-3 '). And, more than 50 percent of all dinucleotides can be methylated. Various evidences suggest that the degree of methylation of certain sequences containing the 5'-CG-3 'dinucleotide may be a determining factor in mammals in regulating the expression of particular genes, inactivation of the X chromosome, oncogenesis (1993 in DNA methylation: Molecular Biology and Biological Significance, Eds Jost and Saluz), and also hereditary diseases (Bâtes et al. 1994 BioEssays 16 p277).

The present invention uses an expression cassette for a DNA methyltransferase making it possible to methylate the cytosine residues on the 5'-CG-3 'dinucleotides. Therefore, within the meaning of the present invention, methyl DNA more particularly means methyl DNA on the cytosine residues of dinucleotides 5 '-CG-3'. Advantageously, the DNA methyltransferase used preferentially methylates the cytosine residues of the 5'-CG-3 'dinucleotides, that is to say almost does not affect the adenine residues, nor the cytosine residues which are present in a context different from 5 '-CG-3' dinucleotides. Advantageously, the term methyl plasmid DNA is understood to mean plasmid DNA of which at least 50% of the cytosine residues of the 5 '-CG-3' dinucleotides are methyl. More preferably, at least 80%, advantageously 90% of said residues are methylated. Plasmid DNA methylation can be checked in several ways. In particular, it can be controlled by digesting the plasmid preparations with restriction enzymes, the cleavage of which is not possible if the cytosine residue of the dinucleotide 5'-CG-3 ', contained in the cleavage site, is methylated. Mention may be made, for example, of the restriction enzymes Hpall. AatlI. BstBI. Methylation can also be determined by chromatography. Thus, the amount of unmethylated plasmid present in the preparation of methylated plasmid was quantified as follows: to the undigested unmethylated plasmid are added 1% or 5% of unmethylated plasmid and fully digested with Hpall. These samples, as well as the methylated plasmid digested with Hpall. are analyzed by anion exchange liquid chromatography and detection at 260 nm, which makes it possible to separate and quantify the undigested DNA from the digested DNA. It is found that the methylated plasmid contains less than 5% of unmethylated plasmid DNA, in other words that more than 95% of the plasmid DNA is methylated.

Advantageously, the method of the invention is characterized in that the DNA methyltransferase preferably methylates the cytosine residues of the 5'-CG-3 'dinucleotide.

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

Several mammalian DNA methyltransferases making it possible to methylate the cytosine residues on the sequences containing any 5'-CG-3 'dinucleotide have been characterized and the corresponding genes have been cloned, for example that of the mouse (Bestor et al. 1988 J. Mol. Biol. 203 ρ971) or that of man (Yen et al. 1992 Nucl. Acids Res. 20 p2287). These enzymes have a molecular weight between 135 and 175 kD. They methylate hemimethylated DNA much faster than unmethylated DNA, suggesting that they are maintenance methylases (Smith 1994 Progress in Nucleic Acid Research and Molecular Biology 49 p65). The enzyme counterpart at £. coli does not exist. On the other hand, the methylase M. SssI of Spiroplasma methylated exclusively and completely the cytosine residues of any dinucleotide 5 '-CG-3' with a comparable speed, whatever the hemimethylated or unmethylated substrate (Razin et al. 1992 FEBS letters 313 ρ243; Baker et al. 1993 Biochim. Biophys. Acta 196 p864). This enzyme was isolated from the Spiroplasma sp. MQ1. Its molecular weight is 42 KD and the gene has been cloned and overexpressed in £. coli (Razin et al. 1990 Nucl. Acids Res. 18 pi 145 and EP0412676A1 derwent 91045812).

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

The DNA methyltransferase expression cassette generally comprises a nucleic acid encoding a DNA methyltransferase making it possible to methylate the cytosine residues of the 5 '-CG-3' dinucleotide under the control of a promoter. The promoter used for this purpose can be any promoter functional in the chosen host cell. In this regard, it may be a promoter as defined above. As regards prokaryotic cellular hosts, there may be mentioned more particularly the promoter of the lactose operon (Plac), of the tryptophan operon (Ptrp), the hybrid Plac / Ptryp promoters, the PL or PR promoter of bacteriophage lambda, the promoter of the tetA gene (in Vectors 1988 pl79 Rodriguez and Denhardt editors), 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 very particularly advantageous to use an inducible promoter, making it possible to control the expression of the methylase. The inducible promoter can for example be the promoter of bacteriophage T7 or the Plac promoter.

Advantageously, the expression cassette also contains end of transcription signals (transcriptional terminators), such as ribosomal terminators. The DNA methyltransferase expression cassette can be carried by a replicative vector, or can be integrated into the genome of the host cell.

Being a replicative vector, a vector compatible with the plasmid TG is advantageously used, that is to say capable of co-residing in the same cell. Two different plasmids can replicate in the same cell if the control of replication of each plasmid is different. Thus compatible plasmids belong to two incompatibility groups. However, there are approximately 30 groups of incompatibility of plasmids replicating in enterobacteria (Maas et al. 1988 Microbiol. Rev. 52 p375). As a result, there are many possibilities for replicating two plasmids in the same cell and several examples are described in the literature. Mention may be made, for example, of the co-replication of the plasmids derived from ColEl with the plasmids having for replicon R6K or pl5A or RSF1010 or RK2; one can also cite the corplication of the plasmids derived from RK2 with plasmids derived from R6K or RSF1010 or pSa or ColEl (in Vectors 1988 p287 Rodriguez and Denhardt editors). This list is not exhaustive and other examples are still described in Vectors 1988 p287 Rodriguez and Denhardt editors. Advantageously, the replicative vector used has a different number of copies in the host cell than the plasmid TG. Thus, the vector carrying the gene coding for methylase, the expression of which may be inducible, has a low copy number (derived for example from pACYC184 or RK2), while the plasmid TG has a high copy number (derived from ColEl). It is also possible to clone in the plasmid TG a sequence making it possible to form with an appropriate oligonucleotide a triple helix sequence so that the plasmid TG can be separated from the other plasmid by affinity purification.

The DNA methyltransferase expression cassette can also be integrated into the genome of the host cell. Integration can be achieved by homologous recombination, insofar as the expression cassette is surrounded by adjacent fragments of a gene, nonessential, of the host genome and cloned on a plasmid which cannot replicate in the host considered. This plasmid can be i) a derivative of ColEl in a strain of E. poli ^ts (Gutterson et al. 1983 Proc. Natl. Acad. Sci. USA 80 p4894); ii) a thermosensitive derivative of pSC101 in any strain of E- £ Ωli (S. Kushner et al. 1989 J. Bacteriol. 171 p4617); iii) a suicide vector such as M13mp10 in the strains of E. CQ_ {sjp ⁺ (Blum et al. 1989 J. Bacteriol. 171 p538) or also iv) a plasmid comprising only the origin g of R6K in any strain of E-coli lacking the J2Î £ 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. As an integrative cassette, it is generally introduced before, and the cells containing said cassette are selected and used for the production of plasmid DNA.

A particular aspect of the invention is to express the gene coding for the methylase M. SssI in bacterial cells (in particular E. coli) containing a plasmid TG. As indicated in the examples, said plasmid is then methylated on the cytosines of dinucleotides 5'-CG-3 '. More specifically, the plasmid TG is transformed into a strain of E-coli mcrA mcrB D (mcrC-mrr) already containing a plasmid carrying the gene coding for the methylase M-SssI and compatible with the plasmid TG. During the growth of the bacterium, the two co-residing plasmids replicate and are 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 those skilled in the art (transformation, transfection, conjugation, electroporation, pulsing, precipitation, etc.). The transformation can in particular be carried out by the Caθ2 transformation technique (Dagert and Ehrlich, Gene 6 (1979) 23), or that perfected by Hanahan et al. (J. Mol. 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 al. 1988 Nucleic Acids Res. 16 p3580). See also the general techniques of Molecular Biology below. The methyl plasmid DNA according to the invention can then be purified by any technique known to a person skilled in the art (precipitation, chromatography, centrifugation, dialysis, etc.). In the particular case of the use of a replicative methyltransferase expression vector, the TG plasmid must also be separated from said vector. Different techniques can be used, based on the differences in size or mass of the two plasmids, or on the digestion of the vector at restriction sites present only in the vector and not in the plasmid TG. A particularly advantageous purification method is based on the affinity between a specific sequence present on the plasmid TG and an immobilized oligonucleotide. This triple-helix purification has been described in detail in applications FR96 03519 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 same unmethylated plasmid DNA. This methylated plasmid DNA should not cause the immune stimulation associated with bacterial DNA and therefore has a definite advantage for being used in non-viral gene therapy.

The methylated plasmid DNAs according to the invention can be used in any vaccination or gene and cell therapy application, for 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 cells in vitro or ≤x vivo, with a view to their 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 combination with different chemical and / or biochemical, synthetic or natural vectors. These may include cations

(calcium phosphate, DEAE-dextran, ...) which act by forming precipitates with DNA, which can be "phagocytosed" by cells. It can also be liposomes in which the DNA molecule is incorporated and which fuse with the plasma membrane. Synthetic gene transfer vectors are generally cationic lipids or polymers which complex DNA and form with it a particle carrying positive charges on the surface. These particles are capable of interacting with the negative charges of the cell membrane, then of crossing it. Examples of such vectors include DOGS (TransfectamTM) or DOTMA (Lipofectin M). Chimeric proteins have also been developed: they consist of a polycationic part which condenses DNA, linked to a ligand which binds to a membrane receptor and drives the complex into cells by endocytosis. The DNA molecules according to the invention can also be used for the transfer of genes into cells by physical transfection techniques such as bombardment, electroporation, etc. In addition, prior to their therapeutic use, the molecules of the invention can optionally be linearized, for example by enzymatic cleavage.

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

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

LEGEND OF FIGURES

Figure 1. Map of plasmid pXL2784

Figure 2. Restriction map of plasmid pXL2784

Figure 3. Digestion profile of plasmids 1-pXL2784, methylated 2-pXL2784, methylated 3-pXL2784 + pAIT2 and methylated 4-pAIT2, digested with the enzymes A-AatH. B-BstBI. C-HindIII. D-Hpall. E-EcoRI (M is the IKB ladder molecular weight marker).

GENERAL CLONING AND MOLECULAR BIOLOGY TECHNIQUES

Conventional molecular biology methods such as centrifugation of plasmid DNA in cesium chloride-ethidium bromide gradient, digestion with restriction enzymes, gel electrophoresis, electroelution of DNA fragments from agarose gels, transformation in E- £ ûli. the precipitation of nucleic acids ete, are described in the literature (Maniatis _i _ [., 1989, Ausubel et al., 1987). The nucleotide sequences were determined by the chain termination method following the protocol already presented (Ausubel ≤î al., 1987).

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

For the ligations, the DNA fragments are separated according to their size on 0.7% agarose or 8% acrylamide gels, purified by electrophoresis then electroelution, phenol extracts, ethanol precipitates and then incubated. in a tampon 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 10 mM DTT, 2 mM ATP, in the presence of phage T4 DNA ligase (Biolabs). The oligonucleotides are synthesized using the chemistry of phosphoramidites protected in b by a cyanoethyl group (Sinha ej al „1984, Giles 1985) with the automatic DNA synthesizer Applied Biosystems 394 DNA / RNA Synthesizer using the manufacturer's recommendations.

LB and 2XTY culture media are used for the bacteriological part (Maniatis ≤l al., 1989).

The plasmid DNAs are also purified according to the alkaline lysis technique (Maniatis ≤î âl-. 1989).

EXAMPLES

Example 1. Description of the plasmid TG

Numerous cassettes of eukaryotic expression carried by replicative plasmids in the bacterium E- £ Ωli are known to those skilled in the art. These cassettes can express reporter genes such as the gene coding for the E. coli b-galactosidase, or the chloramphenicol acetyltransferase of the transposon Tn9, or luciferase, or genes of interest in gene therapy. These cassettes contain a promoter which can be viral or eukaryotic. these expression systems can be specific tissue and / or inducible or else have a ubiquity of expression. The cassette used in this example comprises the gene ___, coding for the luciferase of Photinus pyralis and the promoter pCMV, promoter / enhancer intermediate of human cytomegalovirus. The Ju £ gene has 4.78% of 5 '-CG-3' dinucleotides, and the viral promoter pCMV 5%. These percentages are therefore high compared to the low frequency of 0.8% of 5 '-CG-3' on mammalian sequences. In the presence of a methylase (such as methylase M. SssI or CpG methylases endogenous to mammals), the promoter pCMV and the gene lnς can therefore be highly methylated. This expression cassette was cloned into the replicative plasmid in E-coli pXL2784, the map of which is presented in FIG. 1. The plasmid is 6390 bp in size and contains 5.8% of 5'-CG-3 \ dinucleotides \ The plasmid pXL2784 was constructed from the vector pXL2675 (2.513 kb), a minimal replicon of ColEl derived from pBluescript (ORI) and having as a selection marker the gene for the transposon Tnϋ coding for resistance to kanamycin. The plasmid pXL2784 also contains a sequence TH (GAA) i7 which can bind to an oligomer (CTT) _n where n = l to 17, to locally generate a triple helix structure and allow purification by affinity. Plasmid pXL2784 has the ££ T locus (382 bp) from ColEl; the csi locus contains a specific site sequence of XerC / XerD recombinases and leads to the resolution of plasmid dimers (Summers et al. 1988 EMBO J. 7 p851). The transgene cloned on this plasmid pXL2784 is an expression cassette (3.3 kb) of the read gene coding for Photinus pyralis luciferase (from pomegal basic Promega) under the control of the enhancer / promoter pCMV human cytomegalovirus (from pcDN A3 of In Vitrogen).

Example 2 Construction of a DNA Methyltransferase Expression Cassette

This example describes the structure of an M. SssI methylase expression cassette from Spiroplasma sp. MQ1. It is understood that the same principle can be applied to the construction of an expression cassette for any other enzyme according to the invention.

The expression cassette used comprises the gene coding for methylase M. SssI from Spiroplasma sp. MQ1 which is expressed under the control of the promoter plac. Thus, in the presence of IPTG (isopropylthio-b-D-galactoside) methylase is synthesized and active (Gotschlich et al. 1991 J. Bacteriol. 173 p5793).

This cassette is present in the plasmid pAIT2, which replicates pACYC184 and further carries the transposon gene Tn9Q3 coding for resistance to lividomycin, allowing the selection of transformed host cells. Example 3. Production of plasmid DNA pXL2784 methylated to the cytosine residues of the 5'-CG-3 'dinucleotides.

Plasmid pXL2784 is methylated on the cytosines of all 5'-CG-3 'dinucleotides with methylase M. ≤ssl from Spiroplasma sp. MQl. The methylation mode according to the invention uses this enzyme and the plasmid is methylated during production in the bacteria.

For this, the strain of E-CΩli ER 1821 whose genotype is! ' end Al thi- 1 supE44 mcrA5 D ⁽ mrr-hsdRMS-mcrB ⁾ l - :: IS10. and carrying the plasmid pAIT2, is transformed by the TSB method (Transformation and Storage Buffer; Chung et al. 1988 Nucleic Acids Res. 16 p3580) by the plasmid pXL2784. The transformants are selected on LB medium containing kanamycin 50 mg 1 and lividomycin 100 mg l in order to select pXL2784 which carries the Tn transposon gene coding for resistance to kanamycin and pAIT2 carries the Tn903 transposon gene coding for resistance to lividomycin. When an ER1821 transformant, pAIT2, pXL2784 is cultured in LB medium kanamycin 50 mg / l + lividomycin 100 mg / 1 + 2.5 mM IPTG at 37 ° C. for 15 hours, the extracted plasmid DNA is methylated.

The methylation is verified by digesting the plasmid preparations with the restriction enzymes Hpall. AatH. BstBI. The integrity and the presence of the two plasmids are verified by digesting these preparations with the restriction enzymes HindIII and EcoRI. see figure 2. Hpall restriction enzymes. AatH. BstBI are three enzymes whose cleavage is not possible if the cytosine residue of the dinucleotide 5'-CG-3 ', contained in the cleavage site, is methylated. On the pCMV promoter are located four AatH recognition sites; on the ___Q gene are mapped two recognition sites of fisîBI and eleven recognition sites of Hpall; the latter enzyme cutting the plasmid pXL2784 into 30 fragments. In the photograph of the agarose gel stained with ethanol bromide (FIG. 2), it can be seen 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 Hpall enzymes. AatH. BstBI whereas digestions with the enzymes HindIII and EcoRI lead well to the profile expected from the restriction map. Digests controlled by Hpall enzymes. AatH. BgtBI of plasmid pXL2784 show the expected profile according to the restriction map.

These results demonstrate that the extracted plasmid DNA is methylated, on the cytosine residues of the dinucleotide 5'-CG-3 '. These results also show that methylation concerns more than 90% of these cytosines.

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

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

A protocol for the preparation of solutions used for transfection

Two batches of plasmids are used for comparative studies:

a) pXL2784, b) methylated pXL2784.

The methylated plasmid ρXL2784 is obtained in the form of a mixture with the plasmid pAIT2 which ensured its methylation after bacterial co-transformation. A fractionation by affinity chromatography was carried out to purify the plasmid of interest and the technique used is described in application No. FR 94tl5162. A dialysis step against 0.15M NaCl can be carried out to remove the buffer which constitutes the elution phase from the column.

When the plasmid pXL2784 is used as a reference, it is purified according to the same protocol as that described above. In this example, vectorization of the DNA is ensured by a cationic lipid, RPR120535A, belonging 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 are prepared from a volume-to-volume mixture of DNA at 30 μg / l and aqueous solution of cationic lipid RPR 120535 at 90 μM; the cationic lipid / DNA ratio is therefore 3 nanomoles cationic lipid / μg DNA. After homogenization with a vortex and incubation for at least 15 minutes at room temperature, the DNA / lipofectant solutions are distributed at 4.8% (VV) final in the wells where the cells have been washed with medium devoid of proteins (serum) and returned to growth during the transfection time in a serum-free medium.

B transfection protocol

Samples of Î.IO ^ cells [NIH3T3 (mouse fibroblasts) and Hela (human uterine carcinoma)] in exponential growth phase over 2 cm2 (500 μl of medium without serum / well) are treated with 25 μl of transfection solution ce which corresponds to the contribution of 0.375 μg of DNA / 1.10 ⁵ cells. After a 2 hour incubation at 37 ° C under 5% CO2 in a humid atmosphere, the growth medium is supplemented with final 8% fetal calf serum (V / V).

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

C result?

The results obtained under the conditions described above are shown in the following table. NIH 3T3 cells Hela cells

Plasmid PXL2784 PXL2784CH3 PXL2784 PXL2784CH3 purified by 2.0.1010 +/- 2.2.101 ° +/- 3.1.10 ⁸ +/- 1.7. 10 ⁸ +/- chromatography 4% 10% 15% 11% affinity and dialysis 2.3.1010 +/- 3.1.10 ¹⁰ +/- 3.8.10 ⁸ +/- 2.4. 10 ⁸ +/- 21% 10% 14% 7%

Enzyme activity in RLU / 10 seconds / mg protein (coefficient of variation% [3 transfection experiments per result]

Given the coefficients of variation obtained for this type of experiment, we can conclude that there are no significant differences as to the expression of the two plasmids used under the same transfection conditions. In addition, the luciferase activity obtained is of the same order of magnitude for the two purification steps considered.

Claims

1. A method of producing DNA usable in gene therapy, characterized in that said DNA is produced in a cell containing an expression cassette for a DNA methyltransferase making it possible to methylate the cytosine residues of the 5'-CG-3 'dinucleotide.

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. Method according to claims 1 to 3 characterized in that the expression cassette is carried by a replicative vector.

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

6. Method according to one of the preceding claims, characterized in that the expression cassette comprises a nucleic acid coding for a DNA methyltransferase making it possible to methylate the cytosine residues of the 5'- CG-3 'dinucleotide under the control of a promoter .

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

8. Method according to claim 1 characterized in that the DNA methyltransferase preferably methylated the cytosine residues of the dinucleotide 5 ^* -CG-3 '.

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

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

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

12. Method according to claim 1 characterized in that more than 90% of the cytosine residues of the dinucleotide 5'-CG-3 'of the plasmid DNA are methyl.

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

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

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

16. Process for the preparation of a pharmaceutical composition intended for the therapeutic treatment or diagnosis 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 making it possible to methylate the cytosine residues of dinucleotides 5'-CG-3 ', the recovery of said DNA, and,

- conditioning of said DNA with a pharmaceutically acceptable vehicle.

17. The method of claim 16 characterized in that the DNA is a plasmid DNA comprising a nucleic acid of therapeutic interest.

18. The method of claim 16 characterized in that the DNA is a mini-circle comprising a nucleic acid of therapeutic interest.

19. Composition comprising a bacterial DNA of which at least 50% of the cytosine residues of the 5 '-CG-3' dinucleotide are methylated.