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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
  • C12N9/1007—Methyltransferases (general) (2.1.1.)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

• 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 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.
• 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.
• the DNA macromolecule is therefore called immunogenic.
• 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.
• interferon g 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).
• 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
• CD4 + T cells IL-6 and IFN-g
• 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 ⁇ 29903). As a result, the bacterial plasmid DNA used has a significant potential for stimulating the immune system.
• 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.
• 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).
• 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.
• 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.
• the cell host is a bacterium.
• bacteria there may be mentioned more preferably E, CQli. fi. ___ ⁇ ll_, 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 ⁇ eur ⁇ oniae, Enterobacter aerogenes.
• the cell host used is a non-pathogenic organism and makes it possible to produce large and homogeneous quantities of plasmid DNA.
• E. coli is used as a particularly preferred example.
• 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.
• 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
• 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.
• Plasmids 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.
• the origins of replication from plasmids replicating in E. coli are used.
• 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).
• 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.
• an antibiotic ampicillin, kamamycin, geneticin, hygromycin, etc.
• 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 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).
• all the essentially non-therapeutic regions oil of replication, marker gene, ete.
• 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.
• 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.
• 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, suicide genes
• 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).
• 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.
• 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).
• they may be promoter sequences of eukaryotic or viral genes.
• they may be promoter sequences originating from the genome of the target cell.
• promoters 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).
• ubiquitous promoters promoter of the HPRT, PGK genes, a-actin, tub
• 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.
• these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression.
• 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.
• 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.
• 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 '.
• DNA methyltransferase dam which methylates adenosine residues within the 5'-GATC-3 'sequences
• 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.
• the enzyme M. Hpall methylated the second cytosine residue in the sequence 5'-CCGG-3 '.
• 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'.
• 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.
• 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.
• 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.
• the method of the invention is characterized in that the DNA methyltransferase preferably methylates the cytosine residues of the 5'-CG-3 'dinucleotide.
• 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.
• the DNA methyltransferase is chosen from methylase M.SssI, mouse methylase and human methylase.
• 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.
• 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.
• lac lactose operon
• Ptrp tryptophan operon
• 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
• 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.
• the expression cassette also contains end of transcription signals (transcriptional terminators), such as ribosomal terminators.
• 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.
• 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.
• compatible plasmids belong to two incompatibility groups.
• there are approximately 30 groups of incompatibility of plasmids replicating in enterobacteria (Maas et al. 1988 Microbiol. Rev. 52 p375).
• the replicative vector used has a different number of copies in the host cell than the plasmid TG.
• 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- £ ⁇ li (S. Kushner et al. 1989 J. Bacteriol.
• 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 Ca ⁇ 2 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.
• 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.).
• 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.
• 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.
• 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
• 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.
• the molecules of the invention can optionally be linearized, for example by enzymatic cleavage.
• another object of the present invention relates to any pharmaceutical composition
• 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.
• 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.
• nucleic acid 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 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).
• oligonucleotides are synthesized using the chemistry of phosphoramidites protected in b by a cyanoethyl group (Sinha ej al confuse1984, 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).
• cassettes of eukaryotic expression carried by replicative plasmids in the bacterium E- £ ⁇ li 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.
• a methylase such as methylase M. SssI or CpG methylases endogenous to mammals
• the promoter pCMV and the gene ln ⁇ 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 Tn ⁇ coding for resistance to kanamycin.
• 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).
• 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.
• IPTG isopropylthio-b-D-galactoside
• 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.
• 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.
• 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.
• methyl plasmid DNA according to the invention retains its capacity to transfect cells, to replicate therein, and to express a gene of interest therein.
• the methylated plasmid ⁇ XL2784 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.
• the plasmid pXL2784 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 ⁇ g / l and aqueous solution of cationic lipid RPR 120535 at 90 ⁇ M; the cationic lipid / DNA ratio is therefore 3 nanomoles cationic lipid / ⁇ g DNA.
• 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.
• Î.IO ⁇ cells [NIH3T3 (mouse fibroblasts) and Hela (human uterine carcinoma)] in exponential growth phase over 2 cm2 (500 ⁇ l of medium without serum / well) are treated with 25 ⁇ l of transfection solution ce which corresponds to the contribution of 0.375 ⁇ g 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).
• the cells are washed with PBS and lysed with a buffer containing 1% Triton X-100 and 2 mM DTT.
• 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%

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• 1996
  • 1996-07-04 FR FR9608327A patent/FR2750704B1/fr not_active Expired - Fee Related
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