CA2266123A1 - Method for preparing a plasmid dna - Google Patents

Abstract

The invention concerns a method for preparing a plasmid DNA using a wet cell biomass comprising after resuspension of said biomass the following steps:, alkaline lysis, high ionic strength acidification, elimination of solubles, endotoxin and contaminating RNA reduction, filtering gel chromatography and conditioning. The invention also concerns a pharmaceutical composition containing a plasmid DNA and its use for gene therapy.

Description

WO 98/11208 PCT ~ FR97 / 01594 PROCESS FOR THE PREPARATION OF PLASMID DNA

The subject of the present invention is a new DNA purification protocol.
plasmid making it possible to produce a high-quality DNA
5 pharmaceutical acceptable for use in humans. It also concerns a pharmaceutical composition comprising the DNA thus obtained and its use for the transfer of a nucleic acid into a host cell. The invention has a whole interest particularly for gene therapy perspectives.

The transfer of genes into a given cell is the very basis of therapy gene. This new technology with a wide field of application allows to consider the treatment of serious illnesses for which therapeutic alternatives classics are not very effective or even inexict ~ nt ~ bones and apply to genetic diseases as well (hemophilia, cystic fibrosis, myopathy ....) that acquired (cancer, syndrome 15 of acquired immunodeficiency AIDS, ...). The most common approach is to use a viral vehicle for introducing the therapeutic nucleic acid into the cell to be treated and, in particular, retroviral and adenoviral. Indeed, viruses have developed mechanisms sophisticated to cross cell membranes, escape degradation at the level Iysosomes and penetrate their genome into the nuclei to ensure expression of the 20 therapeutic gene. However, the viral approach has its limitations, notably an ability restricted cloning, a potential production of competent viral particles for the replication likely to spread in the host organism and the environment, a risk of insertional mutagenesis in the case of retroviral vectors and, as regards adenoviral vectors, induction of immune and inflammatory responses in the host 25 which hinder repetitions of treatment. These significant disadvantages in the context of a human use justify the search for alternative nucleic acid transfer systems.
More and more gene transfer methods are using non-vector vectors ~ viral. One of the most used is to deliver therapeutic nucleic acid by means of synthetic vectors, such as cationic lipids that interact spontaneously with ~ 0 nucleic acid to form positively charged complexes capable of fusing with the anionic cell membranes and penetrate the nucleic acid that they ...... . . .

WO 98/11208 PCTtFR97 / 01594 tMnsportent (see for example Behr, Bioconjugate Chemistry (1994) 5: 382). A method called biolistics ("gene gun") by bombardment of cells with microprojectiles DNA-coated metal was recently used in an AIDS trial (Woff'endin et al., 1996, Proc. Natl. Acad. Sci. USA 93, 2 ~ 89-2894). Finally, an approach 5 even simpler can also be envisaged by direct administration of naked DNA, especially in the context of diseases affecting the muscles by intr ~ muscular injection.
These non-viral methods generally use a plasmid vector carrying the therapeutic gene and the elements necessary for its expression.
The conduct of clinical trials based on non-viral methods requires 10 be able to produce large quantities of pharmaceutical grade plasmid DNA.
Classically used methods are not optimal since they use enzymes of animal origin (Iysozyme, proteinase, ribonuclease ...), known organic solvents for their toxicity (phenol, chlorophorme) and mutagenic compounds (ethidium bromide) likely to contaminate the end product. In addition, their implementation at scale 15 industrial is difficult to achieve.
International applications W095 / 21250 and W096 / 02658 describe processes preparation of plasmid DNA in purified form usable for human tests.
However, it is known that various variables can influence the effectiveness of methods preparations, in particular the plasmid to be purified, its size, the microorganism which produces it, 20 the fermentation conditions and medium. In this context, it is advantageous to be able have a new method for producing large amounts of DNA
pharmaceutical grade plasmid.

We have now found a new method of preparing plasmid DNA
25 co- ~ - involving a succession of simple steps to implement, avoiding the use of products of animal origin, toxic and mutagenic such as those mentioned above and adaptable to scale industrial. DNA is produced in high yield, in substantially pure form and of a quality compatible with human use. Residual contamination by proteins, endotoxins, RNA and genomic DNA of the producing microorganism are 30 particularly weak or not detectable by standard detection techniques. The the following examples also show that this process allows the purification in a way W 0 98111208 PCT ~ R97 / 01594 efficacy of a large plasmid incorporating the dystrophin cDNA intended for treatment of Duchenne muscular dystrophy.
This is why the present invention relates to a process for preparing DNA.
plasmid from a wet cell biomass harvested after fermentation of a 5 producer cell comprising said plasmid DNA, characterized in that it comprises the following steps:
a) Alkaline lysis of the biomass resuspended, hanged, after resuspension of the biomass wet cell b) acidification with high ionic strength, l O c) elimination of insolubles, d) reduction of endotoxins and ribonucleic acids (RNA), e) gel filtration chromatography, and f) conditioning.

For the purposes of the present invention "plasmid DNA" denotes an extrachromosomal cell element formed from a generally circular DNA molecule capable of autonomous replication in a producer cell (the cell in which it is amplified).
The choice of plasmids usable in the procl ~ die of the present invention is vast. They can be of any origin (prokaryotic, eukaryotic) or be formed by the assembly of various elements. Generally, the plasmids are known from one skilled in the art. A large number of them are commercially available but, there is also possible to construct them by genetic manipulation techniques (Maniatis et al., 1989, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). It may be a cloning or expression vector derived by Example of pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or p Poly (Lathe el: al., 1987, Gene ~ 7, 193-201).
Advantageously, a plasmid used in the context of the present invention ~ has the genetic elements that allow it to replicate autonomously in the producer cell and, optionally, in a host cell (cell in 30 which e ~ and therapeutic is sought). Such elements can be formed between other by an origin of replication ensuring the initiation of replication in a bacterium, .. _ ~ .... ... .

WO 98/11208 P ~ 'llrn97 / 01594 yeast, fungus or mammalian cell. It can be isolated from a proca ~ ote (ColE1 ...), a eukaryote (2 ~ or ARS for autonomous replication sequence), a virus (SV40 ori from simian virus 40, oriP from Epstein Barr EBV virus ...) or a bacteriophage (flori ..). Choosing the appropriate origin of replication is within the skill of the art.
5 For example, for a plasmid intended to be produced in the microorganism ~ scherichin coli (I ~ coli), we will retain the origin ColE1. In addition, if you want it to be self-replicating in a mammalian host cell, it will also include a functional origin in a eukaryote for example oriP and could include the gene coding for the EBNA-1 protein of EBV virus necessary for replication from the latter (Lupton and Levine, 1985, Mol. Cell. Biol. 5, 2533-2542, Yates et al., Nature 313, 812-815) Furthermore, a plasmid used in the present invention can furthermore understand a selection gene to select or identify cells transfected (producer cells and / or host cells). We can apply the methods of selection based on the principle of deficient producer cells (by mutations 15 auxotrophic or introduction of a lethal gene) unable to grow in the absence of a plasmid carrying a gene complementing this deficiency ~ for example the dap system described in application EP 0 25 ~ 118, complementation of an auxotrophy mutation, use of genes coding for a suppressor tRNA sup E, supF ...). Another common practice employed consists in integrating into the plasmid a gene coding for resistance to a 20 antibiotic (ampicillin, kanamycin, tetracycline ...). Of course, it can include additional elements improving its maintenance and / or its stability in a host cell or producer. In this regard, we can cite the sequence cer whose presence promotes the maintenance monomer of a plasmid (Summers and Sherrat, 1984, Cell 36, 1097-1103) and some sequences of viral origins (retrovirus LTR, ITR of a virus associated with adenovirus ...) or 25 cells allowing integration into the chromosomes of the host cell. In accordance with the aims pursued by the present invention, a plasmid used in the present invention is intended to transport one or more gene (s) of interest ~ therapeutic in a host cell. In general, the gene of interest can code for an antisense RNA, a messenger RNA which will then be translated into the polypeptide of interest, 30 a ribozyme or an RNA conferring a direct therapeutic benefit (RNA VA of a adenovirus capable of suppressing the immune response, RNA activating synthesis WO 98/11208 PCTAFR97 / OlS94 interferon) (Abbas et al. in Cellular and ~ olecular Immunology, WB, Saunders - Company Harcourt Brace Jovanovich, Inc. p. , '28).
The gene of interest can be isolated by any conventional technique such as cloning, PCR (Polymerase Chain Reaction) or even chemically synthesized. He can be S of genomic type (provided with one or more introns) or complementary DNA (cDNA). The polypeptide of interest can consist of a mature protein, a precursor and, in particular a precursor intended to be secreted and comprising a signal peptide, a truncated protein, a chimeric protein originating from the fusion of original sequences various or a mutated protein with improved biological properties 10 and / or modified.
As examples, we can use a gene of interest selected from those coding for the following polypeptides:

- cytokines or lymphokines (interferons a,, B and r, interleukins and in particular IL-2, IL-6, IL- 10 or IL- 12, tumor necrosis factors (TNF), factors colony stimulators (GM-CSF, C - ('SF, M-CSF ...);
- cellular or nuclear receptors, in particular those recognized by organisms pathogens (virus, bacteria, or parasites) and, preferably, by the HIV virus (Virus Human Immunodeficiency) or their ligands;
20 - proteins involved in a genetic disease (factor VII, factor VIII, factor IX, dystrophin or minidystrophin, insulin, protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), growth hormones (hGH);
- enzymes (urease, renin, thrombin, etc.), - enzyme inhibitors (al-antitrypsin, antithrombin III, protease inhibitors viral ..);
- polypeptides with anti-tumor effect capable of at least partially inhibiting initiation or tumor progression or cancer ~ rs (anti-sense RNA, antibodies, inhibitors acting at the level of cell division or transduction signals, products for expression of suppressor genes for l: moods, for example pS3 or Rb, proteins stimulating the immune system ..);
- proteins of the major histocompatibility complex of classes I or II or proteins ~.

WO 98/11208 PCT ~ R97 / OlS94 regulators acting on the pressure of the corresponding genes;
- polypeptides capable of inhibiting a viral, bacterial or parasitic infection or its development (antigenic polypeptides with immunogenic properties, antigenic epitopes, antibodies, trans-dominant variants capable of inhibiting The action of a native protein by competition ..);
- toxins (thymidine kinase from herpes simplex virus I (TK-HSV-I), ricin, toxin cholera, diphtheria ..) or immunotoxins; and - markers (~ -galactosidase, luciferase ....).

Of course, the gene of interest can be traced under the control of the elements necessary for its expression in the host cell. By "elements necessary for its expression ", we designate all the elements allowing its transcription into RNA and the translation of an mRNA into a polypeptide. Among these the promoter is important particular. It can be derived from any gene (eukaryotic, viral, natural promoter of 15 gene of interest in question ...) or can be artificial. Furthermore, it can be constitutive or adjustable. Alternatively, it can be modified so as to improve the promoter activity, delete a transcription inhibiting region, modify its mode of regulation, introduce a restriction site, .... Mention may be made, by way of examples, of the promoters CMV (Cytomegalovirus), RSV (Rous Sarcoma Virus), of the TK gene of the HSV-I virus, 20 early SV40 virus, adenoviral MLP, or eukaryotic gene promoters Murine or hllm ~ in, al-antitrypsin (liver-specific) PGK (Phospho Glycerate kinase), immunoglobulins (Iymphocyte-specific).
Such elements may also include additional elements such as introns, signal sequence, nuclear localization sequence, terminator sequence of the transcription (polyA), initiation site for IRES or other types of translation, etc.
A plasmid used in the present invention is amplified in a cell producer before being purified according to the process of the present invention. We prefer everything particularly gram negative bacteria and, in particular, ~ coli. As an indication, we can cite the DH5 strains (Grant et al., 1990, Proc. Natl. Acad. Sci. USA 87, 4645-4649), MC1061 (Wertman et al., - 1986, Gene i ~ 9, 253-262) and its derivatives as DHIOB (Grant et al., 1990, sllpra). Since this is a widely known technology to date, WO 98/11208 PCT ~ R97 / 01594 only a brief description will be given of how to operate to introduce a plasmid in a bacteria and amplify it. All conventional techniques can be used in the context of the present invention (treatment with chlorides of calcium, rubidium, hexamine cobalt, by reducing agents, by DMSO, S electroporation, transduction, liposomes, ...; i ~ Ianiatis et al., 19g9, ~ llpra). Cells producers thus transformed are then cultivated according to general art practices (continuous fermentation "batch" or fed "fed batch"). Culture conditions can be easily established by those skilled in the art on the basis of knowledge general in this area and the selection system carried by the plasmid. Their harvest is 10 carried out according to the usual techniques, such as filtration or centrifugation at low speed, to generate the wet cellular biomass which can, at this stage, be frozen or stored at 4 ~ C before being subjected to the process according to the invention.
According to the method of the invention, the lysis of the wet cellular biomass is carried out after resuspending him. Usually a resuspension pad is used 15 slightly basic to neutralize the acidity of the cell paste and strength weak ionic with little or no lytic effects on transformed cells. Her composition and its pH can vary in particular depending on the producer cell, the culture medium used or any other parameter. Those skilled in the art are able develop an appropriate resuspension buffer. An example is a buffer 20 containing EDTA (concentration from I to 50 mM, preferably 10 mM) and Tris-HCI
(concentration from 10 to 100 mM, preferably sal mM) buffered to a pH of about 8. The cells can be resuspended by any usual technical means such as shaking rectilinear, repeated pipetting and / or homogenizer (vortex, homogenizer by shear ...).
The alkaline Ise step releases the cell contents and solubilizes them all components. Proteins, RNA and DNA are denatured including plasmid DNA including the two homologous strands remain tangled, unlike genomic DNA. he can be advantageous to perform the alkaline lysis in the presence of a detergent and, preferably, a anionic surfactant. The choice of base and surfactant is not limited. In this regard, the soda and SDS (sodium dodecylsulfate) combination is preferred, in particular to final concentrations around 0.1 M and C, 5% respectively. The final pH of the WO 98/11208 rCT ~ R97 / 01594 Iyse solution is preferably between 1 1 and 13 and, optimally, between 12.2 and 12.4. It is indicated that it is preferable to mix the transformed cells resuspended and the Iyse solution in a gentle manner, for example by inversion, in order to minimize breaks in the genomic cellular A ~ N which would then be likely to 5 contaminate the plasmid DNA preparation. Although this is not a pretended mode, it is nevertheless possible within the framework of the present invention to facilitate the cell lysis by heating at high temperature (see for example international application W096 / 02658) or the use of animal enzymes degrading cell membranes (Iysozyme...).
The second step of the process according to the invention results in high acidification ionic strength of the Iysat obtained previously. The acidification preferably takes place brutally, that is, all at once. Under these conditions, the plasmid DNA is renatured quickly while the vast majority of proteins, genomic DNA
denatured and insoluble RNA species under high flocculent ionic strength.
In the context of the present invention, a solution is implemented comprising a buffer or a strong acid combines with a salt whose pH is between 4.5 and 6.5. According to a advantageous embodiment, a potassium acetate solution is preferably used, at a final concentration close to IM, so as to obtain a final pH of approximately 5.1.
However, one could also have used a sodium acetate solution of pH and 20 concentration as indicated above.
We then proceed to the elimination ~ tion of insolubles formed by cellular debris and macromolecule flocculates. This can be done by any technique conventional filtration or centrifugation. It may be wise to eliminate the majority insolubles first by centrifugation and then continue clarification by filtration. Of Many filters can be used in the context of the present invention provided that that they retain the insolubles and let through the plasmid DNA. Advantageously, the selected filter will have a porosity between I and 100 ~ lm, more preferably 2 and 75 llm, preferably S and 75 ~ Lm, quite advantageously, 3 and 50 ~ lmlet, so quite preferred, 10 and 50 ~ m. It can be made of synthetic material like nylon ~
Organic like cellulose or inorganic like glass. According to a mode of Wo 98/11208 PCT / FRg7 / 01594 advantageous embodiment, successive filtrations are carried out using porosity filters decreasing, for example a first filtration on sintered glass with a porosity included between 100 and 40 ~ m (N ~ 2 sinter, Schott AG), the second on sinter with porosity 16 to 40 llm (sintered N ~ 3, Schott AG) and the last on sintered porosity 10 to l 6 ~ Lm (sintered N ~ 4, Schott 5 AG) According to another variant, it is possible to carry out a single filtration using a polypropylene cartridge Sartopure PP with a porosity of 8 llm (Ref. 552 1302 P9-00-A) or Sartopure PP2 with a porosity of 3 llm (Ref. 559 1: 302 P9-00-A).
According to an optional but particularly advantageous embodiment, the filtrate 10 can be concentrated before the next endotoxin reduction step. The means of concentrating DNA dissolved in an aqueous solution are known to those skilled in the art. We may include ultrafiltration, alcoholic precipitation or a combination of these two techniques.
With regard to ultrafiltration, various membranes can be used from l As long as they do not or hardly adsorb plasmid DNA under the conditions of use.
We will advantageously use members whose cutoff threshold is understood between 20 and 300 kDa, preferably between 30 and l00 kDa. They can be of compositions varied, organic or not (poly (ether) sulfone, cellulose acetate, ...). Membranes particularly suitable are those of the YM type (and in particular, YM30-76, Diaflo and YM30-4208, Centricon), those fitted to the Easy Flow units (reference 14669-OS-lV or 14669-OS-2V, Sartorius) or minipellicon 2 PL300 (Millipore in cellulose regenerated). Ultrafiltration is a powerful way to reduce the contamination of the plasmid DNA preparation with pigments of cell origin or from the culture medium.
It is also possible to concentrate nucleic acids by precipitation alcoholic in the presence of ethanol or isopropanol. Precipitation parameters such as volume of alcohol to be added, temperature, presence of monovalent cations and the recovery of precipitated material is detailed in numerous works accessible to the skilled person. In particular, precipitation with isopropanol has the advantage of further reduce the pigment content, some of them being soluble in the phase alcoholic.

.

According to a preferred mode, the filtrate is first concentrated by ultrafiltration on polysulfone membrane having a cutoff threshold of approximately 100 kDa using a Easy Flow type (Sartorius) for single use or on a minipellicon 2 PL300 membrane (Millipore) with a cutoff threshold of around 300 kDa. Once the volume is reduced by a factor of 5 to 20, the nucleic acids are precipitated by adding 0.7 volume isopropanol. The precipitated material is recovered by centrifugation and can be subjected to a or several washes in ethanol at a concentration of 70 to 80% in order to reduce the cont ~ min ~ nts soluble in alcohol such as salts and, as already mentioned, pigments residual. After drying, the nucleic acids are taken up in an appropriate buffer, by example of Tris-HCI 10 rnM pH 8 containing EDTA at a concentration of about l mM to inhibit nucleases and optionally sodium acetate (at one final concentration of approximately 0.3 M allowing precipitation of nucleic acids after the Triton extraction step). It is interesting to note here that the ultrafiltration stages and isopropanol precipitation are particularly advantageous for eliminating majority of the pigments in the preparation.
At this stage, the plasmid DNA preparation contains significant amounts of RNAs and endotoxins and the next steps are to reduce their levels.
By reduction is meant a significant decrease in the level of endotoxins or RNA between the start and end of the stage, by a factor of at least 100 and, preferably, at least 1000. The RNA and endotoxin concentration can be assessed by tests similar to those described below or by any other methodology disclosed in the literature. even though can be interchanged, we prefer to act first on the endotoxins and then on RNAs.
Endotoxins, because of their pyrogenic nature, must be considerably reduced or even eliminated before considering administration in humans. In what for standard pharmaceutical products, the maximum tolerable quantity has been set by health authorities at 5 units (EU) per dose. However, it has been shown that the processes plasmid DNA preparation currents (ultracentrifugation on chloride gradient 30 cesium, anion exchange chromatography,) Large quantities remain endotoxins (Cotten et al., 1994, Gene Therapy 1, 239-246).

~ For the purposes of the present invention, it is preferable to use an extraction in presence of a non-ionic detergent having a cloud point between 15 ~ C and 35 ~ C, preferably 18 ~ C and 30 ~ C and preferably 20 ~ C and 25 ~ C. A preferred detergent is 5 chosen from polyoxyethylenes. Examples of detergents usable according to the present invention are described in the following table:

Point Density Detergent Formula Suppl.
cloud (20 ~ C) (~ C) Brij58 45 ~ C Cl6H33 (OCH2CH2) 200H HERE
Americas TritonTMX-I 1422 ~ C 1,054 (CH: 3) 3C-CH2-C (CHi) 2-CGH ~ - Sigma (OC'H2CH2) 7 80H

TergitolTM 37 ~ C 1 009 C, 2E ~ 25 (OCH2CH2) ~ 0H Sigma TergitolTM NP7 41 ~ C 1,048 C9H, ~ -C6H ~ - (OCH2CH2) 7.80H Sigma TergitolTM 40 ~ C 0.995 Cll lsH23 31-O (CH2CH20) ~ Sigma Min-Foam IX ~ CH2CH20 / CH2CH (CH3) 0] y CH2CH (CH3) 0H

TergitolTM 20 ~ C 0.978 C ", 5H23 3, -O (CH2CH20) Y Sigma Min-Foam 2X [CH2CH20 / CH2CH (CH3) 0] y CH2CH (CH3) 0H

....

The compounds described above are amphiphilic compounds whose miscibility in the aqueous phase can be controlled by varying the temperature around from their cloud point. Advantageously, according to the process of the invention, the preparation S DNA is cooled to a temperature below 10 ~ C before adding said detergent. The final concentration of detergent to be used can be between 0.5 and 6%, av ~ nt ~ ellsement between 1 and 5% and, quite preferably, around 1%. In these conditions, said detergent is soluble in water and forms micelles complexing the endotoxins. After incubation and centrifugation of melan ~ e DNA pl ~ cmidique / detergent at a temperature significantly higher than the cloud point (for example> 37 ~ C in the case of TritonT: ~ Xl 14), there is separation of two phases: an aqueous phase containing DNA
plasmid and a phase containing the detergent and endotoxins. When the detergent chosen has a density higher than that of the DNA solution, after separation of the phases by centrifugation, the aqueous phase is located in the upper part of the tube and the phase conterl ~ nt the detergent and endotoxins in the lower part, and vice versa when the detergent has a density lower than that of the DNA solution. For some obvious technical reasons, we will choose ple ~ élence a detergent having a density greater than that of the DNA solution. The skilled person also has the necessary knowledge allowing him to adjust, if necessary, the density of the solution of DNA, for example by modifying the salt concentration of said solution. The process according to the invention can comprise one or more (preferably 3) successive extractions as described above.
According to the present invention, a preferred detergent consists of TritonTM Xl 14 (octoxynol or octylphenoxy-poly (ethylene glycol ether) n with n = 7 or 8) with cloud is about 20 ~ C and the density about 1.06.
The present invention also relates to a variant of the method of the invention that the non-ionic detergent chosen has a cloud point outside the recommended range, and according to which said cloud temperature is a ~ ustated by the addition a small amount of anionic detergent (~ Nonionic Surf ~ ct ~ nts ~, Chapter <~ Surfactant and Detersive Systems)) in Kirk-Othmer Encyclopaedia of Chemistry, John WLEY & Sons, 1995).

WO 98/11208 rCT ~ R97 / 015g4 According to an interesting variant of the pn ~ sold according to the invention, the reduction of endotoxins is followed by an alcoholic precipitation step of plasmid DNA by cold incubation (4 ~ C, -20 ~ C or - ~ 0 ~ C) in the presence of 0.3 M sodium acetate and 5 about 70% ethanol. The nucleic acid precipitate is conventionally recovered by centrifugation. It can be washed with a solution of 80% ethanol in water before being dry and redissolved in an aqueous medium as pam ~ xample of Tris-HCI 10 mM pH 8, EDTA
I mM. This precipitation step, which is also optional, offers an effective means to remove traces of TritonT ~ X-1 14 residual.
Reducing RNA contamination can be achieved by any means known in the art, for example enzymatic hydrolysis using an original ribonuclease animal such as bovine pancreatic ribonuclease A. But, in the context of this invention, it is preferred to use a selective pri ~ cipitation of RNA in high ionic strength conditions or in the presence of dehydrating agents. Various salts can 15 to be used and mention will be made, for example, of lithium chloride (Ze'ev Lev, 1987, Analytical Biochemistry 160, 332-336), calcium chloride, ammonium acetate and ammonium sulfate. As such, ammonium sulfate constitutes an embodiment preferred, particularly at a final concentration of between 1 and 3.5 M, of preferably between 1.5 and 3M and, most preferably, between 2 and 2.5 M. According to a In another variant of the invention, calcium chloride can be used at a concentration final between 10 mM and 2 M, advantageous: ment between 20 mM and 0.5 M, and preferred between 50 mM and 0.1 M. Optimally, after adding the salt or saline solution, the mixture is left with gentle stirring, possibly at temperature low, for a variable duration (1 to 120 min), centrifuged and the plasmid DNA recovered 25 in the supernatant.
The method according to the invention comprises. this stage a chromatography step exclusion on gel filtration supports, which allows to perfect the purification of the preparation of plasmid DNA (reduction of ~ RNs and residual proteins) and also to ensure desalting. The choice of support is wide and within the reach of those skilled in the art. We 30 will more particularly retain the supports approved for human or veterinary use by the competent American authorities (FDA for Food and Drug Administration) and / or .. .. ...

WO 98/11208 PCT ~ R97 / 01594 European Union agencies with a high exclusion limit and, in particular, greater than or equal to 20X106 Da (as measured on polymers such as dextrans).
Mention may be made, for example, of the Sephacryl S500 H: R supports (Pharmacia, reference 17-0613-01), S 1000 SF (Pharmacia, reference 17-0476-01) and GF2000 (Biosepra, reference 260651).
The Sephacryl S500 support is preferred in the context of the invention.
The column is initially balanced under saline conditions limiting the hydrophobic interactions between the support and the DNA. Advantageously, the buffer is used TEN (10 mM Tris-HCI pH 8, I mM EDTA, and 100 mM NaCI). The conditions of chromatography can be adapted according to different parameters and in particular 10 of the volume of the column, of the support chosen, of the concentration of the DNA preparation and the size of the latter. Plasmid DNA is excluded from the phase and is eluted before cont ~ min ~ nts of lower molecular weight. The fractions containing it can be analyzed by usual techniques (absorbance at 254 nm, visual analysis after separation by agarose gel electrophoresis ...). It is also possible to connect 15 the column to a detector fitted with a filter (at 254 nm for example) for online detection positive fractions. It will be noted that an advantage of the process according to the invention consists in elimination during this step of the residual salt from the previous step.
According to an optional embodiment, the fractions obtained after the step chromatography can be collected and concentrated according to the indicated methodology 20 above (ultrafiltration and / or alcoholic precipitation).
Finally, the method according to the invention comprises a step of conditioning the preparation of plasmid DNA. The conditioning pads usable within the framework of the present invention are varied. 11 may be a physiological saline solution (NaCl 0.9%), of a Hepes-Ringer solution, of Lactate-Ringer, of TE (10 mM Tris-HCI pH7.5 at 25 8, EDTA 1 mM) or simply H O. Optionally, the preparation can be subjected to sterilizing filtration. Advantageously, 0.22 filters will be used ~ lm of an area adapted to the volume to be treated. We can cite, for example, the units of Minisart type filtration (Sartorius, reference SM16534), Sartolab P20 (Sartorius, reference 18053D), Millex GF (Millipore, reference SLGS025BS), Millex GV (Millipore, reference SLGV025BS), Millex GP (Millipore, reference SLGPR25LS), Anotop 25 W O98 / 11208 PCT ~ FR97 / 01594 (Whatman, reference 20025H-68092122), Anol: op 25 Plus (Whatman, reference 2002AP-68094122), Sartobran 300 (reference 52313071 ~ 5pO0V) or Easy Flow 0.2, um in acetate cellulose (reference 12307-05-1-V). Then, the fi.ltrat is conditioned in doses adjusted to a given concentration.
S The concentration of plasmid DNA can be determined conventional, for example by spectrophotometry at a wavelength of 260 nm. The relative proportion of the different topoisomers can be assessed visually by agarose gel electrophoresis and ethidium bromide staining possibly followed densitometric analysis. The integrity of the pla ~ smide can be verified by digestion by restriction enzymes having one or more; cleavage sites.
The quality of the plasmid DNA prepared by the process according to the invention can be appreciated by standard tests such as those described in the examples which follow. RNA contamination can be assessed visually by gel electrophoresis of agarose and staining with bromide, ethidium OIJ by spectrophotometry after reaction a Hydrochloric orcinol (Bial reagent) (Moulé, 1953, Arch. Science Physiol. 7, 161;
Mejbaum, 1939, Hope Seyler Z. 25 ~, 117). A purified plasmid I ~ DN according to the process of the invention preferably has a residual RNA contamination of less than 5%
(mass / mass), advantageously less than 3 '~ / o, preferably less than 2% and, most preferred, less than I%.
Residual protein contamination can be measured by any technique assay for proteins showing little or no interference from DNA. A
adequate technique is that of the E3CA technique (bicinchoninic acid) based on the spectrophotometric detection at a wavelength of 562 nm of the colored complex between BCA and Cu + ions resulting from the reduction in an alkaline medium of copper ions Cut + by proteins (Smith et al., 1985, Anal. Biochem. ./50, 76-85). Purified plasmid DNA
according to the process of the present invention pre; ~ erence residual contamination in protein less than 3% (mass / mass), advantageously less than 2%, preferably less than I% and, most preferably, less than 0.5%.
The endotoxin assay techniques are known to those skilled in the art. We can for example proceed with a colorimetric test derived from the LAL method (Limulus Amebocyte Lysate) recommended by the pharmacopoeias of the European Union W O98111208 PCTA ~ R97 / 01594 and from the United States, as used in commercial kits (Bio-Whittaker, QCL-1000, reference L50-647-U; Biogenic, COATEST, reference 82 2387) From preferably, the amount of entoxins in the plasmid DNA preparation is less than 50 EU, advantageously less than 20 EU, preferably less than 10 EU and, so 5 most preferred, less than 5 EU per mg of plasmid.
The contaminating chromosomal DNA can be assayed by the PCR technique competitive quantitative based on the amplification of specific sequences of the producing microorganism, by Southern or by "Slot-blot" using a probe specific. A plasmid DNA purified according to the method of the invention exhibits 10 preferably a residual chromosomal DNA contamination of less than 5% (mass / mass), advantageously less than 3%, preferably less than 2% and, from most preferred, less than I%.
A method according to the invention is particularly advantageous with regard to the preparation of plasmid DNA larger than 10 kb in size.
IS The present invention has ~ g ~ lement for a pharmaceutical composition comprising a plasmid DNA purified by the process according to the invention as a therapeutic agent or prophylactic. A pharmaceutical composition according to the invention can be used in various types of host cells. It is preferably a mammalian cell and, in particular of a human cell. Said cell can be a primary or tumor cell 20 of hematopoietic origin (totipotent stem cell, leukocyte, Iymphocyte, monocyte, macrophage, ...), hepatic, epithelial, fibroblast and, in particular, a cell muscle (myoblast, myocyte, satellite cell, cardiomyocyte, ...), a tracheal cell or pulmonary. Furthermore, a composition according to the invention may comprise an element targeting to a particular cell, for example a ligand to a cellular receptor or 25 another antibody. Such targeting elements are known.
A composition according to the invention can be administered systemically or by aerosol, in particular by intragastric, subcutaneous, inl ~ a ~ drdiac, intramuscular, intravenous, intraperitoneal, intrapulmonary, intranasal or intratracheal.
Administration can take place as a single dose or repeat one or more times after 30 certain interval time. The route of delivery and the appropriate dosage will vary depending on various parameters, for example, the individual or the disease to be treated or the or W 0 98tll208 P ~ l / rh57 / 01594 gene (s) of interest to be transferred. In particular, a pharmaceutical composition according to The invention can be formulated in doses comprising between 0.05 and 100 mg of DNA
plasmid purified according to the method according to the invention, advantageously 0.1 and 10 mg and, preferably 0.5 and 5 mg. The formulation can also include other compounds such as 5 pharmaceutically acceptable adjuvant or excipient.
In this context, it can be particularly advantageous to associate DNA
plasmid to a compound improving its diffusion, in particular a polymer or a lipid cationic. By way of examples, mention will be made of 5-carboxyspermylglycine dioctadecylamide (DOGS), 3, B ([N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol), le (2,3-10 droleylocyl-N- [2 (sperminecarboxamido) ethyl] N, N-dimethyl- I -propanaminium trifluoroacetate) (DOSPA), spermine cholesterol and spermidine cholesterol (described in the request fran, caise 96 01 347).
Furthermore, such a composition may also comprise an adjuvant capable of improving its transfecting power. It will preferably be a neutral lipid such as phosphatidyl 15 ethanolamine, phosphatidyl choline, phosphatidyl serine, phosphatidyl glycerol and, in particular in particular, dioleyl phosphatidyl ethanolamine (DOPE). It is also possible to combine the plasmid DNA / lipid complex with other substances to further improve transfection efficiency or the stability of complexes.
A composition according to the invention is in particular intended for preventive treatment 20 or curative of diseases such as genetic diseases (hemophilia, cystic fibrosis, diabetes or Duchenne and Becker's myopathies, ...), cancers, viral diseases (hepatitis, AIDS, ...), and recurrent diseases (infections caused by the herpes virus, the virus human papilloma, ...).
Finally, the present invention relates to therapeutic or prophylactic use 25 of a pharmaceutical composition according to the invention for the preparation of a medicament intended for the treatment of the human or animal body and, preferably, by gene therapy.
According to a first possibility, the medicament can be administered directly i ~ vivo (by example by intravenous, intramuscular injection, into an accessible tumor ~ in lungs by aerosol, ...). We can also adopt the ex vivo approach which consists of 30 take cells from the patient (bone marrow stem cells, blood lymphocytes peripheral, musc ~ ires cells, ...), to transfect them i ~ vi ~ ro according to the techniques of the art WO 98/11208 rCTA ~ R97 / 01594 and readminister them to the patient.
The invention also extends to a treatment method using a Plasmid DNA obtained by a method according to the invention, according to which a therapeutically effective amount of the latter to a patient in need of such S treatment.

The present invention is more fully described with reference to the figures following:
Figure I illustrates a chromatogram after gel filtration on Sephacryl SS00 (70 ml column, diameter 16 mm and length 350 mm) and loading of a sample of 2 ml containing 5 mg of pCHl ION obtained after alkaline lysis, ultrafiltration and treatment with ammonium sulfate. Elution is carried out at 0.5 ml / min (15 cm / h) in a Tris- buffer.
HCI 10 mM, EDTA I mM, Nacl 100 mM, pH 8.0. Optical dentistry is recorded at 254 nm.
Figure 2 is a schematic representation of the vector pTGI 1025 comprising the gene conferring resistance to kanamycin (kana), the origin of replication of ColEI, the CMV promoter (pCMv) of cytomegalovirus, the intron of the gene coding for Hydroxy-Methylglutaryl-Coenzyme A Reductase (HMG), the cDNA coding for dystrophin and a polyadenylation sequence of transcribed RNAs (pA).
Figure 3 illustrates a chromatogram after gel filtration on Sephacryl S500 (column of S liters of diameter 8.9 cm and length 82 cm) and loading of 125 ml of sample containing 85 mg of pTGI 1025. Elution is carried out at approximately 15 ml / min (14.5 cm / h) in 10 mM Tris-HCl buffer, I mM EDTA, 100 mM NaCl, pH 8.0.
Figure 4 illustrates the gradual elimination of endotoxins from an alkaline Iysate by 3 successive extractions with 1% or 3% of TritonTM X-1 14 followed by precipitation at ethanol Figure S illustrates the selective precipitation of contaminating RNAs from an Iysatalcalin in the presence of increasing molarities of ammonium sulfate (0 to 3.2 M final).
Figure 6 illustrates the selective precipitation of contaminating RNAs from an Iysatalcalin in the presence of increasing molarities of calcium chloride (10 to 100 mM). The final molarity in CaC12 is indicated under the relevant lines. S = deposit of a fraction WO 98/11208 PCT / FRg7 / 01594 of the sample of soluble material obtained after treatment with CaCl2; I. = filing of a fraction of the sample of insoluble material obtained after treatment with CaCl2; oc =
Open circle plasmid DNA; sc = plasmid DNA in supercoiled form, a = RNAs.
The examples (follow it illustrate only one embodiment of the present invention.

EXAMPLES

The solutions defined below were prepared from stock solutions or commercially obtained chemical products.

EXAMPLE 1 Purification of the plasmid pCHl ION from the strain 1 ~ coli MC1061 transformed.
1. Pré ~ aratio ~ 1 e ~ amplificalio ~ l des cellllles r ecombi ~ 7at ~ es The strain ~ coli MCilO61 (Wertman et al., 1986, sllpra) and the plasmid pCHI ION. It is an 8.5 kb plasmid which is maintained in L: coli by an origin of replication (ColEI) and a glob of resistance to ampicillin, both from pBR322. The gene of interest consists of the reporter gene, ~ -galactosidase d'l ~
coli whose expression can be easily detected: by staining with X-Gal (4-chloro-5-bromo-3-indolyl-, BD-galactopyranoside). It is provided in its 3 ′ part with a coding sequence for a eukaryotic nuclear localization signal. The nuclear location of the, B-recombinant galactosidase overcomes the background noise problems caused - by the cross-reaction with the endogenous, ~ -galactosidase of the host cell also detectable by Xgal, and therefore to ensure a specific detection of the enzymatic activity resulting from the transfected plasmid. Expression of the reporter gene is driven by the promoter SV40 early.

W 0 98/11208 PCTA ~ R97 / 01594 MC1061 cells are made competent by treatment with chloride of calcium and transformed by the plasmid pCHllON. Recombinant bacteria are selected in a selective environment. A clone is chosen by examining the restriction profiles at from which a primary glycerol stock is built up.
After inoculation of a preculture in a vial, this is used to seed a fermenter. The fermentation was carried out continuously (batch) in 18 liters of LB medium

2 times concentrated (LB2x) without addition of carbon substrate, at 37 ~ C and in the presence of ampicillin (100 lag / ml). The culture is collected after 2 hours in the stationary phase. In these conditions and for a final OD600 optical density of 7.5, 180 g of biomass are obtained total.

2. Harvesting of hllmid cell biomass The contents of the fermenter are distributed in clean centrifuge pots and sterile (Nalgene, ref 3122- 1000, 3122-1010, 3120- 1000 or 3120-1010) and cells transformed recovered by low speed centrifugation (5000 rpm (revolutions per min) for 30 min) and at 4 ~ C. You can use a Sorvall RC3 centrifuge with a H6000-A rotor with a capacity of 6x 1 liter and carry out three successive centrifugations in the same pots to harvest the entire crop. After elimination of the medium, the weight of cell pellets is estimated by weighing and these can be frozen at -20 ~ C
before being treated by the process details below. The cells thus collected constitute wet cell biomass.

3. Preparaliotl dll Iysal acidified The frozen pellet is fragmented and the amount of biomass that we want to process. Thereafter, the volumes of the solutions used are given for 27 g of wet biomass. The cells are taken up in 320 ml of buffer.
resuspension (10 rnM EDTA, 50 mM Tris-HCI, pH 8) previously balanced at 4 ~ C and 0 rernises in suspension using a shear homogenizer (Ultra Turrax-25 with WO 98/11208 PCTA ~ R97 / 01594 of a probe with a diameter of 18 mm) before being lyzed in the presence of 320 ml of buffer Iyse (I% SDS, 0.2 M NaOH) balanced at 20 ~ C ,. Let the Iyse continue S min at room temperature, gently shaking the preparation by inversion, then add 320 ml of acid solution (CH, COOK 3M pH 5.5) l balanced at 4 ~ C. The acidified cell iysate 5 is left 20 min at 0 ~ C by regularly carrying out gentle stirring by inversion Le final pH is 5.1.

. ~. Elinti ~ a ~ io ~ d ~ s io ~ olllbl ~ * ~ co ~ 7c ~ ratio ~ fil ~ ral The flocculate is first coarsely removed by low speed centrifugation (5000 rpm for 30 min at 4 ~ C in a GSf ~ rotor, Sorvall). The supernatant is subjected to two successive filtrations on frits of controlled porosity (16 to 40 μm then 10 to 16 μm;
sintered N ~ 3 and 4; Schott AG) using a vacuum flask connected to a water pump or a equivalent vacuum source.
The filtrate is subjected to a concentration step by ultrafiltration on a cartridge Easy Flow (Sartorius) with a dl membrane. polysulfone with a cutoff threshold of 100 kDa (Sartorius, 0.1 m reference 14669-OS ~ or 0.2 m ~ reference 14669-OS-2-V according to the volume to be treated). ~ a cartridge is connected to a peristatic pump and the flow of recirculation applied is approximately 400 ml / min. Ultrafiltration is carried out until 20 the final volume is reduced by a factor of 8 to 16. We work at room temperature in order to reduce the duration of the operation.
The nucleic acids contained in the filtrate are precipitated by adding 0.7 volume isopropanol maintained at 20 ~ C. The mixture is homogenized by successive reversals, incubated for 5 min at room temperature and the precipitate collected by centrifugation at 10,000 rpm 25 for 30 min at 4 ~ C (GSA rotor, Sorvall). f ~ near elimination of the supernatant, the pellet of nucleic acids is washed twice in succession with 50 ml of an 80% ethanol solution in water (balanced at around -20 ~ C) and again recovered by centrifugation at 10,000 rpm for 15 min at 4 ~ C.

CA 02266l23 l999-03-09 WO 98tll208 PCTA ~ R97 / 01594 Lx ~ r ~ lc ~ io ~ l ~ se ~ lotoxi ~ es The pellet is dried and dissolved in 18 ml of a sodium acetate solution (CH3COONa 0.3 M in 10 mM Tris-HCI, EDTA I mM) and preserved about thirty S minutes at 0 ~ C. 2 ml of a solution of TritonT ~ Xl 14 (Sigma; reference Xl 14TM) are added at 10% (weight / volume) in 0.3 M CH3COONa at pH 5.5 (final concentration of Triton 1%) and the mixture is homogenized by manual stirring. After 10 min incubation on ice then 25 min at 52 ~ C, the lower phase obtained after centrifugation (SLA 1500 rotor, Sorvall) at 10,000 rpm for 10 min at 35 ~ C is removed and eliminated. We do two 10 additional extractions under the same conditions in the presence of 2.2 ml and 2.4 ml of Triton T: V'XI 14 respectively. The upper phase is precipitated by adding 2.5 volumes absolute ethanol at -20 ~ C. After incubation at least 45 min at -20 ~ C, the precipitate is recovered by centrifugation at 10,000 rpm (rotor SLA 1500, Sorvall) for 30 min and at

4 ~ C and subjected to I or 2 successive washes with a solution of 80% ethanol in water 15 stored at -20 ~ C. The centrifugation base can be frozen before proceeding to next step.

~ limi ~ 1atio ~ of RNAs The centrifuge pellet is dried under vacuum and taken up in 9.4 ml of Tris-HCl 10 mM, EDTA I mM, pH 8 at room temperature. Add ammonium sulfate (NH4), solid SO4 so as to have a final concentration of approximately 2 M. After mixing by inversion and incubation 20 min on ice, centrifuging for 30 min at 7,000 rpm (SLA rotor 1500, Sorvall) and at 4 ~ C. A second centrifugation under identical conditions is e ~ carried out on the supernatant recovered from the first in order to perfect the elimination of insoluble.

Chromcltogrclphie sl ~ r Séphcrcryl S500 The centrifuge supernatant is carefully removed and subjected to WO 98/11208 P ~ h57 / 01594 gel filtration chromatography on Sephacryl S500 matrix (Pharmacia; reference 17-0613-01). Under the above conditions, a column having a capacity of I liter is used (length 622 mm, diameter 44 mm) developed at 3.8 mUmin (15crn / h) but, of course a person skilled in the art is able to adapt the capacity of the column as a function of the volume to treat.
The column is balanced in 2 volumes of TEN before applying a volume of er.h ~ ntillon of nucleic acids representing 4 to 5% of the volume thereof. Collecting and Fraction detection is automated (L ~ CB 2212 fraction collector and detector LKB 2158 Uvicord SD fitted with a 254 nm filter). The 3 min fractions are taken and frozen before being analyzed. Figure I illustrates a chromatogram obtained at more small scale but representative of the process. Plasmid DNA comes out in volume exclusion while RNA and proteins are retained and only appear later.
We will note the clear separation, with a return practically to the baseline, obtained between the peak of plasmid DNA eluted first and the peak of RNAs eluted second.

Coodi ~ io ~ eme ~ t The fractions containing the plasmid DNA are combined and concentrated approximately 8 × using a Sartocon-Micro type ultrafiltration unit with a polysulfone membrane.
having a cutoff threshold of 100 kDa (Sartorius, reference 15669-00-1). Alternately, and when a larger amount of sample needs to be processed, a EasyFlow ultrafiltration unit with cellulose acetate membrane (20 kDa threshold cut, Sartorius, reference 1 4549-0S-IV).
After concentration, the sample of purified plasmid DNA is precipitated by adding a solution of Na acetate (3M, pH 5.5) to the final concentration of 0.3 M and addition of 2.5 volumes of pure ethanol (99.95 ~ / 'o) at -20 ~ C. After incubation at -20 ~ C (30 min), the plasmid DNA is recovered by centrifugation at 10,000 rpm for 30 min at 4 ~ C
(SLA 1500 rotor, Sorvall). The pellet is washed with 80% ethanol at around -20 ~ C, then dried and taken up in the appropriate conditioning buffer (TE, 0.9% NaCI, Hepes Ringer, Lactate Ringer, H2O; about 20 ml per aliquol: e of 2 ~ g of treated cells).

WO 98111208 PCTA ~ R97 / 01594 After measuring the optical density at 260 nm, the DNA concentration is calculated taking as a base an OD260 corresponding to 50 μg / ml. It can then be adjusted to 1.0 mg / ml by dilution with the conditioning buffer, and we typically obtain approximately 20 mg of plasmid DNA per aliquot of 27 g of initial biomass.

EXAMPLE 2 Purification of the plasmid pTGI 1025 from the L ~ strain: c ~> li DHIOB transformed.

The 1 ~ Coli DHIOB Electro Max strain is supplied by Gibco BRL (Reference 18290-015). The 18.7 kb plasmid pTGI 1025 (Figure 2) carries a marker gene which gives bacteria the capacity to resist kanamycin (gene coding for aminoglycoside 3 'phosphotransferase transforming the antibiotic into an inactive derivative) and the origin of ColEl replication, these two elements ensuring the maintenance of the plasmid in the 15 producing strain. It also includes a cDNA expression cassette coding for dystrophin under the control of the CMV promoter associated with the HMG intron.
Competent DHIOB cells are transformed by the plasmid in conditions recommended by the supplier and a primary glycerol stock is stored at -80 ~ C after selection of a recombinant clone. One batch of primary seed is used to 20 constitute a preculture in a flask on LB2x medium in the presence of kanamycin SO! Lg / ml. The culture is incubated at 30 ~ C in a thermostatically controlled shaker (180 rpm) for 14 to 16 hours.
After transferring the preculture into a seeding flask, the transformed strain is propagated in a 20 liter fermenter. Growth is carried out at 30 ~ C in a medium complex culture (Hycase SF 37.5g / 1, Yeast Extract 9g / 1 supplemented with 25 growth and mineral salts) using glycerol as carbonaceous substrate (20 g / l) and presence of kanamycin (50! lg / ml) for the selection pressure. The pH = 7.0 is reduced by automatic addition of soda (NaOH, 30%) and sulfuric acid (H2SO ~, 2 M). Oxygen dissolved is maintained at a saturation greater than or equal to 25% (aeration rate of Iv.vm (10 I min ~ ') and a variable stirring speed. It may be beneficial to add 30 kanamycin during culture.

WO 98/11208 rCT ~ R97 / 01594 The culture is stopped by cooling to 4 ~ C when the bacteria reach the ~ stationary growth phase. The crop is racked and the biomass harvested by centrifugation (Sorvall RC3B centrifuge, I; min, 4 ~ C, 5000 rpm). The cell pellet is stored at -20 ~ C until the implementation of the plasmid purification process pTG 11025.
This is similar to the process described in. Example 1, with the exception of the modifications following:
- The purification is carried out from 360 g of wet biomass taken up in 3840 ml of resuspension buffer and lysed with an equivalent volume of Iyse buffer then acid solution.
- The insolubles are eliminated by filtration on Sintered N ~ 4 (16-10 llm, Schott AG) before concentration 15 times on Easy Flow.
- The filtrate is separated into 6 aliquots. Isopropanol precipitation, washes with 80% ethanol, the endotoxin extraction steps with TritonTM X-114, ethanol precipitation and subsequent ethanol 80 ~ vages are made on each of the aliquots c, as described in Example 1.
- The samples are pooled for precipitation with ammonium sulfate at a final molarity of 2 M.
- The centrifugation supernatant collected after the sulfate precipitation step amrnonium is loaded on a coloMe of Sephacryl S500 filtration gel of volume of 5 liters (length 82 cm, diameter 8.9 cm) developed at a flow rate of 15 ml / min (14.5 cm / h). The chromatogram obtained (Figure 3) shows that the plasmid DNA is eluted in the exclusion volume, while the Ai ~ Ns cont ~ min: ~ nts and other compounds of small molecular weights are retained on the coionne.
- The fractions containing the purified plasmid DNA are pooled and concentrated by ultrafiltration (factor 10.9). The plasmid DNA is then precipitated by adding sodiium acetate to a final concentration of 0.3 M and 2.5 volumes of 99.95% ethanol a-20 ~ C.
- The precipitate is collected by centrifugation (10,000 rpm at 4 ~ C, rotor SLA 1500 WO 98/11208 PCT ~ R97 / 01594 Sorvall, 30 min) and washed with 200 ml of 80% ethanol.
- After drying under vacuum, the DNA is taken up in the conditioning buffer TE (10 mM Tris-HCI, I mM EDTA, pH 7.5), its concentration measured by UV spectrometry and adjusted to I mg / ml in the same buffer. We obtain S typically 145 mg of purified plasmid DNA.

The integrity of the plasmid is evaluated by restriction enzyme mapping and the profiles obtained correspond to what is expected. Furthermore, the presence of contaminants is also determined in the final preparation and the results presented below.

Contaminant Method Result ~ t 0.49% BCA protein (n = 3) RNA Bial colorimetric reaction 2.48% (n = 3) Endotoxin Colorimetric method LAL 2.34 UElmg (n = 5) Furthermore, the functionality of the purified plasmid pTG11025 is verified by transfection of cell lines and demonstration of recombinant dystrophin by immunofluorescence.

Ten llg of purified plasmid DNA are combined with 40 ~ g of Lipofectin (one mixture of compounds facilitating the transfection of eukaryotic cells) according to the instructions from the supplier (Life Technologies, Bethesda, USA, reference 18292-0011), then are added, in 2 ml of DMEM medium (Life Technologies, Bethesda, USA, reference 11963), 2.5 x 10 6 A549 cells (human pulmonary adenocarcinoma) seeded ia watch in 35 mm diameter dish, cultivated 24 hours in the presence of DMEM medium supplemented with 10% (vol / vol) of fetal calf serum (Life Technologies ~ Bethesda, USA, reference 10101-061) and previously rinsed with a physiological buffer (PBS) WO 98 / lltO8 PCTIFRg7 / OlS94 Ix). Four hours after addition of the DNA, the medium is supplemented with 10% (voVvol) of fetal calf serum, then the culture is continued for 48 hours. The cells are then fixed by treatment at -20 ~ C in a methanol / acetone mixture (1/1) (vol / vol), dried at air, and incubated in the presence first of a mouse anti-dystrophin monoclonal antibody S (Novocastra, Newcastle / Tyrne, UK, reference NCL-DYS2) then an anti-rabbit antibody mouse (ICN, Costa Mesa, USA, reference 651713) coupled to FITC (thio- fluorescein isocyanate); the detailed conditions of these operations are known to those skilled in the art. The dystrophin produced during the expression of the gene encoded by the vector pTGI 1025 is detected by fluorescence microscopy examination of immune complexes. Simultaneous examination of 10 cells transfected in the presence of a pTGI 1025 plasmid already functional check demonstrated purified according to a standard protocol (~, ~ Ianiatis et al., 1989, sll ~ ra) (positive control) or in the absence of DNA (negative control) makes it possible to evaluate the functional character of the preparation of plasmid DNA pTGI 1025. The level of fluorescent cells expressing the recombinant dystrophin is comparable after transfection with pTGI 1025 control and IS by the plasmid purified by the method according to the invention. Of course, cells not transfected show no fluorescence.

EXAMPLE 3 Elimination of endotoxins by TritonT ~ 'Xl 14.
~ 0 Triton TM X-114 extraction trials were carried out on an alkaline Iysate such as that obtained in Example I in order to determine the optimal concentrations of TritonT '' X- 1 14 to be used and the number of extractions to be carried out to reduce the level of endotoxins below the tolerated threshold (<2 EU / dose).
To do this, the initial alkaline Iysate is divided into 2 lots subjected to 3 extractions consecutive in the presence of TritonTM X-114 at a final concentration of I and 3%
respectively. The mixture is homogenized, incubated on ice (0 ~ C) for a few minutes, centrifuged for 5 minutes (12,000 rpm, iEppendorf centrifuge, reference 5414) at temperature 30 ambient After centrifugation, the lower phase (Tritonr 'Xl 14 and extracted endotoxins) WO 98/11208 PCTA ~ R97 / 01594 of each batch is eliminated, and the upper aqueous phase (Extr. I; nucleic acids and remaining endotoxins) treated again as described by I% or 3% of Triton '' ~ 114 after removal of an aliquot used for the dosage of endotoxins (Extr. 2 and 3). After the third extraction, the upper phase is precipitated by adding 2.5 volumes of ethanol

5 absolute. The precipitate is taken up in 10 mM Tris-HCl, EDTA ImM, pH 8 (Final).
The level of endotoxins is measured by a colorimetric test derived from the method LAL, using the Biogenic COATEST kit (reference 822387) and the results presented in Figure 4. There is a significant reduction in the endotoxin level after two extractions in the presence of Triton 1%. A similar result is obtained when 3% of Triton'M Xl 14 10 are used. In both cases, the endotoxin level measured in the final product is compatible with pharmaceutical use for an average dose of I mo of DNA
plasmid.

IS EXAMPLE 4: Selective precipitation of contaminating RNAs.

Selective precipitation tests were carried out on an alkaline Iysate as obtained in Example I in order to determine the optimum ammonium sulfate concentrations for precipitate contaminating RNAs. To do this, the alkaline Iysate is divided into 7 aliquots 20 subjected to precipitation in the presence of increasing quantity of ammonium sulphate (0.5M, 1M, 1.5M, 2M, 2.5M, 3Mand3.2Mfinal).
The precipitated material recovered by centrifugation and the soluble material are analyzed by 0.4% agarose gel electrophoresis. After staining with ethidium bromide and revealed by UV fluorescence, the plasmid DNA appears in the form of clear bands 25 corresponding to the different topoisomers. On the contrary, the RNAs form a very band diffuse migrating in the lower part of the gel.

The results illustrated in Figure 5 show that beyond a final concentration 1.5 M ammonium sulfate, the vast majority of RNAs are found selectively 30 precipitated while the plasmid DNA remains soluble. Note also the elimination in the precipitate of large nucleic acid species retained in the gel pockets WO 98/11208 PCTA ~ R97 / 01594 analysis.

EXAMPLE 5 Selective precipitation of RNAs contaminating with chloride of calcium.
s The selective precipitation of RNAs was also tested in the presence of chloride.
calcium (CaC12). An alkaline Iysate prepared according to the method described in Example 1, including the TritonTU Xl 14 treatment step, was aliquoted into identical samples to which different volumes of a concentrated solution of ~ CaC12 (IM in this specific case, but a less concentrated solution, 0.5M for example, can be used) are added to to obtain a final calcium chloride concentration of 10, 20, 30, 50, 75 or 100 mM, respectively. The soluble and insoluble fractions are separated by centrifugation, and the insoluble pellet is resuspended in a low ionic strength buffer in the absence of CaC12. The soluble and insoluble samples are analyzed by gel electrophoresis.
of agarose and staining with ethidium bromide of a sample fraction in order to separate plasmid DNA (open circular and overwound forms) and contaminating RNAs.

The results (Figure 6) show that above a concentration of 30 mM in CaC12 a detectable precipitation of RNAs is observed. This precipitation becomes significant then massive beyond 50 and 75 m ~ \ / l respectively, then almost total at 100 mM. Plasmid DNA in its open circulatory forms (Open-Circle, least migrating quickly) and supercoiled (Super-Coiled, rnigrant more quickly) remains for its part mostly soluble in each case.

EXAMPLE 6 Elimination of colored pigments.

Effectiveness of elimination of colored pigments present after the Iyse stage alkaline (cf. Examples 1, 2 and 5) was evaluated during the concentration steps by ultrafiltration, precipitation with isopropanol and washing with 80% ethanol.

WO 98/11208 PCTA ~ R97 / 01594 The presence of these pigments results in a yellow coloration of the raw alkaline Iysate before its concentration by ultrafiltration which can be analyzed by measuring the density optical at 340 nm. This absorbance was therefore measured on the samples obtained after the stages of initial ultrafiltration, precipitation and lava, ~, e by isopropanol and by 5 80% ethanol. Two series of experiments were carried out, one involving filtration alkaline lysate on glass frit, the other on Sartopure PP polypropylene cartridge. The Initial Iysates are obtained from 150 g of cellular biomass from two couple 1 ferments: coli strain DH5 x plasmid pTG11025 (~ 19 kbp).

Experiment N ~ 1: Macrofiltration carried out on glass frit N ~ 3 (16 to 40 ~ m of porosity - Schott AG

Stage Final volume Density Yield Factor (ml) Cumulative optics in elimination at 340 nm pi cumulated items (X) Lysatbrutinitial 5340ml 0.29 100% IX

Post macrofiltration 5260 ml 0.28 95.1% 1.1 X

Post concentration per 795 ml 0 38 19.5% 5.1 X
ultrafiltration (Easy-Flow 300kDa, Sartorius) Post precipitation 432 ml 0 39 10.1 ~~ / ~ 9.2 X
alcoholic W O98 / 11208 P ~ llrh97lol594 Experiment N ~ 2: Macrofiltration carried out on SartopurePP2 cartridge (8 mm porosity- Sartorius) Stage Final volume Density Yield Factor (ml) Cumulative optics in elimination 340 nm cumulative pigments (X) Initial crude lysate 5200 ml 0;; 2 100% 1 X

Post macrofiltration 5000 ml 0.31 93.1% 1.1 X

Post concentration per 790 ml 0.57 27.0% 3.7 X
ultrafiltration (Easy-Flow 300kDa, Sartorius) Post precipitation 432 ml 0.62 16.1% 6.2 X
alcoholic These results show that the ultrafiltration and precipitation steps at isopropanol contributes to the removal of pigments from a 4 to 5 x and 1.5 to 2 x tactor, respectively. Combined, these two stages make it possible to achieve an elimination factor from 6 to 10 X, ie a total elimination of 80 to 90% of the colored pigments.
EXAMPLE 7 Purification of the plasmid pTGI 1025 from the r coli DH5 strain Library Efficiency.

The L ~ strain: coli DH5 Library Eff ~ ciency (Life Technologies; ref. 18262-014) is transformed with the plasmid pTG11025 (see example 2 and FIG. 2). The stages of WO 98/11208 PCTA ~ R97 / 01594 processing, preparation and harvesting of biomass are identical to those described in example 2.
The raw Iysat is filtered on a Sartopure PP2 filter cartridge (Sartorius ~ ref:
5591302P9--0) at a flow rate of 700 ml / min using a peristaltic pump. The filtrate is then concentrated by ultrafiltration on a Millipore Minipellicon 11 membrane in regenerated cellulose type PL300 with a surface area of 0.1 I m2 (ref. P2C300C01) (flow rate recirculation ~ 400 ml / min., withdrawal rate between 60 and 30 ml / min. so that maintain an average transmembrane pressure less than or equal to 0 ~ 5.10S Pa).
The nucleic acids thus concentrated (815 ml) are treated with isopropanol and Ethanol as previously described in Example 2. The endotoxins are eliminated by three successive extractions with Tritonr ~ 'X-114. The nucleic acids are precipitated. The residual nonionic detergent and salts are rinsed off with aqueous ethanol.
The nucleic acids are taken up in a 10 mM Tris buffer, EDTA I rnM, pH 7.5 and the RNAs are specifically precipitated in the presence of final 100 mM CaCl 2. These RNAs are removed by centrifugation (10,000 rpm, Sorvall rotor, SLA1500, temperature 20 ~ C, 40 min.). The plasmid DNA present in the soluble phase is precipitated by addition isopropanol and resolubilized in a 10 mM Tris buffer, 1 mM EDTA, 250 rnM NaCl, pH 8.
An aliquot of the sample corresponding to 180 g of the initial biomass is deposited on a column of Sephacryl S500 (volume of 6 I, length 92 cm, diameter 8.9 cm, flow rate 5 ml / min or ~ 5 cm / hour). The absorbance of the eluate is monitored at 254 nm. Fractions containing the plasmid DNA are harvested just after elution of the dead volume of the column (~ 2100 ml), while those containing the RNAs are harvested after approximately 2 dead volumes (~ 4100 ml).
The fractions containing the DNA are then pooled (volume 1480 ml). DNA
is concentrated by ultrafiltration on an EasyFlow membrane unit in cellulose triacetate with cutoff threshold 20 kDa (Sartorius, ref. 14549-OS-IV). After precipitation with ethanol in the presence of sodium acetate, the plasmid DNA is washed with 80% ethanol and dried under vacuum then taken up in a Tris conditioning buffer 10 mM, 1 mM EDTA, pH 7.5 and stored frozen at -20 ~ C.

WO 98/11208 rCT ~ R97 / 01594 In order to analyze the quality of this DNA, the following parameters were measured:

Initial biomass: 360 g (wet weight) of pTGI 1025 / DHSLE
approximately 1.1 mg DNA / OD 600 ml) Final quantity: 120 mg (post Ul ~: final re-filtration) Packaging: I mg / ml in 10 mM Tris, I mM EDTA, pH 7.5 Contaminating proteins: <0.20% (n = l) (*) Contaminating RNAs: <1 0% (n = l) (*) Endotoxins: 3.7 EU / mg DNA
Functionality: Active in transfecl: ion i ~ 1 vilro Structure: Compliant by restriction analysis Yield: ~ 30%
(*) values ~ at the limits of quantification of dosages.

Claims (31)

1. Method for preparing plasmid DNA from wet cell biomass harvested after fermentation of a producer cell comprising said DNA
plasmid, characterized in that it comprises the following steps:
a) alkaline lysis of the resuspended biomass, after resuspension of the biomass wet cell, b) acidification at high ionic strength, c) removal of insolubles, d) reduction of endotoxins and ribonucleic acids (RNA), e) gel filtration chromatography, and f) packaging. 2. Method according to claim 1, characterized in that the alkaline lysis step is carried out in the presence of sodium hydroxide and sodium dodecyl sulphate (SDS). 3. Method according to claim 1 or 2, characterized in that the acidification step is carried out in the presence of potassium acetate at a final pH of about 5.1. 4. Method according to one of claims 1 to 3, characterized in that the step of eliminating insolubles comprises at least one macrofiltration step. 5. Method according to claim 4, characterized in that it comprises two to three steps successive macrofiltration on filters with decreasing porosities of less than 100 µm, 40 µm and/or 16 µm. 6. Method according to claim 4, characterized in that it comprises a single step of macrofiltration carried out on a cartridge with a porosity equal to 8 µm or 3 µm. 7. Method according to one of claims 1 to 6, characterized in that the reduction step endotoxins comprises at least one extraction step in the presence of a detergent having a cloud point of between 15°C and 35°C, advantageously 18°C and 30°C and, preferably 20°C and 25°C. 8. Method according to claim 7, characterized in that the step of reducing the endotoxins comprises 1 to 3 successive extractions carried out in the presence of 1 to 3%
end of said detergent. 9. Method according to claim 7 or 8, characterized in that the detergent is chosen from polyoxyethylenes. 10. Method according to claim 9, characterized in that the detergent is Triton TM
X-114. 11. Method according to one of claims 7 to 10, characterized in that the reduction step endotoxins is followed by an alcoholic precipitation step. 12. Method according to one of claims 1 to 11, characterized in that the reduction step of RNAs includes selective precipitation of RNAs under conditions of high ionic strength. 13. Process according to claim 12, characterized in that the selective precipitation is carried out in the presence of ammonium sulphate at a final concentration of between 2 and 2.5 m. 14. Process according to claim 12, characterized in that the selective precipitation is carried out in the presence of calcium chloride at a final concentration of 10 mM and 2M, advantageously between 20 mM and 0.5 M, and more preferably between 50 mM and 0.1 M. 15. Method according to one of claims 1 to 14, characterized in that the step of gel filtration chromatography is performed on a support having a limit exclusion greater than or equal to 20X10 6 Da. 16. Method according to claim 15, characterized in that the support is selected from Sephacryl S500, Sephacryl S1000 and GF2000 supports. 17. Method according to one of claims 1 to 16, characterized in that the packaging includes a sterilizing filtration step. 18. Method according to one of claims 1 to 17, characterized in that it further comprises between steps c) and d), d) and e) and/or e) and f), a concentration step followed by optionally, by a step of alcoholic precipitation and resuspension in phase watery. 19. Process according to claim 18, characterized in that the concentration step is carried out by ultrafiltration on a membrane having a cut-off threshold between 20 and 300 kDa. 20. Process according to claim 19, characterized in that the concentration step is carried out by ultrafiltration on a membrane having a cut-off threshold between 30 and 100 kDa. 21. Method according to one of claims 1 to 20, characterized in that the plasmid DNA
comprised in said producer cell is a recombinant plasmid DNA
comprising a gene selected from genes encoding a cytokine, a receptor cellular or nuclear, a ligand, a coagulation factor, the CFTR protein, insulin, dystrophin, a growth hormone, an enzyme, an enzyme inhibitor, a polypeptide with antitumor effect, a polypeptide capable of inhibiting an infection bacterial, parasitic or viral and, in particular to HIV, an antibody, a toxin, a immunotoxin and a marker. 22. Method according to one of claims 1 to 21 for the preparation of a plasmid DNA
larger than 10 kb. 23. Method according to one of claims 1 to 22, characterized in that the biomass wet cell is harvested after fermentation of a strain of Escherichia coli comprising recombinant plasmid DNA. 24. Method according to claim 23, characterized in that the strain of Escherichia coli is selected from strains DH5, DH10B and MC1061. 25. Method according to one of claims 1 to 24, characterized in that it comprises the steps following:
a) resuspension of the wet cell biomass, b) alkaline lysis of the resuspended biomass obtained in step a) in the presence of sodium hydroxide and SDS, c) acidification of the alkaline lysate obtained in step b) in the presence of potassium acetate at a final pH of about 5.1, d) removal of insolubles by macrofiltration of the acidified lysate obtained in step c), e) concentration of the filtrate obtained in step d) by ultrafiltration on a membrane having a cut-off threshold between 20 and 300 kDa, preferably between 30 and 100 kDa, and alcoholic precipitation followed by resuspension of the precipitate in medium aqueous, f) reduction of the endotoxins of the precipitate resuspended in step e) by extraction in presence of Triton TM X-114 followed by an alcoholic precipitation and a resuspension of the precipitate in an aqueous medium, g) reduction of the RNAs of the resuspended precipitate obtained in step f) by precipitation selective in the presence of ammonium sulphate or calcium chloride, h) gel filtration chromatography of the supernatant obtained in step g) on a support Sephacryl S500, i) concentration of the fractions containing said plasmid DNA obtained in step h) by ultrafiltration on a membrane having a cut-off threshold between 20 and 300 kDa, preferably between 30 and 100 kDa, and alcoholic precipitation, and j) conditioning by resuspension of the precipitate obtained in step i) in a buffer pharmaceutically acceptable followed by sterilizing filtration and of a dose distribution. 26. Pharmaceutical composition comprising a plasmid DNA purified by the method according to one of claims 1 to 25. 27. A pharmaceutical composition according to claim 26, further comprising a lipid compound. 28. Pharmaceutical composition according to claim 27, characterized in that the lipid compound is selected from 5-carboxyspermylglycine dioctadecylamide (DOGS), 3.beta.([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), (2,3-droleylocyl-N-[2(sperminecarboxamido)ethyl] N,N-dimethyl-1-propanaminium trifluoroacetate) (DOSPA), spermine cholesterol and spermidine cholesterol. 29. Pharmaceutical composition according to one of claims 26 to 28, comprising in in addition to an adjuvant capable of improving the transfecting power of said composition pharmaceutical. 30. Pharmaceutical composition according to claim 29, characterized in that said adjuvant is a neutral lipid and, in particular, dioleyl phosphatidyl ethanolamine (DOPE). 31. Use of a pharmaceutical composition according to one of claims 26 to 30, for the preparation of a medicament intended for the treatment of the human or animal body by gene therapy.