FR2765241A1 - Combination of nucleic acid and electric field - Google Patents

Abstract

A combination of a nucleic acid and an electric field with an intensity of 1-800 V/cm for simultaneous, separate or sequential administration to a tissue in vivo and for gene therapy based on the electrotransfection of tissues in vivo, is new. PREFERRED ELECTRIC FIELD - The electric field intensity is 4-400 (preferably 30-300) V/cm. It is applied for a total of more than 10 msec in several regular pulses, preferably 1-100,000 pulses at a frequency of 0.1-10,000 Hz, or it is applied in irregular pulses and the function describing the intensity of the field dependent on time of a pulse is variable. The integral of the function describing the variation of the electric field with time is > 1 (preferably \- 5) kV.msec/cm. PREFERRED ELECTRIC PULSES - The electric pulses are in the form of square waves, exponential decay waves, oscillating unipolar waves of limited duration, oscillating bipolar waves of limited duration, or other wave forms. PREFERRED NUCLEIC ACID - The nucleic acid is administered together with at least some of the components of the organism of origin or the synthesis system. The nucleic acid encodes an RNA (catalytic or antisense) or a protein of interest.

Description

 The present invention relates to a very remarkable improvement in the transfer in vivo of nucleic acids into the cells of multicellular eukaryotic organisms or of nucleic acids associated with products making it possible to increase the yield of such transfers by using weak electric fields. between I and 600 V / cm, and the combination of a nucleic acid and the transfer method according to the invention for their use in gene therapy.

 The transfer of genes into a given cell is the basis of gene therapy. However, one of the problems is to succeed in getting a sufficient quantity of nucleic acid into cells of the host to be treated, in fact, this nucleic acid, generally a gene of interest, must be expressed in transfected cells. One of the approaches adopted in this regard has been the integration of nucleic acid into viral vectors, in particular into retroviruses, adenoviruses or viruses associated with adenoviruses. These systems take advantage of the cellular penetration mechanisms developed by viruses, as well as their protection against degradation. However, this approach has drawbacks, and in particular a risk of production of infectious viral particles capable of dissemination in the host organism, and, in the case of retroviral vectors, a risk of insertional mutagenesis. In addition, the ability to insert a therapeutic or vaccine gene into a viral genome remains limited.

 In any event, the development of viral vectors usable in gene therapy requires the use of complex techniques of defective viruses and complementary cell lines.

 Another approach (Wolf et al. Science 247, 1465-68, 1990, Davis et al.

Proc. Natl. Acad. Sci. USA 93, 7213-18, 1996) therefore consisted in administering into the muscle or into the circulation a nucleic acid of a plasmid nature, associated or not with compounds intended to promote its transfection, such as proteins, liposomes, charged lipids or cationic polymers such as polyethyleneimine, which are good transfection agents in vitro (Behr et al. Proc.

Natl. Acad. Sci. USA 86, 6982-6, 1989, Felgner et al. Proc. Natl. Acad. Sci. USA 84, 7413-7, 1987, Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297-301, 1995).

With regard to muscle, since the initial publication by JA Wolff et al. showing the capacity of muscle tissue to incorporate DNA injected in the form of free plasmid (Wolff et al. Science 247, 1465-1468, 1990) many authors have attempted to improve this process (Manthorpe et al., 1993, Human Gene Ther. 4, 419-431, Wolff et al., 1991, BioTechniques 11, 474-485). Some trends emerge from these tests, such as the use of mechanical solutions to force the entry of DNA into cells,
by adsorbing DNA on beads then propelled onto tissues (gene gun)
(Sanders Williams et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2726-2730; Fynan
et al., 1993, BioTechniques 11, 474485). These processes have proven to be effective
in vaccination strategies, but only affect the upper layers
fabrics. In the case of muscle, their use would require a surgical approach
to allow access to the muscle because the particles do not pass through the tissue
skin,
DNA injection, no longer in the form of a free plasmid, but associated with
molecules capable of serving as a vehicle facilitating the entry of complexes into
cells. Cationic lipids, used in many other methods of
transfection have so far been disappointing because those who have been
tested were shown to be transfection inhibitors (Schwartz et al., 1996, Gene
Ther. 3, 405-411). The same is true for cationic peptides and polymers
(Manthorpe et al., 1993, Human Gene Ther. 4, 419-431). The only case of association
favorable seems to be the polyvinyl alcohol or polyvinylpyrrolidone mixture with
DNA. The increase resulting from these associations is only one factor
less than 10 compared to naked injected ADS (Mumper et al., 1996, Pharmaceutical
Research 13, 701-709),
pretreatment of the tissue to be injected with solutions intended to improve the
DNA diffusion and / or stability (Davis et al., 1993, Hum. Gene Ther. 4, 151
159), or to favor the entry of nucleic acids, for example the induction of
cell multiplication or regeneration phenomena. Treatments have
concerned in particular the use of local anesthetics or cardiotoxin,
vasoconstrictors, endotoxin or other molecules (Manthorpe et al., 1993,
Human Gene Ther. 4, 419-431, Danko et al., 1994, Gene Ther. 1, 114-121;
Vitadello et al., 1994, Hum. Gene Ther. 5, 11-18). These preprocessing protocols
are difficult to manage, the bupivacaine in particular requiring to be effective
to be injected in doses very close to lethal doses. Pre-injection of sucrose
hyperosmotic, intended to improve diffusion, does not increase the level of
transfection into muscle (Davis et al., 1993).

Other tissues were transfected in vivo either using plasmid DNA alone, or in combination with synthetic vectors (reviews of Cotten and Wagner (1994), Current Opinion in Biotechnology 4, 705; Gao and Huang (1995),
Gene Therapy, 2, 710; Ledley (1995), Human Gene Therapy 6, 1129). The main tissues studied were the liver, the respiratory epithelium, the vessel wall, the central nervous system and tumors. In all these tissues, the expression levels of the transgenes have proved to be too low to foresee a therapeutic application (for example in the liver, Chao et al. (1996) Human Gene Therapy 7, 901), although certain encouraging results have recently been presented for the transfer of plasmid DNA into the vascular wall (Iires et al. (1996) Human Gene Therapy 7, 959 and 989). In the brain, the transfer efficiency is very low, as in tumors (Schwartz et al. 1996, Gene Therapy 3,405; Lu et al. 1994, Cancer
Gene Therapy 1, 245; Son et al. Proc. Natl. Acad. Sci.USA 91.12669)
Electroporation, or the use of electric fields to permeabilize cells, is also used in vitro to promote the transfection of DNA into cultured cells. However, it has so far been accepted that this phenomenon responds to a threshold-dependent effect and that this electro-waterproofing can only be observed for electric fields of relatively high intensity, of the order of 800 to 1,200 volts. / cm for animal cells. This technique has also been proposed in vivo to improve the efficacy of antitumor agents, such as bleomycin, in solid tumors in humans (US Patent No. 5,468,228, LM Mir). With very short pulses (100 microseconds), these electrical conditions (800 to 1,200 Volts / cm) are very well adapted to the intracellular transfer of small molecules. These conditions (pulses of 100 microseconds) were applied without improvement for the transfer of nucleic acids in vivo in the liver, or fields below 1000 Volts / cm have been shown to be totally ineffective, and even inhibitory compared to DNA injection in the absence of electrical pulses (patent WO 97/07826 and Heller et al FEBS Letters, 389, 225-8, 1996).

 This technique also presents difficulties of application in vivo, because the administration of fields of such intensity can cause more or less extensive tissue damage, which does not represent a problem for the treatment of cancer patients but which can represent a major disadvantage for the healthy subject or for the sick subject when the nucleic acid is administered into tissues other than tumor tissues.

 While all the studies cited mention the need for high electric fields, of the order of 1000 Voltslcm, to be effective in vivo, in a truly unexpected and remarkable way, the applicants have now shown that the transfer of nucleic acids into tissue in vivo could be increased very significantly, without undesirable effects, by subjecting the tissue to electrical pulses of low intensity, for example 100 or 200 Volts / cm, and of relatively long duration. In addition, the applicants have found that the great variability of expression of the transgene observed in the prior art of DNA transfer was notably reduced by the method according to the invention.

 This is why, the present invention relates to a method for transferring nucleic acids in vivo, in which the cells of the tissues are brought into contact with the nucleic acid to be transferred, by direct administration into the tissue or by topical or systemic administration. , and in which the transfer is ensured by applying to said tissues one or more electric pulses of an intensity between I and 600 Volts / cm.

 According to a preferred embodiment, the method according to the invention applies to tissues whose cells have specific geometries such as for example large cells and / or of elongated shape and / or responding naturally to electric action potentials and / or having a specific morphology.

 Preferably, the intensity of the field is between 4 and 400 Volts / cm and the total duration of application is greater than 10 milliseconds. The number of pulses used is for example from 1 to 100,000 pulses and the frequency of the pulses is between 0.1 and 10,000 Hertz. The pulses can also be delivered irregularly and the function which describes the intensity of the field as a function of time can be variable. The integral of the function describing the variation of the electric field over time is greater than 1 kVxmsec / cm. According to a preferred embodiment of the invention, this integral is greater than or equal to 5 kVdnsec / cm.

 According to a preferred embodiment of the invention, the field strength of the pulses is between 30 and 300 Volts / cm.

 The electrical pulses are chosen from square wave pulses, electric fields generating exponentially decreasing waves, oscillating unipolar waves of limited duration, oscillating bipolar waves of limited duration, or other waveforms. According to a preferred embodiment of the invention, the electrical pulses are square wave pulses.

The administration of electrical pulses can be carried out by any method known to a person skilled in the art, for example:. system of external electrodes placed on either side of the tissue to be treated,
in particular non-invasive electrodes placed in contact with the skin,
system of electrodes implanted in the tissues,
electrode / injector system for simultaneous administration of acids
nucleic acid and the electric field.

 The administration being carried out in vivo, it is sometimes necessary to have recourse to intermediate products ensuring electrical continuity with non-invasive external electrodes. It will, for example, be an electrolyte in the form of a gel.

 The nucleic acids can be administered by any suitable means, but are preferably injected in vivo directly into the tissues or administered by another route, local or systemic and in particular by means of a catheter, which makes them available at the place of application of the electric field. The nucleic acids can be administered with agents allowing or facilitating the transfer, as mentioned previously. In particular, these nucleic acids can be free in solution or associated with synthetic agents, or carried by viral vectors. The synthetic agents can be lipids or polymers known to a person skilled in the art, or alternatively targeting elements allowing the fixation on the membrane of the target tissues. Among these elements, there may be mentioned vectors carrying sugars, peptides, antibodies or hormone receptors.

 It is conceivable, under these conditions of the invention, that the administration of nucleic acids can precede, be simultaneous or even follow the application of electric fields.

This is why, the present invention also relates to a nucleic acid and an electric field with an intensity of between I and 600 volts / cm, as a combination product for their simultaneous, separate or spread over time, administration to the cells. mammals and in particular human cells, in vivo. Preferably, the intensity of the field is between 4 and 400 Volts / cm and, even more preferably, the intensity of the field is between 30 and 300
Volts / cm.

 The method according to the present invention can be used in gene therapy, that is to say therapy in which the expression of a transferred gene, but also the modulation or blocking of a gene, makes it possible to ensure the treatment of a particular pathology.

Preferably, the tissue cells are treated for the purpose of gene therapy allowing:
either the correction of the dysfunctions of the cells themselves (for example
for the treatment of diseases linked to genetic deficiencies such as
cystic fibrosis),
either the safeguard and / or the regeneration of the vascularization or the innervation of
tissues or organs by trophic, neurotrophic and angiogenic factors
produced by the transgene,
either the transformation of the tissue into a secretory organ of products leading to an effect
therapeutics such as the gene product itself (e.g.
regulation of thrombosis and hemostasis, trophic factors, hormones) or such
than an active metabolite synthesized in the tissue through the addition of the gene
therapeutic,
either a vaccine or immunostimulatory application.

 Another object of the invention is the association of electrical pulses from a field with compositions containing the nucleic acids formulated for any administration allowing access to the tissue by topical, cutaneous, oral, vaginal, parenteral, intranasal route. , intraarterial, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for any other administration. They may be sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. The doses of nucleic acid used for the injection as well as the number of administrations and the volume of the injections can be adapted according to different parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to express, or the duration of the treatment sought.

 The nucleic acids can be of synthetic or biosynthetic origin, or extracted from viruses or from prokaryotic cells or from eukaryotic cells originating from unicellular organisms (for example, yeasts) or pluricellular ones. They can be administered in combination with all or part of the components of the original organism and / or of the synthesis system.

 The nucleic acid can be a deoxyribonucleic acid or a ribonucleic acid. They may be sequences of natural or artificial origin, and in particular gene DNA, cDNA, mRNA, tRNA and rRNA, hybrid sequences or synthetic or semi-synthetic sequences of modified oligonucleotides or not. These nucleic acids can be obtained by any technique known to a person skilled in the art, and in particular by targeting of banks, by chemical synthesis or also by mixed methods including chemical or enzymatic modification of sequences obtained by targeting of banks. They can be chemically modified.

 In particular, the nucleic acid can be a DNA or a RNA sense or antisense or catalytic property such as a ribozyme. By antisense is meant a nucleic acid having a sequence complementary to a target sequence, for example an mRNA sequence whose expression is sought to block by hybridization on the target sequence. By sense is meant a nucleic acid having a sequence homologous or identical to a target sequence, for example a sequence which binds to a protein transcription factor and is involved in the expression of a given gene.

According to a preferred embodiment, the nucleic acid comprises a gene of interest and elements allowing the expression of said gene of interest. Advantageously, the nucleic acid fragment is in the form of a plasmid.

Deoxyribonucleic acids can be single or double stranded, as can short oligonucleotides or longer sequences. They can carry therapeutic genes, transcription or replication regulatory sequences, or regions of binding to other cellular components, etc. Within the meaning of the invention, the term “therapeutic gene”) in particular any gene coding for a
RNA or for a protein product having a therapeutic effect. The encoded protein product can be a protein, a peptide, etc. This protein product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter presents no pathology). In this case, the expression of the transgene makes it possible, for example, to compensate for an insufficient expression in the cell or for the expression of an inactive or weakly active protein due to a modification, or also makes it possible to overexpress said protein.

The therapeutic gene can also code for a mutant of a cellular protein, having increased stability, modified activity, etc. The protein product can also be heterologous towards the target cell. In this case, an expressed protein can, for example, supplement or bring about a deficient activity in the cell (treatment of enzymatic deficits), or make it possible to fight against a pathology, or stimulate an immune response for example for the treatment of tumors. It may be a suicide gene (Thymidine Kinase Herpes) for the treatment of cancer or restenosis.

Among the therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes including enzymes for biosynthesis of vitamins, blood derivatives, hormones such as growth hormone, lymphokines: interleukins, interferons, TNFα, etc. . (French patent no. 92 03120), growth factors, for example angiogenic factors such as VEGF or
FGF, neurotransmitters or their synthetic precursors or enzymes, trophic factors, in particular neurotrophics for the treatment of neurodegenerative diseases, traumas which have damaged the nervous system, or retinal degenerations: BDNF, CNTF, NGF, IGF, GMF, aFGF , NT3, NT5,
HARP / pleiotrophin, or bone growth factors, hematopoietic factors, etc., dystrophin or a minidystrophin (French Patent No. 9111947), the genes coding for factors involved in coagulation: factors VII, VIII, M, genes suicides (thymidine kinase, cytosine deaminase),.

genes for hemoglobin or other protein transporters, the genes corresponding to the proteins involved in lipid metabolism, of the apolipoprotein type chosen from apolipoproteins AI, A-II, A-IV, B, CI, C-II, C -III, D, E, F, G, H,
J and apo (a), metabolism enzymes such as lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, 7 alpha cholesterol hydroxylase, phosphatidyl acid phosphatase, or lipid transfer proteins such as protein transfer of cholesterol esters and the phospholipid transfer protein, an HDL binding protein or a receptor chosen for example from LDL receptors, chylomicronsonnective receptors and scavenger receptors, etc. It is also possible to add leptin for the treatment of obesity. We can also add antioncogens like p53 or other tumor suppressors or the protein GAX limiting the proliferation of cells in smooth muscles (treatment of restenosis).

 Among the other proteins or peptides which can be secreted by a tissue or produced by this tissue, it is important to underline the antibodies, the variable fragments of single chain antibody (ScFv) or any other fragment of antibody having recognition capacities for its use in immunotherapy, for example for the treatment of infectious diseases, tumors, autoimmune diseases such as multiple sclerosis (antiidiotype antibody). Other proteins of interest are, without limitation, soluble receptors, such as for example the soluble CD4 receptor or the soluble receptor for TU'FI for anti-HIV therapy, the soluble acetylcholine receptor for treatment myasthenia gravis; enzyme peptides or inhibitors, or peptides agonists or antagonists of receptors or adhesion proteins such as for example for the treatment of asthma, thrombosis and restenosis; artificial, chimeric or truncated proteins. Among the hormones of essential interest, there may be mentioned insulin in the case of diabetes, growth hormone and calcitonin.

 The many examples which precede and those which follow illustrate the potential scope of the scope of the present invention.

 The therapeutic nucleic acid 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 RNA complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in European patent No. 140 308. The therapeutic genes also include the sequences encoding for ribozymes, which are capable of selectively destroying target RNAs (European Patent No. 321,201).

As indicated above, the nucleic acid can also contain one or more genes coding for an antigenic peptide, capable of generating in humans or animals an immune response. In this particular mode of implementation, the invention therefore makes it possible to produce either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers. They may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis virus
B (European Patent No. 185,573), the pseudo-rabies virus, the syncitia forming virus, other viruses or even tumor specific antigens such as the MAGE proteins (European Patent No. 259,212).

 Preferably, the nucleic acid also comprises sequences allowing and / or promoting the expression in the tissue of the therapeutic gene and / or of the gene coding for the antigenic peptide. These may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the transfected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to transfect. Likewise, they may be promoter sequences originating from the genome of a virus. In this regard, mention may be made, for example, of the promoters of the EIA, MLP, CMV, RSV, etc. genes. In addition, these expression sequences can be modified by adding activation, regulation sequences, etc. It can also be a promoter, inducible or repressible.

 Furthermore, the nucleic acid can also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.

The nucleic acid may also include a signal sequence directing the synthesized therapeutic product to a particular compartment of the cell.

Other genes of interest have been described in particular by
McKusick, VA Mendelian (Inheritance in man, catalogs of autosomal dominant, autosomal recessive, and X-linked phenotypes. Eighth edition. John Hopkins
University Press (1988)), and in Stanbury, JB et al. (The metabolic basis ofinberited disease, Fith edition. McGraw-Hill (1983)). The genes of interest cover the proteins involved in the metabolism of amino acids, lipids and other constituents of the cell.

 Mention may therefore be made, without limitation, of the genes associated with diseases of the metabolism of carbohydrates such as, for example, fructose-1-phosphate aldolase, fructose-1, 6-diphosphatase, glucose-6-phosphatase, a-1,4-glucosidase lysosomal, amylo-1, 6-glucosidase, amylo- (1,4: 1,6) -transglucosidase, muscle phosphorylase, muscle phosphofructokinase, phosphorylase-b-kinase, galactose-lphosphate uridyl transferase, all enzymes of the pyruvate dehydrogenase complex, pyruvate carboxylase , 2-oxoglutarate glyoxylase carboxylase, Dglycerate dehydrogenase.

We can also cite:
- genes associated with diseases of the metabolism of amino acids such as phenylalanine hydroxylase dihydrobiopterin synthetase tyrosine aminotransferase tyrosinase histidinase, fumarylacéto-acétase, glutathione synthetase, synthase y-glutamylcysteine, ornithine4 aminotransferase, synthase carbamoylphosphate, ornithine transcarbamylase , argininosuccinate synthetase, argininosuccinate Iyase, arginase, L-lysine dehydrogenase, L-lysine ketoglutarate reductase, valine transaminase, leucine isoleucine transaminase, branched chain 2-ketoacid decarboxylase, isovaleryl Cohen dehydrogenase, hydroxyacidase hydrogenase 3 -methylglutaryl-C oA lyase, acetoacetyl-CoA 3ketothiolase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase,
ATP: cobalamin adenosyltransferase, dihydrofolate reductase, methylene tetrahydrofolate reductase, cystathionine B-synthetase, sarcosine dehydrogenase complex, proteins belonging to the glycine cleavage system, ss-alanine transaminase, serum carnosinase, homocarnosinase.

 - Genes associated with diseases of fat and fatty acid metabolism, such as, for example, lipoprotein lipase, apolipoprotein C-II, apolipoprotein E, other apolipoproteins, lecithin cholesterolacyltransferase, LDL receptor, liver sterol hydroxylase, phytanic acid a -hydroxylase.

- Genes associated with lysosomal deficiencies, such as for example lysosomal a-Liduronidase, lysosomal iduronate sulfatase, heparan lysosomal N-sulfatase, N-acetayl-aD-lysosomal glucosaminidase, acetyl-CoA: a-glucosamine
Lysosomal N-acetyltransferase, Lysosomal N-acetyl-D-glucosamine 6-sulfatase, lysosomal galactosamine 6-sulfate sulfatase, lysosomal ss-galactosidase, arylsulfatase
Lysosomal B, lysosomal ss-glucuronidase, N-acetylglucosaminylphosphotransferase, lysosomal aD-mannosidase, lysosomal a-neuraminidase, lysosomal asparrylglycosaminidase, lysosomal lL-fucosidase, acid lipase

 The advantage of using electrotransfer in gene therapy lies in the safety provided by the local treatment linked to the use of local and targeted electric fields.

 Due to the safety linked to the use of weak fields, the present invention could be applied to the heart muscle for the treatment of heart diseases, for example by using a suitable defibrilator. It could also be applied to the treatment of restenosis by the expression of genes inhibiting the proliferation of smooth muscle cells such as the GAX protein.

 The combination of weak fields and long administration times applied to tissues in vivo improves the transfection of nucleic acids without causing significant tissue damage. These results improve the efficiency of DNA transfers in the context of gene therapy using nucleic acids.

Consequently, the method according to the invention makes it possible, for the first time, to envisage producing, by gene therapy, an agent at physiological and / or therapeutic doses, either in the tissues, or secreted in their vicinity or in the blood circulation. or lymphatic. In addition, the method according to the invention allows, for the first time, fine modulation and control of the effective amount of transgene expressed by the possibility of modulating the volume of the tissue to be transfected, for example with multiple administration sites. , or the possibility of modulating the number, shape, surface and arrangement of the electrodes. An additional element of control comes from the possibility of modulating the efficiency of the transfection by the variation of the field intensity, the number of the duration and the frequency of the pulses, and obviously according to the state of the art, the quantity and volume of administration of the nucleic acids. It is thus possible to obtain a level of transfection appropriate to the desired level of production or secretion. Finally, the method allows additional safety compared to chemical or viral methods of gene transfer in vivo, for which damage to organs other than the target organ cannot be totally excluded and controlled. Indeed, the method according to the invention allows the control of the localization of the transfected tissues (strictly linked to the volume of tissue subjected to local electrical impulses) and therefore brings the possibility of a return to the initial situation by total ablation or partial tissue when this is made possible by the non-vital nature of this tissue and by its regenerative capacities as in the case of the liver or muscle. This great flexibility of use makes it possible to optimize the process according to the animal species (human and veterinary applications),
The subject's age, physiological and / or pathological state.

 The method according to the invention also makes it possible, for the first time, to transfect large genes and / or introns and / or small or large regulatory elements, unlike viral methods which are limited by the size of the capsid. This possibility is essential for the transfer of very large genes such as that of dystrophin or of genes with large introns and / or regulatory elements, which is necessary for example for a physiologically regulated production of hormones. This possibility is essential for the transfer of episomes or artificial chromosomes of yeast or minichromosomes.

 The examples which follow are intended to illustrate the invention without limitation.

In these examples, reference will be made to the following figures:. Figure 1: Effects of high field strength electrical pulses on the
transfection of pxl 2774 plasmid DNA into the cranial tibial muscle in
mouse; average values + SEM,. Figure 2: Effects of electrical pulses of intermediate field strength on the
transfection of pxl 2774 plasmid DNA into the cranial tibial muscle in
mouse; average values + SEM,
Figure 3: Effects of electrical pulses of weak field strength and
different durations on the transfection of plasmid DNA pxl 2774 into the muscle
cranial tibial in mice; average values + SEM,
Figure 4: Effects of electrical pulses with weak field strength and
different durations on the transfection of plasmid DNA pxl 2774 into the muscle
cranial tibial in mice; mean values + SEM, Figure 5: Efficiency of electrotransfection of pxl 2774 plasmid DNA in the
Mouse cranial tibial muscle with weak electric field intensities:
average values + SEM.

EXAMPLE 1
Experience carried out under the conditions of the prior art in
which the electric fields show inhibitors of the transfection
In this example, the following products were used:
DNA pxl 2774 (PCT patent / FR 96/01414) is a plasmid DNA comprising the reporter gene for luciferase. The other products are available from commercial suppliers. Ketamine, Xylazine, Physiological serum (0.9% NaCl).

 A commercial oscilloscope and electric pulse generator (rectangular or square) (Electro-pulsator PS 15, Jouan, France) were used. The electrodes used are flat stainless steel electrodes 5.3 mm apart.

 The experiment is carried out in the C57 BV6 mouse. Mice from different cages are randomly assigned before the experiment ("randomization").

 The mice are anesthetized with a ketamine, xylazine mixture. The plasmid solution (30 μl of a solution at 500 tg / ml of 0.9% NaCl) is injected longitudinally through the skin into the cranial tibial muscle of the left and right legs using a hamilton syringe . The two electrodes are coated with a conductive gel and the injected tab is placed between the electrodes in contact with them.

 The electrical pulses are applied perpendicular to the axis of the muscle using a square pulse generator, one minute after the injection. An oscilloscope makes it possible to control the intensity in Volts (the values indicated in the examples represent the maximum values), the duration in milliseconds and the frequency in hertz of the pulses delivered, which is 1 Hz. 8 consecutive pulses are delivered.

 For the evaluation of the transfection of the muscle, the mice are euthanized 7 days after the administration of the plasmid. The cranial tibial muscles of the left and right legs are then removed, weighed, put in lysis buffer and ground. The suspension obtained is centrifuged in order to obtain a clear supernatant. The measurement of the luciferase activity is carried out on 10 μl of supernatant using a commercial luminometer in which the substrate is automatically added to the solution. The intensity of the light reaction is given in RLU (Relative Luminescence Unit) for a muscle knowing the total volume of suspension. Each experimental condition is tested on 10 points: 5 animals injected bilaterally. Statistical comparisons are made using non-parametric tests. Two figures, the scale of which is linear or logarithmic, illustrate the results.

 In this first experiment we tested the effects of an electric field of 800 to 1200 Volts / cm which allows the electroporation of tumors (Mir et al. Eur. J. Cancer 27, 68, 1991).

It can be seen from FIG. 1 that, relative to the control group, where the DNA is injected without an electrical pulse:
with 8 pulses of 1200 Volts / cm and a duration of 0.1 msec, the average value
luciferase activity is much lower,
with pulses of 1200 Volts / cm and 1 msec, 3 animals died, the
mean value of luciferase activity is much lower,
with pulses of 800 Volts / cm and 1 msec the average value of the activity
luciferase is also significantly reduced.

 Most of the muscles that have undergone the action of the electric field are visibly altered (brittle and whitish in appearance).

EXAMPLE 2
Experiment with electrotransfer of nucleic acids to moderate electric fields
This experiment is carried out with C57 BV6 mice. Aside from the electric field intensity of the pulses and their duration, the conditions of production are those of Example I.

 The results are shown in FIG. 2. The result of Example 1 is reproduced, that is to say the inhibitory effect of a series of 8 pulses at 800 Volts / cm with a duration of 1 msec over luciferase activity detected in muscle. With a field of 600 Volts / cm, we observe the same inhibition and the same alteration of muscle tissue.

On the other hand, remarkably and surprisingly, the reduction in voltage makes it possible to no longer visibly alter the muscles, and, moreover, at 400 and 200 volts / cm the level of transfection of the muscles is on average higher than that obtained on the muscles. not subject to a field. It should be noted that, relative to the control group (not subjected to an electric field), the dispersion of the values of luciferase activity is reduced to 200 Volts / cm (SEM = 20.59% of the average value against 43.32 % in the absence of an electric field (Figure 2A)).

EXAMPLE 3
Nucleic acid electrotransfer experiment with pulses of weak field intensity showing a very strong stimulation of the expression of the transgene
This experiment is carried out with C57 BV6 mice. Aside from the electric field intensity of the pulses and their duration, and the fact that the pulses are delivered 25 seconds after the injection of the DNA, the conditions of realization are those of the previous examples.

 The results are shown in FIG. 3. The mean value of the expression of the luciferase transgene is markedly increased with a pulse duration of 20 msec at 100 volts / cm, and from a pulse duration of 5 msec at 200 Volts / cm.

This experiment also clearly shows that the average value of luciferase activity obtained by electrotransfection of DNA in the muscle is a function of the duration of the electrical impulses, when voltages of 200 and 100 are used.
Volts / cm. It should also be noted that the dispersion of the values is notably reduced for the groups of electrotransfected muscles (FIG. 3A). In the absence of electrical pulses (control), the SEM represents 77.43% of the average value while the
Average relative SEM is reduced to 14% (200 Volts / cm, 5 msec), 41.27% (200
Volts / cm, 20 msec) and between 30% and 48% for electrotransfer at 100 Volts / cm of electric field.

 In the best condition of this experiment, the expression of the transgene is improved by a factor of 89.7 compared to the control injected in the absence of electrical pulses.

EXAMPLE 4
Experiment of electrotransfer of nucleic acids in muscle at 200 Volts / cm showing an increase in the expression of the transgene by a factor greater than 200
This experiment is carried out in DBA 2 mice, with electrical pulses with a field intensity of 200 Volts / cm and of variable duration, the other conditions of this experiment being those of Example 3.

This example confirms that at 200 volts / cm the transfection of luciferase activity is increased from a pulse duration of 5 msec and then continues to increase for longer durations (FIGS. 4 and 5). Again, we observe with electrotransfection a reduction in inter-individual variability indicated by the
SEM compared to non-electrotransfected control (the relative value of SEM is 35% for the control and 25, 22, 16, 18, 16 and 26% for series of pulses of 1, 5, 10, 15, 20 and 24 msec respectively).

 In the best condition of this experiment, the expression of the transgene is improved by a factor of 205 compared with the control injected in the absence of electrical pulses.

EXAMPLE 5
Efficiency of electrotransfer of nucleic acids as a function of the product number of pulses x field intensity x duration of each pulse
FIG. 5 exemplifies the importance of the parameter corresponding to the product number of pulses x intensity of the field x duration of each pulse. This parameter corresponds in fact to the integral as a function of time of the function which describes the variation of the electric field.

 The representation in FIG. 5 of the results obtained during experiments 2, 3 and 4 with electric field intensities of 200 V / cm, 100 V / cm or in the absence of electric fields shows that the transfection efficiency increases as a function of the product of the total duration of exposure to the electric field by the field intensity.

A stimulation effect is obtained for a value greater than 1 kVxmsec / cm of the product electric field x total duration of the pulses. According to a preferred mode, stimulation is obtained for a value greater than or equal to 5 kVxmsec / cm of the product electric field x total duration of the pulses.

Claims (42)

Claims  1) Combination product characterized in that a nucleic acid and an electric field of an intensity between 1 and 600 volts / cm are combined, for their simultaneous, separate or spread over time in vivo administration to a tissue and, for gene therapy based on in vivo electrotransfection in tissues.  2) Combination product according to claim 1, characterized in that the intensity of the field is between 4 and 400 volts / cm.  3) Combination product according to claim 1, characterized in that the intensity of the field is between 30 and 300 volts / cm.  4) Combination product according to one of claims I to 3, characterized in that the total duration of application of the electric field is greater than 10 milliseconds.  5) Combination product according to one of claims 1 to 4, characterized in that the application to the fabric of the electric field comprises one or more pulses of regular frequency.  6) Combination product according to claim 5, characterized in that the application to the fabric of the electric field comprises between 1 and 100,000 pulses of frequency between 0.1 and 10,000 hertz.  7) Combination product according to one of claims I to 5, characterized in that the electrical pulses are delivered irregularly with respect to each other and in that the function describing the intensity of the electric field as a function of time of a pulse is variable.  8) Combination product according to one of claims I to 7, characterized in that the integral of the function describing the variation of the electric field with time is greater than 1 kVxmsec / cm.  9) Combination product according to claim 8, characterized in that this integral is greater than or equal to 5 kVxmsec / cm.  10) Combination product according to one of claims 1 to 9, characterized in that the electrical pulses are chosen from square wave pulses, the electric fields generating waves with exponential decay, oscillating unipolar waves of limited duration, limited-time oscillating bipolar waves, or other waveforms.  11) Combination product according to one of claims 1 to 10, characterized in that the electrical pulses comprise square wave pulses.  12) Combination product according to one of claims 1 to 11, characterized in that the electrical pulses are applied externally.  13) Combination product according to one of claims I to 11, characterized in that the electrical pulses are applied inside the fabric.  14) Combination product according to one of claims 1 to 13, characterized in that the nucleic acid is injected into the tissue.  15) Combination product according to one of claims 1 to 13, characterized in that the nucleic acid is injected systemically.  16) Combination product according to claim 15, characterized in that the nucleic acid is injected intra-arterially or intravenously.  17) Combination product according to one of claims I to 13, characterized in that the nucleic acid is administered by topical, cutaneous, oral, vaginal, intranasal, intramuscular, subcutaneous or intraocular route.  18) Combination product according to one of claims 1 to 17, characterized in that the nucleic acid is present in a composition containing, in addition, pharmaceutically acceptable excipients for the different modes of administration.  19) Combination product according to one of claims I to 13, characterized in that the nucleic acid is present in a composition, suitable for parenteral administration.  20) Combination product according to one of claims I to 13, characterized in that the nucleic acid is a deoxyribonucleic acid.  21) Combination product according to one of claims I to 13, characterized in that the nucleic acid is a ribonucleic acid.  22) Combination product according to one of claims 1 to 13, characterized in that the nucleic acid is of synthetic or biosynthetic origin, or extracted from a virus or from a unicellular or pluricellular prokaryotic or eukaryotic organism.  23) Combination product according to claim 22, characterized in that the administered nucleic acid is associated with all or part of the components of the original organism and / or of the synthesized system.  24) Combination product according to one of claims I to 23, characterized in that the nucleic acid codes for an RNA or a protein of interest.  25) Combination product according to claim 24, characterized in that the RNA is a catalytic or antisense RNA.  26) Combination product according to claim 24, characterized in that the nucleic acid codes for a protein chosen from enzymes, blood derivatives, hormones, lymphokines, growth factors, trophic factors, angiogenic factors, neurotrophic factors, bone growth factors, hematopoietic factors, coagulation factors, antigens and proteins involved in the metabolism of amino acids, lipids and other essential components of the cell.  27) Combination product according to claim 26, characterized in that the nucleic acid codes for the angiogenic factors VEGF and FGF, the neurotrophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, NT3, NT5, Gax protein, growth hormone, calcitonin, leptin and apolipoproteins, vitamins biosynthesis enzymes, hormones and neuromediators.  28) Combination product according to claim 24, characterized in that the nucleic acid codes for an antibody, a variable fragment of single chain antibody (ScFv) or any other fragment of antibody having recognition capabilities for the purpose of immunotherapy, or code for a soluble receptor, for an agonist or antagonist peptide for a receptor or an adhesion protein, for an artificial, chimeric or truncated protein.  29) Combination product according to claim 28, characterized in that the nucleic acid codes for an antiidiotype antibody, a soluble fragment of the CD4 receptor or of the TNFα receptor or of the acetylcholine receptor.  30) Combination product according to one of claims 1 to 13, characterized in that the nucleic acid codes for a precursor of a therapeutic protein.  31) Combination product according to one of claims 20 to 30, characterized in that the nucleic acid is in the form of a plasmid.  32) Combination product according to one of claims 20 to 30, characterized in that the nucleic acid contains a large gene and / or introns and / or regulatory elements of small or large.  33) Combination product according to one of claims 20 to 30, characterized in that the nucleic acid is an episomal DNA or an artificial yeast chromosome or a minichromosome.  34) Combination product according to one of claims 20 to 33, characterized in that the nucleic acid contains sequences allowing and / or promoting the expression of the transgene in the tissue.  35) Combination product according to one of claims 1 to 34, characterized in that the acid is associated with any type of vector or with any combination of vectors making it possible to improve the transfer of nucleic acid such as viruses, synthetic or biosynthetic agents, or even propelled or non-propelled beads.  36) Combination product according to one of claims 1 to 35, characterized in that the tissue is subjected to a treatment aimed at improving the transfer of discomfort, a treatment of pharmacological nature in local or systemic application, or an enzymatic treatment, permeabilizing, surgical, mechanical, thermal or physical.  37) Combination product according to one of claims I to 14, characterized in that it allows the tissue to produce an agent at physiological and / or therapeutic doses, either in the cells of the tissue, or secreted.  38) Combination product according to one of claims 1 to 19, characterized in that it allows to modulate the amount of transgene expressed by modulating the volume of transfected tissue.  39) Combination product according to claim 38, characterized in that it allows the volume of transfected tissue to be modulated by the use of multiple administration sites.  40) Combination product according to one of claims 1 to 13, characterized in that it makes it possible to modulate the quantity of transgene expressed by modulating the number, the shape, the surface and the arrangement of the electrodes, and by varying the field strength, number, duration, frequency and shape of the pulses, as well as the quantity and volume of administration of the nucleic acid.  41) Combination product according to one of claims 1 to 13, characterized in that it makes it possible to control the location of the tissues transfected by the volume of tissue subjected to local electrical impulses.  42) Combination product according to one of claims 1 to 13, characterized in that it allows a return to the initial situation by the ablation of the area of transfected tissue.