SK70999A3 - Transfecting composition usable in gene therapy combining a recombinant virus incorporating an exogenous nucleic acid, a non-viral and non-plasmid transfecting agent - Google Patents

Abstract

The invention concerns a transfecting composition usable in gene therapy characterised in that it combines one or several non-coated recombinant viruses and comprising in their genome at least an exogenous nucleic acid, at least a non-viral and non-plasmid transfecting agent.

Description

A transfection preparation suitable for gene therapy, which comprises a recombinant virus and a transfection agent

Technical field

The present invention relates to the field of gene therapy and in particular relates to the transfer of genetic material in vitro, ex vivo and / or in vivo. In particular, the invention provides novel compositions useful for efficient transfection of cells.

BACKGROUND OF THE INVENTION

Chromosome deficits and / or anomalies (mutations, aberrant expression, etc.) are the source of many diseases of hereditary or non-hereditary nature. Medicine remained powerless for a long time. With the development of gene therapy, it can now be hoped that this type of chromosome aberration can be repaired or prevented in the future. This novel method of treatment consists in introducing genetic information into the affected cell or organ to correct this deficiency or anomaly, or alternatively express the protein of interest herein.

The major obstacle to the penetration of a nucleic acid into a cell or target organ lies in the size and polyanionic nature of the nucleic acid, preventing its passage through the cell membrane.

In order to overcome this obstacle, various techniques are now proposed, including specifically transfecting naked DNA across the plasma membrane in vivo (WO90 / 11092) and transfecting DNA using a transfection vector.

With regard specifically to this latter technique, two strategies are proposed in principle:

The first uses naturally transfection vectors, which are viruses, and the second is the use of chemical or biochemical vectors.

With respect to vectors of viral origin, they are now widely used for cloning, gene transfer and expression in vitro (for recombinant protein production, screening tests, regulatory protein studies, etc.), ex vivo or in vivo (for animal modeling or in animal models). gene transfer procedures with therapeutic significance). Of these viruses, particular adenoviruses, adeno-associated viruses (AAVs), retroviruses, herpesviruses or even vaccinia virus may optionally be mentioned.

The Adenoviridae family is widespread in mammals and birds, and includes more than a hundred different viral serotypes that consist of double-stranded DNA that encapsulates and forms a capsid with icosahedral symmetry (Horwitz, In: Fields BN, Knipe, DM, Howley PM, ed. Virology Third Edition ed. Philadephia: Raven Publishers, 1996: 2149-2171). Their genome contains inverted terminal repeat (ITR) at each end, encapsidation sequence (Psi), early and late genes. The essential early genes are found in the E1, E2, E3 and E4 regions. Of these genes, those found in the E1 region are essential for virus propagation. Essential late genes are found in the L1 to L5 regions. The entire genome of the Ad5 adenovirus has been sequenced and is accessible in databases (see, in particular, Genebank M73260). Also, parts or even all other adenoviral genomes (Ad2, Ad7, Ad12, etc.) were sequenced in the same manner.

In addition to being harmless, adenovirus has a broad cellular tropism. Unlike a retrovirus, whose life cycle is dependent on cell division, adenovirus can advantageously infect actively dividing cells, but also non-dividing cells and its genome is maintained in episomal form. In addition, it can be produced at a very high titer (10 ¹¹ pfu / ml). These major properties predetermine it as a vector for the cloning and expression of heterologous genes. The C group of adenoviruses, especially types 2 and 5, as well as the CAV-2 type adenoviruses, whose molecular biology is best known, are the sources of the vectors commonly used today. For these reasons, adenoviral vectors have been used to clone and express genes in vitro (Gluzman et al., Cold Spring Harbor, New York 11724, p. 187), to generate transgenic animals (WO95 / 22616), to transfer genes to cells ex vivo (WO95 / 14875; WO95 / 06120) and for gene transfer into cells in vivo (see in particular WO93 / 19191, WO94 / 24297 and WO94 / 08026).

For adeno-associated viruses (AAVs), they are relatively small DNA viruses that integrate into the genome of cells that infect in a stable and relatively site-specific manner. They are able to infect a wide range of cells. The AAV genome was cloned, sequenced and described. It contains about 4700 bases and has an inverted terminal repeat (ITR) region of approximately 145 bp at each end, which serves as a replication origin for the virus. The remainder of the genome is divided into two essential parts carrying encapsidation functions: the left part of the genome, which contains the rep gene involved in viral replication and viral gene expression, and the right part of the genome, which contains the cap gene, encoding the viral capsid proteins. The use of AAV-derived vectors for gene transfer in vitro and in vivo has been described (in particular see WO91 / 18088; WO93 / 09239; US 4,797,368, US 5,139,941 and EP 488 528).

These vectors, especially adenoviruses, have been shown to be very advantageous in terms of transfection levels. Conventionally, infection of cells with a viral transfection vector is performed in two steps. In a first step, cell surface target receptors are recognized which allow recombinant virus to be attached. The nature of these receptors, as well as their distribution depending on the cell type, is poorly documented, but in the particular case of adenovirus, it appears to be a fiber, a protein on the outer surface of the adenovirus that may be involved in this attachment. The second step consists in the interaction of the viral penton protein at the root of the strand via the RGD peptide motif with the cellular integrins avb3 and / or avb5, which allow the internalization of the virion.

The efficacy with which a recombinant virus can infect a cell (transduction efficiency) is highly variable and depends on the type of cells studied in tissue culture or tissues whose testing is important for in vivo treatment. In all cases, this is the first attachment step that determines the efficiency of the internalization of the virion. Thus, the number of receptors on the cell surface is a limiting factor in the infection process and it is clear that any approach to bypassing or facilitating this attachment step will make it possible to remove this barrier.

In particular, the first object of the present invention was to increase the transfection efficiency of these viral vectors by optimizing their internalization at the level of the target cell to be treated.

A second object directed and achieved by the present invention relates to the reduction or even suppression of the immune response, usually expressed by the treated cells, against a viral vector. In fact, as with all known viruses, administration of recombinant viruses such as recombinant adenoviruses that are defective in replication (Yang et al., PNAS (1994) 4407) elicits a significant immune response. One of the main tasks of the immune system is to destroy elements that are not intrinsic or intrinsic but altered. Administration of a gene treatment vector of viral origin introduces motifs that are not intrinsic to it. Similarly, cells infected with such a vector, resulting in the expression of an exogenous gene, will become intrinsic altered elements. Thus, the immune response developed against these infected cells represents a major impediment to the development of these viral vectors, because i) by inducing and destroying infected cells limits gene expression time and hence therapeutic effect, ii) induces simultaneous and significant inflammatory response and iii) rapidly eliminates infected cells after repeated injections.

SUMMARY OF THE INVENTION

The present invention advantageously enables these two objectives to be achieved by combining the desired recombinant virus with a chemical or biochemical compound that effectively facilitates its transduction and protects it from immune system responses elicited upon encountering the virus.

As mentioned above, non-viral transfection vectors have also been developed at the level of gene therapy along with viral vectors. More specifically, they are chemical or biochemical vectors. These synthetic vectors have two main functions, to condense the DNA to be transfected and to promote its cellular attachment as well as its passage through the plasma membrane and, if necessary, through the two nuclear membranes.

Although these chemical vectors are not as effective as viral vectors, they are, on the other hand, more advantageous from an immune point of view. They do not appear pathogenic, the risk of DNA multiplication in these vectors is zero and have virtually no theoretical limit as to the size of the DNA to be transfected.

/

Of these synthetic vectors developed, cationic polymers of the polylysine and DEAE-dextran type or even lipofectants are most preferred. They are capable of condensing DNA and promoting its association with the cell membrane. Recently, a method for targeted receptor-mediated transfection has been developed. This technique relies on the principle of DNA condensation by the cationic polymer, while at the same time controlling the attachment of the complex to the membrane through chemical bonding between the cationic polymer and the membrane receptor ligand present on the cell type surface that is desired to bind. Thus, targeting of the transferrin receptor, the insulin receptor, or the hepatocyte asialoglycoprotein receptor has been described.

More specifically, the main object of the present invention lies in the association of at least one of these chemical or biochemical vectors with a recombinant virus containing in its genome at least one exogenous nucleotide sequence with a view to more efficiently promoting cellular transduction of this recombinant virus.

The author unexpectedly demonstrated that it was possible to exploit simultaneously the respective properties of these two types of transfection vectors to effectively induce the expression of therapeutic genes.

The present invention preferably proceeds simultaneously from the properties of carriers of liposome or lipofectant-type chemical vectors that are capable of fusing with a variety of cell types and the efficiency of internalizing recombinant viruses, such as, in particular, adenovirus. The following is a synergy between the virion, which is the transducing agent of the gene of the desired therapeutic effect, and the lipofectant as a transfection aid. This synergism is preferably manifested in two levels.

Initially, it exhibits a very significant increase in the local concentration of the recombinant virus. This support is such that in cell type, such as vascular smooth muscle cells, where the filament receptor is very poorly expressed. the transfection efficiency of the recombinant adenovirus associated with the liposome is 50 to 100x higher than that of the recombinant adenovirus alone. Moreover, this observation has been confirmed in different adenoviruses. It has been shown that in the presence of a chemical or biochemical vector, reduced amounts of recombinant viruses with the same, if not higher, efficacy can be used.

Finally, it has been unexpectedly shown that the association of a chemical vector has been effectively attenuated by a recombinant adenovirus system allowing the phenomenon of neutralization, usually induced by viruses. If when encountered with recombinant adenovirus in isolated form neutralizing containing anti-Ad reduced transduction efficiency.

contacts antibodies,

This immune transduction with human serum observed to be strong is unexpectedly significantly elevated lipofectant. According to the presence of the lipofectant dose type chemical vector, the transduction efficiency is either restored or increased 10 to 20-fold compared to the adenovirus transduction under favorable conditions.

Thus, as a first object, the present invention has a composition useful in gene therapy, characterized in that it is associated with one or more recombinant non-encoded viruses containing in their genome at least one exogenous nucleic acid and at least one transfection agent of non-viral or plasmid origin.

For the non-viral transfection agent, it is preferably selected from cationic polymers and lipofectants.

More specifically, with respect to lipofectants, it is to be understood that within the meaning of the present invention, this designation covers any lipid compound and already presented as an active agent with respect to cell transfection with nucleic acids. In general, they are amphiphilic molecules comprising at least one lipophilic region linked or not linked to a hydrophilic region. Representative of the first family may be specific lipids capable of forming liposomes such as POPC, phosphatidylserine, phosphatidylcholine, cholesterol, lipofectamine, maleimidophenylbutyrylphosphatidylethanolamine, lactosylceramide in the presence or absence of polyethylene glycol liposome-forming or liposome-forming liposomes .

In a particular method of the invention, the lipid reagent used has a cationic region. This cationic region, preferably a polyamine charged as a cation, is likely to reversibly associate with the recombinant virus.

By way of illustration of this type of cationic lipids constructed in the following structural model: the lipophilic group associated with the amino group via a spacer branch, mention may be made specifically of DOTMA, as well as those lipids which contain two fatty acids or cholesterol derivative as lipophilic group; a quaternary ammonium group such as an amino group. Representative examples of this category of cationic lipids may be mentioned in particular

DOTAP, DOBT, or ChOTB. Other compounds, such as DOSC and ChOSC, are characterized by the presence of a choline group in place of the quaternary ammonium group.

Lipofectants suitable for the invention may also advantageously be selected from lipopolyamines whose polyamine region corresponds to the general formula

H ₂ N - (- (CH) _m -NH-) _n -H

While m is an integer equal to or greater than 2 and n is an integer equal to or greater than 1, and m can vary between different carbon groups located between two amines, this polyamine moiety is covalently linked to a lipophilic portion of the hydrocarbon chain that is saturated or unsaturated, cholesterol, or a natural or synthetic lipid capable of forming lamellar or hexagonal phases. This polyamine region is most preferably represented by spermine, thermin, or one of their analogs, which retains the nucleic acid binding properties.

Patent application EP 394 111 describes lipopolyamines of this family that can be used in the sense of the present invention. Examples of such lipopolyamines include, in particular, dioctadecylamidoglycyl-spermine (DOGS) and palmitoylphosphatidylethanolamine-5-carboxyspermylamide (DPPES).

The lipolyamines described in patent application WO96 / 17823 can also be advantageously used according to the invention. They are represented by the general formula

H ₂ N - (- (CH) _m -NH-) _n -H wherein R represents

wherein X and X 'are, independently of one another, an oxygen atom, a methylene group - (CH 2) _q - sq equal to 0, 1, 2 or 3, or an amino group, -NH- or -NR'-, wherein R' is alkyl a group containing 1 to 4 carbon atoms (a C 1 to C 4 alkyl group),

Y and Y 'independently of one another represent a methylene group, a carbonyl group or a C = S group,

R ³ , R ⁴ and R ^5, independently of one another, represent a hydrogen atom or a substituted or unsubstituted C 1 -C 4 alkyl group, wherein p may range from 0 to 5,

R ⁶ represents a cholesterol derivative or an alkylamino group -NR ¹ R ² , wherein R ¹ and R ² , independently of one another, represent a saturated or unsaturated, linear or non-linear or branched aliphatic group having 12 to 22 carbon atoms (C12 to C22 aliphatic group) ).

Representative examples of these lipopolyamines include, in particular, 2-5-bis- (3-amino-propylamino) pentyl (dioctadecylcarbamoylmethoxy) acetate and 2- [1,3-bis (3-aminopropylamino)] propyl (dioctadecylcarbamoylmethoxy) acetate.

Recently, novel lipopolyamines have been developed which have been described in patent application FR95 / 13490 and may also be useful in the sense of the present invention. These are compounds having the following general formula:

wherein R ¹ , R ² and R ³ represent, independently of one another, a hydrogen atom or a group - (CH 2) _q -NRR ', provided that q may have values of 1, 2, 3, 4, 5 and 6, independently for different R ¹ , R ² and R ³ ,

R and R 'independently of one another represent a hydrogen atom or a group - (CH 2) _q -NH 2, wherein q may have values of 1, 2, 3, 4, 5 and 6, independently for the various groups R and R'.

m, n and p represent, independently of one another, an integer which may be between 0 and 6, if n is greater than 1, m may have different values and R ³ may have different meanings in the general formula, and

R ⁴ represents a group of the general formula

wherein R 6 and R 7 independently of one another are H or a saturated or an unsaturated Cio-C ₂ 2 aliphatic radical having 10 to 22 carbon atoms (Cio-C22), wherein at least one of the two groups is hydrogen, u is an integer between 0 to 10, and if u is an integer greater than-1, R ⁵ , X, Y and r may have different meanings in different themes [X- (CHR ⁵ ) _r -Y],

X represents an oxygen or sulfur atom or an optionally monoalkylated amino group,

Y represents a carbonyl group or a methylene group,

R ⁵ represents a hydrogen atom or a natural amino acid side chain, if necessary substituted, ar is an integer ranging from 1 to 10, and when r is 1, R ⁵ represents a substituted or unsubstituted natural amino acid side chain, and if r is greater than 1, R ⁵ represents a hydrogen atom.

As a representative example of these lipopolyamines, in particular, the following may be appropriate:

{H ₂ N (CH ₂ ) ₃ } N (CH ₂ ) ₄ N {(CH ₂ ) ₃ NH ₂ } (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) 17 -CH ₃ ] ₂ (RP120525)

H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) _{χ 8} ] 2 (RP120535)

H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COAr ₉ N [(CH ₂ ) ₁₈ ] ₂ (RP120531)

In another particular embodiment of the invention, the non-viral vector is represented by at least one cationic polymer and, more preferably, a compound of the general formula

N --- _{(CH2) n}

I

R in which R can be a hydrogen atom or a group according to formula (CH ₂ ) _n - N wherein n wherein n is molecular is an apparent mass number between 2 compounds

WO96 / 02655 are that the sum of the p polymer is given specifically in up to 10, p and q are such that 100 are integers, + q between that

10 ⁷ average

these

D.

patent application

Particularly preferred properties are polymers of polyethyleneimine (PEI) and polypropyleneimine (PPI). Preferred polymers for carrying out the present invention are those whose molecular weight is between 10 ³ and 5 x 10 ⁶ . An example is polyethyleneimine with an average molecular weight of 50,000

D (PEI50K) or polyethyleneimine with an average molecular weight of 800000 D (PEI800K). PEI50K and PEI800K are available commercially. With respect to the other polymers represented by the general formula above, they can be prepared according to the procedure described in patent application FR 94 08735.

Lipofectamine, dioctadecylamidoglycylspermine (DOGS), palmitoylphosphatidylethanolamine-5-carboxyspermylamide (DPPES), 2-5-bis (3-aminopropylamino) pentyl (dioctadecylcarbamoylmethoxy) acetate can be used particularly preferably for the purposes of the invention.

2-1,3-bis (3-aminopropylamino)] propyl (dioctadecylcarbamoylmethoxy) acetate, {H ₂ N (CH ₂ ) ₃ } N (CH ₂ ) ₄ N {(CH ₂ ) ₃ NH ₂ } (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₇ -CH ₃ ] ₂ , H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₈ ] 2,

H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COAr ₉ N [(CH ₂ ) ₁₈ ] ₂ and / or

H ₂ N (CH _{2) 3} NH (CH _{2) 4} NH (CH _{2) 3} NHCH ₂ COGlyN [(CH _{2) 7} CH _{3] 2}

The recombinant viruses used according to the present invention are defective, i. unable to replicate autonomously in the target cell. In general, the genome of the defective viruses used according to the invention thus has minimally deleted sequences necessary for the replication of said virus in the infected cell. These regions can either be eliminated (in whole or in part) or rendered inoperative or substituted by other sequences, in particular the inserted nucleic acid. The defective virus preferably contains sequences of its genome which are necessary for encapsidation (packaging) of viral particles.

The recombinant virus used in the context of the present invention is derived from an encapsidated virus. Adenoviruses and adeno-associated viruses (AAVs) are particularly suitable as non-limiting examples of this type of virus. According to a preferred embodiment, the virus is an adenovirus.

There are various adenovirus serotypes whose structure and properties vary somewhat. Of these serotypes, the use of human adenovirus type 2 or 5 (Ad2 or Ad5) or adenoviruses of animal origin is preferred for the purposes of the present invention (see application WO94 / 26914). Of the adenoviruses of animal origin which may be mentioned in the context of the present invention, mention may be made of canine, bovine, murine (e.g.: Mavl, Beard et al., Virology, 75, 81, 1990), sheep, swine, avian or, optionally, monkey (for example: SAV). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26 / 61 strain (ATCC VR-800). Preferably, adenoviruses of human or canine or mixed origin are used in the invention.

Preferably, at least one E1 region in the adenovirus genome is disabled. The viral gene contemplated may be rendered inoperative by any technique known to one skilled in the art, namely complete suppression, substitution, partial deletion or addition of one or more bases to the genes under consideration. Other regions may also be modified, in particular the E3 (WO95 / 02697), E2 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697) regions.

According to a preferred method of use, the adenovirus comprises a deletion in the E1 and E4 regions. According to another preferred embodiment, it comprises a deletion in the E1 region at the level in which the E4 region and the coding sequences are inserted. In a virus according to the invention, the deletion in the E1 region preferably occupies the nucleotide region 455 to 3329 of the Ad5 adenovirus sequence. According to another preferred embodiment, the exogenous nucleic acid sequence is inserted at the deletion level of the E1 region.

The defective recombinant adenoviruses of the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia an exogenous nucleic acid. Homologous recombination occurs after co-transfection (cotransfection) with said adenoviruses and a plasmid of the respective cell line. The cell line used must preferably (i) be transformable with said elements, and (ii) contain sequences capable of complementing portions of the defective adenovirus genome, preferably in integrated form, to avoid the risk of recombination. An example of a line is the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59), which specifically comprises the left portion of the genome of Ad5 adenovirus (12%) integrated into its genome, or a line capable of complementing E1 and E4 functions, as specifically described in applications Nos. WO94 / 26914 and WO95 / 02697 '. Adenoviruses that have been propagated are obtained and purified by conventional molecular biology techniques as exemplified.

The adenoviruses used according to the invention can similarly be obtained by the original procedure described in patent application WO96 / 25506, which uses as a shuttle plasmid a prokaryotic plasmid containing a recombinant adenovirus genome flanked by one or more restriction sites which are not present in said genome.

As an illustrative and non-limiting example of the compositions of the invention, it is particularly useful to suggest that association of lipofectamine in sufficient quantity with a recombinant adenovirus containing at least one exogenous nucleic acid in its genome will improve its cellular transduction.

In the compositions of the present invention, deoxyribonucleic acid as well as ribonucleic acid can be introduced as a nucleic acid at the recombinant virus genome level. They may be sequences of natural or artificial origin, in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences, or synthetic or semi-synthetic sequences of modified or unmodified oligonucleotides. These nucleic acids may be of human, animal, plant, bacterial, viral, and the like origin. They can be obtained by any technique known to the person skilled in the art, namely by screening, chemical synthesis or alternatively by mixed methods, including chemical or enzymatic modification of sequences obtained by screening libraries.

More specifically, deoxyribonucleic acids may be single or double stranded, as well as short oligonucleotides or longer sequences. These deoxyribonucleic acids can carry therapeutic genes, regulatory sequences for transcription or replication, modified or unmodified antisense sequences, binding regions for other cellular components, and the like.

For the purposes of the invention, a therapeutic gene essentially refers to any gene encoding a protein product having a therapeutic effect. The protein product encoded in this way may be a protein, a peptide and the like. This protein product may be homologous to the target cell (i.e., a product that is normally expressed in a target cell that exhibits no pathology). In this case, protein expression allows to overcome, for example, under-expression in a cell or expression of a protein that is inactive or poorly active due to modification, or alternatively, said protein is overexpressed. The therapeutic gene may also encode a mutant cellular protein having enhanced stability, modified activity, and the like. The protein product may also be heterologous to the target cell. In this case, the expressed protein may produce or contribute to an activity that is deficient in the cell, thus allowing to combat the pathology or stimulate the immune response.

Among the therapeutic products within the meaning of the present invention, it is particularly useful to mention enzymes, blood derivatives, hormones, lymphokines, interleukins, interferons, TNF, and the like. (FR9203120), growth factors, neurotransmitters or their precursors or enzyme synthesis, trophic factors: BDNF, CNTF, NGF, IGF, GMF, bFGF, NT3, NT5, HARP / pleiotrophin, etc., dystrophin or minidystrophin (FR 9111947), protein CFTR associated with mucoviscidosis, genes associated with cell division arrest, specifically the gax gene, tumor suppressor genes: p53, Rb, RapIA, DCC, k-rev and the like. (FR 93 04745), genes coding for factors involved in coagulation: factors VII, VIII, IX, genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), hemoglobin or other protein transporter gene, genes corresponding to proteins involved in the lipid metabolism of apolipoproteins , A-II, A-IV, B, CI, C-II, C-III, D, E, F, G, H, J and apo (a), metabolic enzymes such as lipoprotein lipase, liver lipase, lecithincholesterolacyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidic acid phosphatase, or alternatively lipid transfer proteins such as an HDL binding protein or alternatively a receptor selected from, for example, LDL receptors, residual chylomicron receptors, etc.

scavenger receptors,

The therapeutic nucleic acid may also be a gene or an antisense sequence (a sequence with a sense of normal transcription orientation) whose expression in the target cell allows expression of the gene or suppression of transcription of cellular mRNA. Such sequences may, for example, be transcribed in a target cell into RNA complementary to cellular mRNA and thereby block its translation into a protein, according to the method described in EP 140 308. Therapeutic genes also contain ribozyme coding sequences capable of selectively destroying target RNAs (EP 321 201).

As mentioned above, the nucleic acid may also comprise one or more genes encoding antigenic peptides capable of eliciting an immune response in a human or animal. Thus, in this particular mode of use, the invention allows the production of either vaccines or immunotherapeutic drugs administered to humans or animals, in particular against microorganisms, viruses or cancer. These may be, in particular, specific antigenic peptides of Epstein-Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudorabies virus, syncytic virus or other viruses, or alternative tumor specific peptides (EP 259 212).

Preferably, the nucleic acid also comprises sequences allowing the expression of the therapeutic gene and / or the gene encoding the antigenic peptide in the desired cell or organ. These may be sequences which are naturally responsible for the expression of the gene of interest, if such sequences are capable of functioning in the infected cell. They may 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 derived from the genome of the virus. In this regard, for example, the promoters of the E1A, MLP, CMV, RSV, and the like genes may be mentioned. In addition, these expression sequences can be modified by the addition of activation or regulatory sequences, and the like. They may also be promoter, inducible or repressible sequences.

The nucleic acid may also additionally contain, in particular upstream of the therapeutic gene, a signal sequence for directing the synthesized therapeutic product into 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 comprise a signal sequence directing the synthesized therapeutic product towards a particular compartment of the cell.

In another method of the invention, the formulations of the invention may additionally comprise the adjuvant dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl or -myristoylphosphatidylethanolamine type, as well as their methyl derivatives, which are 1-3 times N; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (particularly galactocerebrosides), sphingolipids (particularly sphingomyelins), or alternatively asialogangliosides (particularly asialoGM1 and GM2).

The author has recently shown that a compound which directly or indirectly at the level of nucleic acid condensation has also been used as an adjuvant in transfection preparations. This compound consists wholly or partially of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of motifs may be between 2 and 10. Such agent may also originate in whole or in part from histones, nucleolin, protamine and / or one of their derivatives (WO96 / 25508).

Such an adjuvant may be useful for improving the intracellular movement of the recombinant virus in the compositions of the invention.

One or more targeting elements that allow viral and non-viral vectors to be directed against cell surface receptors or ligands may also be used in the composition of the invention. By way of example, the composition of the present invention may comprise one or more antibodies directed against cell-surface molecules or alternatively one or more membrane receptor ligands such as insulin, transferrin, folic acid or any other growth factor, cytokines or vitamins. Modified or unmodified lectins may be advantageously used in the formulation to target particular cell surface polysaccharides or adjacent extracellular matrix. Thus, proteins with an RGD motif, cyclic or acyclic peptides containing a pair of RGD motifs, as well as polylysine peptides can be used. These targeting agents may be conjugated to either a recombinant virus and / or a non-viral transfection vector.

The doses of virus used to administer the function of various parameters, in particular the administration used, relating alternatively to the length of treatment required, according to the invention are generally formulated at doses between 10 ⁴ to 10 ¹⁴ pfu / ml. Term pfu

can be set as than features way on the pathology or recombinant viruses and administered in the form

The plaque-forming unit corresponds to the infectious strength of the virion suspension and is determined by infection of the respective cell culture and by measuring, generally after 48 hours, the number of plaques of the infected cells. Methods for determining the pfu titer of a viral solution are well documented in the literature.

Of course, the amount of associated non-viral transfection vector varies according to its nature, the nature of the viral vector with which it is associated, and also the cell type it is targeted to. Generally speaking, an amount in the range of 50 ng to 1 pg is used in vitro. For ex vivo applications, the amount of associated non-viral transfection vector varies according to the amount and type of cell tissue to be transfected. This amount may be between 60 ng / ml and 500 μς / ιηΐ.

The compositions of the invention may be formulated for topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, and the like routes of administration. The pharmaceutical preparations of the invention preferably comprise a pharmaceutically acceptable carrier for an injectable preparation, in particular for direct injection at the desired organ level or for topical administration (to the skin and / or mucous membranes). In particular, they may be sterile isotonic solutions or dry, in particular lyophilized formulations, which allow the solution to be injected by the addition of sterile water or physiological serum as appropriate. The doses of nucleic acid used for injection, as well as the number of administrations, can be set as a function of various parameters, in particular as a function of the methods of administration used, related to the pathology, the gene to be expressed or alternatively to the length of treatment desired. More specifically, the mode of administration may be either direct injection into the tissue or circulation, or processing of cells in culture followed by their reimplantation in vivo, injection or graft. The present invention also relates to cells treated with a composition of the invention for use ex vivo.

The present invention will be more fully described by way of examples and figures which follow, which are to be considered illustrative and not limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Effect of lipofectamine on the efficiency of AdPGal transduction at VSMC level.

Figure 2: Comparison of Ad-luc transduction efficiency in the presence and absence of lipofectamine.

Figure 3: Detection of crystal violet CMV cells transduced with Ad-TK and treated with GCV, in the presence and absence of 0.125 gg lipofectamine.

Figure 4: Evaluation of the effect of lipofectamine on Ad-luc neutralization in FCS or AHS medium for doses ranging from 0 to 0.5 gg lipofectamine (Figure 4A) and from 1 to 10 µg lipofectamine (Figure 4B).

Figure 5: Inhibition of smooth muscle cell proliferation by overexpressing the Gax protein via adenovirus alone (I) or using CLAAT (II).

Figure 6: Ex vivo transfection of adenovirus on isolated artery in combination with increasing doses of lipofectamine.

DETAILED DESCRIPTION OF THE INVENTION

Material and methods

Hereinafter, various methods used in the examples showing the synergy between recombinant adenoviruses and cationic liposomes in transduction of vascular smooth muscle cells in primary culture are described.

A. Primary vascular smooth muscle cell culture (VSMC)

VSMCs are obtained by enzymatic cleavage of rabbit aorta according to the modified method of Chamley et al. (Cell Tissue Res. 197, 177, 503-522) and then seeded in primary culture. Rabbit aorta was removed for this purpose and then incubated for 45 minutes in the presence of collagenase (Collagenase II, Cooper Biomedical) at 37 ° C. The second cleavage in the presence of collagenase and elastase (Biosys) is carried out for two hours at 37 ° C. These two cleavages allow a cell suspension formed essentially with VSMC to be obtained. VSMCs are maintained in culture in DMEM (Gibco) containing 20% fetal calf serum '. (FCS, Gibco) and for all subsequent experiments were used before passage 10. VSNC phenotyping was performed at each passage by immunofluorescence using antibodies directed against specific alpha-actin smooth muscle cells (Sigma).

B. Recombinant Adenoviruses (Ad)

Different recombinant adenoviruses were used for various examples of VSMC transduction. All recombinant Ad used are first generation and defective in replication with a deletion of the E1 region.

Ad-Luc carries an expression cassette containing the luciferase gene under the control of the CMV promoter. This expression cassette is derived from the commercial plasmid pUT650 (Caylam Toulouse, France) and contains a luciferase gene fused to the zeo resistance gene under the control of the CMV promoter. The expression cassette was inserted in the E1 region of the In340 adenovirus (Feldman et al., Improvement in Arterial Gene Transfer by Poloxaner 407 as a Vehicle for Adenonoviral Vectors, Gene Ther., 1997. 4: 189-99; Robert, JJ et al. , Gene Neurochemistry, 1997, Vol. 68, pp 2152-2160; Hearing et al. 1983, Cell 33, pp 695-703).

Ad-pGal carries a cassette encoding the Escherichia coli β-galactosidase gene fused with its N-terminal portion to a nuclear localization sequence (NLS), identical to the SV40 virus T antigen sequence, under the control of the promoter. LTR-RSV (StratfordPerricaudet et al., Widespread long-term gene transfer to mouse skeletal muscles and heart, J. Clin. Invest., 1992, 90: 626630).

Ad-tk encodes the Herpes simplex virus thymidine kinase gene under the control of LTR-RSV (Maron et al., Gene therapy of rat C6 glioma using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene: long-term follow-up by magnetic resonance imaging. Gene Ther., 1996, 3: 315-22).

C. Transduction of VSMC by recombinant Ad alone or complexed with cationic liposomes

VSMCs are inoculated on a 24-well culture dish (Falcon) at a density of 50,000 cells per well in 1 ml DMEM containing 10% FCS (DMEM-FCS). After 20-24 hours, DMEM-FCS of VSMCs is replaced with 300 μΐ serum-free DMEM. The cells are then infected with recombinant viruses, alone or in complex with liposomes, with a multiplicity of infection (MOI) varying according to experiments. This adenoviral preparation was added after incubation for 30 minutes at ambient temperature to a suspension of 100 μΐ of water or phosphate buffered saline (PBS) containing either Ad alone with the desired MOI or Ad with varying amounts of cationic liposomes (Lipofectamine, Gibco). 100 μΐ Ad or Ad-lipofectamine complex was added directly to VSMC in 300 μΐ DMEM. For experiments designed to study the effect of neutralizing human serum (AHS) compared to FCS on transduction efficiency, the transduction mixture was replaced with DMEM 0.5% FCS after 1 hour at 37 ° C. 24 hours after infection, cell proliferation is induced by the addition of serum-rich medium (DMEM 10% FCS). Cell viability (viability) is determined 72 hours after infection by Alamar Blue (Biosource) assays.

In the case of VSMC transduced with Ad-tk, cells are contacted with 25 µg of ganciclovirus (GCV) after 48 hours of culture. The same cells uninfected or cells transduced with Ad-pGal serve as controls untreated or treated with GCV.

D. Transmission of an adenovirus encoding a luciferase gene in isolated arteries

Abdominal arteries of New Zealand White rabbits are collected following euthanasia with an overdose of sodium pentobarbital. Arterial abrasion is performed using a 3F Fogarty catheter inflated with 0.15 ml air. The arteries are cut into 5 mm long fragments, which are then opened longitudinally. Each fragment is placed in a well of a 12-well plate in 1 ml serum free medium.

The infection takes place on the same day as the collection. Adjust the volume of adenovirus mixture (AVi.oCMVLuc or AVi.oCMVPGal, 10 ⁸ pfu) and lipofectamine to 80 μΐ per well with PBS. The solution is then incubated for 30 minutes at ambient temperature and then placed on the artery fragments found in 1 ml serum free medium in one well of a 12-well plate. The fragments are then incubated for 1 hour at 37 ° C and then transferred to a new 12-well plate and incubated in 2 ml DMEM 20% FCS medium. Transmission evaluation is done by measuring luciferase activity (AVi.oCMVLuc) or by histochemistry (AVi. ₀ CMV3Gal) 3 days after infection.

Several doses of lipofectamine for constant MOI were tested

AVi.oCMVLuc.

E. Analysis of VSMC transduction efficiency by recombinant Ad associated or unassociated with liposomes.

Determination of luciferase activity

AdMC-transduced VSMCs were collected in 200 µL of luciferase assay buffer (Prom'ega). After 15 minutes lysis, 10 μΐ of each sample was used to determine luciferase activity using the Luciferase assay kit (Promega) and the luminometer (Berthold). Luciferase activity is expressed in RLU (Relative Light Units).

Measurement of luciferase activity on isolated arteries requires that the artery fragments be crushed in 500 μΐ lysis buffer containing anti-proteases and then incubated for 20 minutes with stirring at ambient temperature. Subtraction is performed on 10 μΐ of extract in the presence of 50 μΐ of substrate for 10 s.

Determination of β-galactosidase activity in cells β-galactosidase activity in VSMCs transduced with Αϋ-βΘβΙ is determined by chemiluminescence using the Galacto-Light plus kit (from Tropix, Inc.). The cells are washed in PBS and then transferred to a 250 μΐ lysis solution containing 100 μΜ potassium phosphate pH 7.8 and 0.2% Triton X-100. The lysates are centrifuged for 2 minutes at 10,000 g and then 5 μΐ of each sample is mixed with 50 μΐ of Galactom-Plus ™ reaction buffer and incubated for 1 hour at ambient temperature. To measure luminescence, the mixtures are placed in a luminometer (light emission accelerator) and the luminescence measured in RLU is measured.

Histochemical detection of β-galactosidase activity in cells

VSMCs, on culture broth or separated by trypsin treatment, are washed with PBS and fixed in PBS containing 4% formaldehyde. The fixation solution is removed at the end of 10 minutes and then cells are stained for 1-4 hours at 37 ° C in PBS containing 4 mM potassium ferrocyanide, 4 mM potassium ferrocyanide, 200 mM magnesium chloride and 0.4 mg / ml X-Gal substrate. The percentage of blue cells is counted from the VSMC in suspension, while the VSMC on the culture medium is photographed.

Histochemical detection of β-galactosidase activity in tissue sections

3 days after infection, the fragments were taken tissues (arteries) fixovali 3 hours in 4% PBS-formole. then rinsed and were treated as follows: PBS 3 x 20 minutes _PBS-MgCl2 30 minutes _PBS-MgCl2 30 minutes at 4 ° C Permeabilizing solution 30 minutes at 4 ° C

(2 mM PBS-MgCl ₂ , 0.01% sodium deoxycholate and 0.02% NP40)

Staining solution 4 to 22 hours at 37 ° C (permeabilization solution plus 35 mM K ₄ Fe (CN) 6 · 3H ₂ 0 and 35 mM K ₃ Fe (CN) ₆

They were then rinsed in PBS and placed in cuvettes and dehydrated as follows:

60% ethanol hour

80% ethanol 1 hour 100% ethanol 1 hour 100% ethanol 1 hour 100% ethanol 1 hour xylene 1 hour xylene 1 hour xylene 1 hour xylene 2 hours paraffin 2 hours paraffin 3 Hours

They were then embedded in paraffin and cut on a microtome.

Evaluation of cell viability by crystal violet staining

To evaluate the efficiency of Ad-tk transduction, VSMCs were treated with GCV as described above. The living cells selected after this treatment were rinsed with PBS and then fixed in PBS containing 4% formaldehyde. At the end of 15 minutes the fixation solution was removed and the cells were stained for 20 minutes in 0.1% crystal violet in water. After staining, the cells were washed three times with water and then photographed.

Example 1

This example illustrates VSMC transduction when Ad-PGal is associated with a cationic liposome such as lipofectamine.

VSMCs are transduced according to the protocol described in Material and Methods using Ad-pGal at an amount corresponding to an MOI of 10 in the presence of increasing doses of lipofectamine ranging from 0 to 0.5 µg. Histochemical detection of β-galactosidase activity clearly showed that from a dose of 0.125 gg lipofectamine significantly increases the efficiency of VSMC transduction with the Ad-PGal vector.

To quantify the efficiency of transduction, the percentage of cells positive for β-galactosidase activity after histochemical staining of cells in suspension was determined according to the protocol outlined in Material and Methods. The results are shown in FIG.

First

When Ad? Gal alone with an MOI of 10 is used, only 1 to 2% of VSMC is positive, but immediately after the addition of lipofectamine from a dose of 0.125 gg, the number of positives exceeds 40%. This transduction efficiency is perfectly correlated with the β-galactosidase activity measured by the chemiluminescence method (Figure 1). A slight decrease in this activity was observed when the lipofectamine concentration increased. This reduction is likely due to poor toxicity, as shown by cell counting (dotted line in Figure 1). However, this toxicity, manifested only at the highest lipofectamine dose of 0.5 μς, does not affect more than 25% of the cells.

The results clearly show an improvement in transduction due to the fact that Ad-Gal was combined with a cationic lipofectant.

Example 2

In this example, the lipofectamine dose was determined by transducing the VSMC with an Ad-Gal vector with an MOI increasing up to 10. Ad-Luc with different MOI values was used for VSMC transduction, and luciferase activity was determined as described in Materials and Methods. . Luciferase activity increased almost linearly as a function of the MO-Ad-Luc used for VSMC transduction (Figure 2, open circles). Interestingly, at any of the MOIs used, the addition of 0.125 gg of lipofectamine to Ad-Luc (Figure 2, solid diamonds) caused a 100-fold increase in the recorded luciferase activity. This shows that up to an MOI of 100 Ad-Luc, a small amount of lipofectamine, i. 0.125 gg is sufficient to significantly improve VSMC transduction.

Example 3

The adenovirus used in this example is the Ad-tk adenovirus used herein for the cytotoxic properties of the herpes simplex virus thymidine kinase gene in the presence of ganciclovir (GCV). VSMCs were transduced with Ad-TK with an MOI of 1, 10, and 1000 and then treated with GCV as described in Material and Methods. Ad-pGal with an MOI of 100 was used as a control.

Ad-TK and Ad-pGal were used alone or in complex with 0.125 gg of lipofectamine. Figure 3 shows VSMC staining with crystal violet after transduction and GCV treatment.

GCV was expected to exhibit no toxicity to cells transduced with Ad-QGal either in the presence or absence of lipofectamine. VSMC Ad-TK transduction followed by treatment with GCV showed toxicity (viability 30-50%) only at the highest MOI, i. j. Interestingly, when Ad-TK was complexed with 0.125 gg lipofectamine, the efficacy after treatment with GCV at MOI 1 was comparable to that when using Ad-TK alone with MOI 100. At MOI 10 or 100, Ad-TK is Ad- TK. BP associated with lipofectamine is highly toxic in the presence of GCV. This example provides evidence of the meaning and composition of the invention. At the same time, it shows that it is possible to significantly reduce the amount of recombinant Ad-TK virus administered, while achieving the same or higher therapeutic efficacy as compared to that achieved using Ad-TK alone.

Example 4

In addition to the efficacy of transduction, which, however, can be improved, as shown in the previous example, another major obstacle to the optimal efficacy of a recombinant adenovirus is its neutralization by the immune system. Indeed, after cell lysis after the first transfer to the animal or after the first human contact with the adenovirus, the immune system can very effectively neutralize the virus in the bloodstream and thus reduce the possibility of repeated injection.

This example reproduces in vitro neutralization of the recombinant adenovirus by a set of human sera and preferably shows that the association of the recombinant adenovirus with liposomes allows the neutralization to be abolished, so that transduction, depending on the dose of the liposome, may be higher than that achieved using the virus alone.

Figures 4A and 4B show VSMC cells transduced with Ad-Luc at MOI 100 in the presence of calf fetal serum (FCS) or adult human serum (AHS) in DMEM culture medium.

Although 10% FCS does not significantly alter the efficiency of Ad-Luc transduction relative to Ad-Luc alone in DMEM, luciferase activity is significantly reduced in the presence of 10% AHS. Increased doses of lipofectamine added to Ad-Luc affect luciferase activity, so that it is consistent or even at the highest doses of lipofectamine significantly higher than when using virus alone (Figure 4B).

Thus, this example has shown that the preparation of the invention, on the one hand, makes it possible to completely suppress the neutralization of recombinant adenovirus by antibodies and, on the other hand, makes it possible to increase the efficiency of transduction in the presence of neutralizing serum.

Example 5

This example demonstrates an increase in the transduction of the therapeutic gene (gax) into rabbit smooth muscle cells by exemplifying the adenovirus AVi, oCMVrGAX, and a cationic lipid such as lipofectamine (CLAAT).

Rabbit smooth muscle cells in DMEM with 10% FCS were seeded in the MW48 microplate at 5 x 10 ⁴ cells per well. Infection was performed 24 hours later in serum-free DMEM medium. Different dilutions of adenovirus were mixed with 60 ng of lipofectamine and made up to a total volume of 25 μΐ with PBS. This solution was incubated at room temperature for 30 minutes. The culture medium was then replaced in each well with 175 μΐ serum free medium and adenovirus-lipofectamine mixture was added to the cells. The actual infection was 1 hour at 37 ° C. The virus-containing medium was then replaced with DMEM with 0.5% FCS. 24 hours after infection, cell proliferation was induced by the addition of serum-rich medium (DMEM with 10% FCS). Cell viability was evaluated using the Alamar Blue assay (Biosource).

This example showed that lipofectamine alone and the dose necessary for CLAAT had no effect on cell viability. However, the MOI necessary to obtain 50% inhibition of SMC growth is 10x less when using a mixture of AVi ₍ oCMVrGAX and lipofectamine compared to AVi, oCMVrGAX alone. Figure 5 shows a comparison of inhibition of smooth muscle cell proliferation by overexpressing GAX protein by adenovirus (I) or using CLAAT (II) Cells were infected with an MOI of 0 to 10 ⁵ PV / cell, either with AVi ₍₀ CMV (a) or alone, AV ₁₀ CMVrGAX (b). Using CLAAT, the MOI was from 0 to 3x10 ⁴ PV / cell of the same viruses (II-a, II-b) After infection, the cells were incubated for 24 hours in DMEM with 0.5% FCS, and the medium was then exchanged for serum-rich medium. hours after infection.

This example shows the increased transduction of the therapeutic gene to the cells of rabbit smooth muscle of example the combination of adenovirus AVi, ₀ CMVrGAX and lipofectamine. The example has shown that a lower dose of virus can be used in the case of a combination of a cationic lipid and an adenovirus.

Example 6

This example demonstrates the transfer of genes to isolated artery fragments using a combination of an adenovirus encoding luciferase or β-galactosidase and lipofectamine (CLAAT).

Artery fragments were obtained and infected as described in Material and Methods (D).

To prepare cellular extracts and measure luciferase activity, the artery fragments were crushed in 500 μl lysis buffer containing antiproteases and then incubated for 20 minutes with stirring at room temperature. They were then settled for 5 minutes at 10,000 rpm. Evaluation was performed with 10 μ 10 of extract in the presence of 50 μΐ of substrate for 10 seconds using the Luciferase assay kit (Promega) and the luminometer (Berthold).

Based on the results presented in Figure 6, it can be concluded that on the one hand luciferase activity is clearly increased and, on the other hand, that it is dose-dependent when the adenovirus is transferred to isolated arteries by CLAAT.

Detection of β-galactosidase activity was performed as described in Material and Methods (E). The histochemical results obtained show that cells that express β-galactosidase after transfer by CLAAT are located in adventitia (outer wall envelope) and are fibroblast-like cells. In addition, the number of transduced cells appears to be higher than when adenovirus alone with the same MOI is transmitted.

This example shows that gene transfer to an isolated artery by CLAAT is possible. Moreover, this ex vivo method makes it possible, on the one hand, to improve gene transfer and, on the other hand, to obtain cells from adventitiousness. Thus, it can be envisaged to use this method to treat vascular grafts with the desired genes, e.g. FGF, carried by adenoviral vectors.

Claims (31)

A transfection preparation suitable for gene therapy, characterized in that it associates one or more packaging-free recombinant viruses comprising in their genome at least one exogenous nucleic acid with at least one transfection agent other than a virus or a plasmid. Second The composition of claim 1, wherein the non-viral transfection agent is selected from cationic polymers or lipofectants. The composition of claim 1 or 2, wherein the non-viral transfecting agent is a lipofectant. The composition of claim 3, wherein the composition is a lipid capable of forming liposomes, mock liposomes, immunoliposomes, or targeted liposomes. The composition of claim 3, wherein the lipofectant comprises at least one polyamine stretch of the general formula: H ₂ N - (- (CH) _m -NH-) _n -H Where m is an integer equal to or greater than 2 and n is an integer equal to or greater than 1, wherein m may vary for different carbon groups, between two amines covalently linked to a lipophilic portion of a saturated or unsaturated hydrocarbon chain of cholesterol, or natural or synthetic lipid capable of forming lamellar or hexagonal phases. The composition of claim 5, wherein the polyamine region is sperm, thermin, or one of their analogs, having retained nucleic acid binding properties. The composition of claim 3, wherein the lipofectant has the general formula H ₂ N - (- (CH) _m -NH-) _n -HIR wherein R represents a lipophilic region of the general formula Y ----- R ⁶ wherein X and X 'represent, independently of one another, an oxygen atom, a methylene group - (CH ₂ ) _q - sq equal to 0, 1, 2 or 3, or an amino group, -NH- or - NR'-, wherein R 'represents an alkyl group having 1 to 4 carbon atoms. Y and Y 'independently of one another represent a methylene group, a carbonyl group or a C = S group, R ³ , R ⁴ and R ^5, independently of one another, represent a hydrogen atom or a substituted or unsubstituted C 1 -C 4 alkyl group, wherein p may range from 0 to 5, R ⁶ represents a cholesterol derivative or an alkylamino group -NR ¹ R ² , wherein R ¹ and R ² , independently of each other, represent a saturated or unsaturated, linear or non-linear or branched aliphatic group containing from 12 to 12 carbon atoms; 22 carbon atoms. A composition according to claim 3, wherein the lipofectant has the general formula having the values wherein R ¹ , R ² and R ³ represent, independently of one another, a hydrogen atom or a group - (CH 2) _q -NRR ', provided that: q can be 1, 2, 3, 4, 5 and 6, independently for different R ¹ , R ² and R ³ , R and R 'independently from each other represent or - (CH 2) _q -NH _{2 /,} wherein q may have values of 5 and 6, independently for different R and R' groups. hydrogen atom 1, 2, 3, 4, m, n and p represent, independently of one number, which can be between 0 and 6, if n 1, m can have different values and R ^{3 have} different meanings of the formula, and the other, whole t is larger than in general R ⁴ represents a group of the general formula to X wherein R6 and R7 independently of one another represent a hydrogen atom or a saturated or unsaturated aliphatic group containing 10 to 22 carbon atoms, wherein at least one of the two groups is not hydrogen, X represents an oxygen or sulfur atom or an optionally monoalkyl amino group, Y represents a carbonyl group or a methylene group, R ⁵ represents a hydrogen atom or a natural amino acid side chain, if necessary so substituted, ar is an integer ranging from 1 to 10, and when r is 1, R ⁵ represents a substituted or unsubstituted natural amino acid side chain, and r is greater than 1, R ⁵ represents a hydrogen atom. The composition of claim 1 or 2, wherein the non-viral transfection agent is selected from polyalkylenimines. is an acacia cationic polymer The formulation according to claim 9, which is polyethyleneimine (PEI) characterized by polypropyleneimine Composition according to claim 1, characterized in that i comprising a molecular effect or 2 characterized by a non-viral transfection agent is selected from lipofectamine, polyethyleneimine having a mass of 50,000 (PEI50K), 800 palmitoyl phosphatidyl-5-bis (3-aminopropyl-1,3-bis (3-acetate), molecular weight group average s polyethyleneimine 000 (PEI800K), the average dioctadecylamidoglycylspermine (DOGS), ethanolamine-5-carboxyspermylamide (DPPES) amino) pentyl (dioctadecylcarbamoylmethoxy methoxy) acetate, -aminopropyloamino) Jpropyl (dioctadecylcarbamoylmethoxy methoxy) {H ₂ N (CH ₂₎ 312N ( CH ₂ ) ₄ N (CH ₂ ) ₃ NH ₂ } (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₇ -CH ₃ ] ₂ , H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₈ ] 2, H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COArgN [(CH ₂ ) ₁₈ ] 2 and H ₂ N (CH _{2) 3} NH (CH _{2) 4} NH (CH _{2) 3} NHCH ₂ COGlyN [(CH _{2) 7} CH _{3] 2} Composition according to any one of the preceding claims, characterized in that the nucleic acid found in the recombinant virus is deoxyribonucleic acid. 13. The composition of any one of claims 1 to 11, wherein the nucleic acid present in the recombinant virus is a ribonucleic acid. 14. The composition of claim 12 or 13, wherein the nucleic acid is chemically modified. 15. The composition of claim 12, 13 or 14, wherein the nucleic acid is an antisense nucleic acid. The composition of any preceding claim, wherein the nucleic acid comprises a therapeutic gene. The composition of claim 16, wherein the therapeutic gene encodes the product derivatives, TNF, NGF precursors, IGF,, dystrophin gene encoding enzymes, blood interleukins, interferons, neurotransmitters or trophic factors: BDNF, NT5, HARP / pleiotrophin and CFTR protein. their CNTF, further, is by the group of lymphokines: factors, syntheses, GMF, aFGF, bFGF, NT3, or minidystrophin and selected hormones, growth or enzymes of aFGF: The composition according to claim 16, characterized in that the therapeutic gene is selected from genes associated with cell division arrest, in particular from the group comprising the gax gene, tumor suppressor genes: p53, Rb, Rapla, DCC, k-rev, genes encoding factors involved in coagulation: factors VII, VIII, IX, genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), hemoglobin or other protein transporter genes, genes responsive to proteins involved apolipoprotein-type lipid metabolism selected from apolipoproteins A1, A-II, A-IV, B, CI, C-II, C-III, D, E, F, G, H, J and apo (a) metabolic enzymes, such as lipoprotein lipase, liver lipase, lecithincholesterolacyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidic acid phosphatase, or alternatively lipid transfer proteins, such as cholesterol ester transfer protein and phospholipid transfer protein, LDL receptor binding protein, or alternatively a receptor selecting HDL, residues of chylomicrons, or scavenger receptors. A composition according to any one of the preceding claims, characterized in that the packaging-free recombinant virus does not contain at least such regions of its genome as are necessary for its replication in the infected cell. The composition according to any one of the preceding claims, characterized in that the packaging-free recombinant virus is derived from adenovirus or AAV. The preparation according to any one of the preceding claims, characterized in that the packaging-free recombinant virus is preferably derived from an adenovirus of animal or human origin. Composition according to any one of the preceding claims, characterized in that the packaging-free recombinant virus is derived from an Ad5 or Ad2 type adenovirus. A composition according to any one of claims 1 to 4 and 12 to 22, characterized in that it associates a recombinant adenovirus containing in its genome one exogenous nucleic acid and lipofectamine in an amount sufficient to improve cellular transduction. A composition according to any one of the characterized in that it is dioleoylphosphatidylethanolamine phosphatidylethanolamine (POPE) or that (DOPE) myristoylphosphatidylethanolamine adjuvant 3 times N-methylated; glycosyldiacylglycerols, (especially derivatives 1 to diacylglycerols, galactocerebrosides), sphingolipids, alternatively asialogangliosides. The foregoing claims additionally comprises an adjuvant or oleyl palmitoyldi stearoyl, -palmitoyl type, as well as their phosphatidylglycerols, cerebrosides (in particular sphingomyelins) or The composition of any preceding claim, further comprising a compound affecting, directly or indirectly, the level of nucleic acid condensation. Composition according to claim 25, characterized in that the compound consists, in whole or in part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of which motifs can range from 2 to 10. A composition according to any one of the preceding claims, characterized in that, in addition to the aforementioned vectors, it is additionally associated with a targeting element. 28. The composition of claim 27, wherein the targeting element is selected from the group consisting of antibodies directed against cell surface molecules, ligands or membrane receptors such as insulin, transferrin, folic acid or other growth factor, cytokines or vitamins, modified or unmodified lectins. , an RGD motif protein, cyclic or acyclic peptides comprising a pair of RGD motifs, as well as polylysine peptides. Use of a composition according to any one of claims 1 to 28 for transferring nucleic acids into cells. 30. A recombinant cell obtained by contacting a composition according to any one of claims 1 to 28. A method of transferring nucleic acids into a cell interior, wherein the cell is contacted with a composition according to any one of claims 1 to 28. RLU / well