AU737846B2 - Transfectant composition useful in gene therapy associating with a recombinant virus incorporating an exogenous nucleic acid and a non-viral and non-plasmid transfection agent - Google Patents

Description

TRANSFECTANT COMPOSITION USEFUL IN GENE THERAPY ASSOCIATING WITH A RECOMBINANT VIRUS INCORPORATING AN EXOGENOUS NUCLEIC ACID AND A NON-VIRAL AND NON-PLASMID TRANSFECTION AGENT.

The present invention relates to the field of gene therapy and is involved more particularly in the in vitro, ex vivo and/or in vivo transfer of genetic material. It especially proposes a novel composition useful for effectively transfecting cells.

Chromosomal deficiencies and/or anomalies (mutation, aberrant expression, etc.) are at the origin of numerous illnesses, of hereditary or non-hereditary character. For a long time, conventional medicine has oooo• remained powerless with regard to them. Today, with the 15 development of gene therapy, it is hoped to be able in goo future to correct or prevent this type of chromosomal aberration. This novel medication consists in introducing genetic information into the affected cell or organ, with a view to correcting this deficiency or anomaly or alternatively of expressing a protein of interest there.

The major obstacle to the penetration of a nucleic acid into a cell or a target organ lies in the size and the polyanionic nature of this nucleic acid which opposes its passage through the cell membranes.

To remove this difficulty, various techniques are proposed today including more particularly the transfection of naked DNA through the plasmic membrane in vivo (WO 90/11092) and the transfection of DNA via a transfection vector.

As far as this second technique is concerned more particularly, it principally proposes two strategies: The first employs the natural transfection vectors which are viruses and the second lies in the employment of chemical or biochemical vectors.

As far as the vectors of viral origin are concerned, they are today widely used for cloning, the transfer and the expression of genes in vitro (for the production of recombinant proteins, the carrying-out of screening tests, the study of the regulation of genes, etc.), ex vivo or in vivo (for the creation of animal models, or in gene transfer approaches of therapeutic interest). Among these viruses, it is possible especially to mention adenoviruses, adeno-associated viruses (AAV), retroviruses, herpesviruses or even vaccinia viruses.

The family of the Adenoviridae is widely distributed in mammals and birds and gathers together more than one hundred different serotypes of nonenveloped double-stranded DNA viruses having a capsid of icosahedral symmetry (Horwitz, In: Fields BN, Knipe DM, Howley PM, ed. Virology. Third edition ed.

Philadelphia: Raven Publishers, 1996: 2149-2171). Their genome especially comprises an inverted terminal repeat 3 (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes. These principal early genes are contained in the El, E2, E3 and E4 regions. Among these, the genes contained in the El region are necessary for viral propagation. The principal late genes are contained in the L1 to L5 regions. The genome of the Ad5 adenovirus has been entirely sequenced and is accessible in databases (see especially Genebank M73260). In the same way, parts, or even the entirety, of other adenoviral genomes (Ad2, Ad7, Adl2, etc.) have likewise been sequenced.

In addition to its innocuousness, the adenovirus has a very wide cellular tropism. On the contrary to the retrovirus whose cycle is dependent on cell division, it can advantageously infect cells in active division as well as quiescent cells and its genome remains in episomal form. In addition, it can be produced with high titres (101 pfu/ml). These major assets have made it a vector of choice for the cloning and the expression of heterologous genes. The adenoviruses of group C, especially of type 2 and 5, as well as the canine adenoviruses of type CAV-2, whose molecular biology is the best known, are at the origin of the vectors actually used. It is for this reason that adenoviral vectors have been used for the cloning and expression of genes in vitro (Gluzman et al., Cold Spring Harbor, New York 11724, p. 187), for the creation of transgenic animals (WO 95/22616) for the 4 transfer of genes into cells ex vivo (WO 95/14785; WO 95/06120) or alternatively for the transfer of genes into cells in vivo (see especially WO 93/19191, WO 94/24297, WO 94/08026) Concerning the adeno-associated viruses (AAV), they are DNA viruses of relatively reduced size, which are integrated into the genome of the cells which they infect, in a stable and relatively site-specific manner. They are capable of infecting a wide spectrum of cells. The genome of the AAV has been cloned, sequenced and characterized. It comprises approximately 4700 bases, and contains at each end an inverted terminal repeat (ITR) of approximately 145 bases, serving as a replication origin for the virus. The rest of the genome is divided into 2 essential regions carrying the encapsidation functions: the left part of the genome, which contains the rep gene involved in the viral replication and expression of the viral genes; the right part of the genome, which contains the cap gene coding for the capsid proteins of the virus. The use of vectors derived from AAV for the transfer of genes in vitro and in vivo has been described in the literature (see especially WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528).

These vectors and more particularly the adenoviruses turn out to be impressive on the transfection level. Conventionally, the process of infection of cells by a viral transfection vector generally takes place in two steps. In a first stage, there is recognition of target receptors on the surface of the cells to allow the attachment of the recombinant virus. The nature of these receptors as well as their distribution according to the cell types remain little documented, however in the particular case of the adenovirus, it seems that it is the fibre, a protein on the external surface of the adenovirus, which may be involved for this attachment step. The second step then consists in an interaction of the viral protein penton, at the root of the fibre, via an RGD peptide motif with the cell integrins avb3 and/or avb5 leading to the internalization of the virion.

The efficacy with which the recombinant viruses can infect a cell (efficacy of transduction) is very variable according to the types of cells studied in culture or the tissues whose screening is important for an in vivo treatment. In all cases, it is the first attachment step which determines the efficacy of internalization of the virion. The quantity of receptors on the surface of the cell thus forms the limiting factor in the infection process and it is clear that any approach aiming at bypassing this attachment step or making it more easy will allow this obstacle to be cleared.

A first objective of the present invention aims precisely at increasing the efficacy of transfection of these viral vectors by optimizing their 6 internalization at the level of the target cell to be treated.

Another objective aimed at and achieved by the present invention relates to the reduction or even the suppression of the immune response conventionally manifested by treated cells against the viral vector.

In fact, as for all the known viruses, the administration of recombinant viruses such as recombinant adenoviruses which are defective for replication (Yang et al., PNAS (1994) 4407) induces a significant immune response. One of the major roles of the immune system consists in fact in destroying nonself or self-altered elements. The administration of a gene therapy vector of viral origin introduces non-self motifs into the organism. In the same way, cells infected by such a vector and as a result of this expressing an exogenous gene become self-altered elements. The immune response developed against these infected cells thus forms a major obstacle to the development of these viral vectors since in inducing a destruction of the infected cells it limits the duration of expression of the gene and thus the therapeutic effect, (ii) it induces in parallel a significant inflammatory response, and (iii) it involves the rapid elimination of infected cells after repeated injections.

Advantageously, the present invention allows these two objectives to be achieved by associating with the recombinant virus under consideration a chemical or biochemical compound which effectively assists in its transduction and protects it from reactions of the immune system manifested on encountering it.

As has been mentioned previously, parallel to the viral vectors, non-viral transfection vectors have likewise been developed at the gene therapy level. More precisely, they are chemical or biochemical vectors.

These synthetic vectors have two principal functions, to compact DNA to be transfected and to promote its cellular attachment as well as its passage through the plasmic membrane and, if necessary, through the two nuclear membranes.

Not as impressive as the viral vectors, these chemical vectors, on the other hand, are more advantageous on the immune plane. They do not manifest pathogenic power, the risk of multiplication of DNA in its vectors is zero and no theoretical limit is attached to them as far as the size of the DNA to be transfected is concerned.

Among the synthetic vectors developed, cationic polymers of polylysine and DEAE-dextran type or even lipofectants are the most advantageous. They have the property of condensing DNA and of promoting its association with the cell membrane. More recently, the concept of targeted transfection, mediated by a receptor, has been developed. This technique profits f4 from the principle of condensing DNA, owing to the 4j) cationic polymer, while at the same time directing the attachment of the complex to the membrane with the aid of a chemical coupling between the cationic polymer and the ligand of a membrane receptor, present at the surface of the cell type which it is wished to graft.

The targetings of the receptor for transferrin, for insulin or of the receptor of asialoglycoproteins of hepatocytes have thus been described.

The principal object of the present invention lies precisely in the association of at least one of these chemical or biochemical vectors with a recombinant virus comprising in its genome at least one exogenous nucleic sequence with a view to promoting more efficiently the cellular transduction of this recombinant virus.

Unexpectedly, the Applicant has demonstrated that it was possible to take advantage simultaneously of the respective properties of these two types of transfection vectors to efficiently induce the expression of therapeutic genes.

The present invention advantageously profits at the same time from the property of vehicles of the chemical vectors of the liposome or lipofectant type capable of fusing with a wide range of cell types and the efficiency of internalization of the recombinant viruses such as more particularly the adenovirus. A synergy between the virion as transduction agent of a gene of therapeutic interest and the lipofectant as transfection assistant follows. This synergism is advantageously manifested at two levels.

Initially, it is expressed by a very significant increase in the local concentration of recombinant viruses. Assistance is thus with a recombinant adenovirus associated with a liposome with an efficiency of transfection 50 to 100 times higher than that observed with the recombinant adenovirus alone, in a cell type such as the vascular smooth muscle cells where the fibre receptor is very little expressed. This observation has additionally been confirmed with different recombinant adenoviruses. It thus proves possible, in the presence of a chemical or biochemical vector, to employ more reduced quantities of recombinant viruses for an efficiency which is at least equivalent if not higher.

Finally, it has been unexpectedly noted that the association of a chemical vector with a recombinant adenovirus allows the phenomenon of neutralization usually induced by the immune system on encountering recombinant viruses to be attenuated efficiently. When an adenovirus is brought together, in an isolated form, with a human serum containing anti-Ad neutralizing antibodies, a severe decrease in the efficiency of transduction is observed. Unexpectedly, this neutralization is increased significantly in the presence of a chemical vector of lipofectant type.

According to the dose of lipofectant employed, the transduction is either restored or increased from 10 to times by comparison with that of the adenovirus under favourable conditions.

The present invention thus provides a transfectant composition useful in gene therapy, which comprises one or more recombinant non-enveloped viruses comprising in their genome at least one exogenous nucleic acid and at least one non-viral and non-plasmid transfection agent chosen from a lipofectant and polyalkylenimine.

Accordingly, the present invention also provides: a recombinant cell which is obtained by transfection with a composition of the invention; a method of transfer of a nucleic acid to a..

the interior of a cell wherein the cell is contacted with a composition of the invention; and 20 the use of one or more non-enveloped recombinant viruses which comprises in their genome at least one exogenous nucleic acid and at least one nonviral and non-plasmid transfection agent chosen from a lipofectant and polyalkylenimine for the manufacture of a composition for the transfer of nucleic acids to cells wherein the composition is a composition of the invention.

More particularly, as far as lipofectants are A 1'oncerned, they are understood as covering in the sense lOa of the invention, under this designation, any compound with lipid character and already proposed as active agent with respect to the cellular transfection of nucleic acids. Generally speaking, they are amphiphilic molecules comprising at least one lipophilic region associated or unassociated with a hydrophilic region.

As representative of the first family of compounds, it is possible especially to propose lipids capable of forming liposomes, such as POPCs, phosphatidylserine, phosphatidylcholine, cholesterol, lipofectamine, maleimidophenylbutyrylphosphatidylethanolamine, lactosylceramide in the presence or absence of polyethylene glycol to form stealth liposomes or, with or without antibodies or ligands, to form o *oo* oo* *o 11 immunoliposomes or targeted liposomes.

According to a particular mode of the invention, the lipofectant employed has a cationic region. This cationic region, preferentially polyamine, charged cationically, probably associates reversibly with the recombinant virus.

As illustrative cases of this type of cationic lipid constructed on the structure model: lipophilic group associated with an amino group via a "spacer" arm, it is possible more particularly to mention DOTMA and likewise those comprising as lipophilic group two fatty acids or a cholesterol derivative, and having, in addition, if necessary, as amino group, a quaternary ammonium group. DOTAP, DOBT or ChOTB can especially be mentioned as representative *cases of this category of cationic lipids. Other compounds, such as DOSC and ChOSC, are characterized by the presence of a choline group in place of the 20 quaternary ammonium group.

Advantageously, lipofectants suitable for the invention can likewise be chosen from lipopolyamines whose polyamine region corresponds to the general formula

H

2 in which m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, m being able to vary among the different carbon fgroups contained between 2 amines, this polyamine 12 region being associated covalently with a lipophilic region of hydrocarbon chain type, which is saturated or unsaturated, of cholesterol, or a natural or synthetic lipid capable of forming lamellar or hexagonal phases.

This polyamine region is more preferentially represented by spermine, thermine or one of their analogues which has conserved its bonding properties to the nucleic acid.

The Patent Application EP 394 111 describes lipopolyamines of this family capable of being employed in the context of the present invention. As representative of these lipopolyamines, it is possible more particularly to mention dioctadecylamidoglycylspermine (DOGS) and palmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).

The lipopolyamines described in the Patent Application WO 96/17823 can likewise be used advantageously according to the invention. They are represented by the general formula H2N-(-(pH)m-NH-)n-H

R

in which R represents R3 Y'

R

H)p X Y R 6 R4 where -X and X' represent, independently of one another, an oxygen atom, a methylene group -(CH 2 q with q equal to 0, 1, 2 or 3, or an amino group -NH- or with R' representing a Ci 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, to C 4 alkyl radical, with p being able to vary between 0 and 5, R 6 represents a cholesterol derivative or an amino-alkyl group -NRjR 2 with R, and R 2 independently of one another representing a saturated or unsaturated, linear or branched C 12 to C 22 aliphatic radical.

As representative cases of these lipopolyamines, it is especially possible to mention 2- 5-bis(3-amino-propylamino)pentyl (dioctadecylcarbamoylmethoxy)acetate and 2-[1,3-bis(3-aminopropylamino)]propyl (dioctadecyl-carbamoylmethoxy)-acetate.

Finally, more recently novel lipopolyamines, which can likewise be developed in the context of the present invention, have been described in the Patent Application FR 95/13490. They are compounds of general formula as follows R3 0 RI R4 N (CH

(CH,)

/m n P R2 in which RI, R2 and R3 independently of one another represent a hydrogen atom or a -(CH 2 )q,-NRR' group with q being able to vary between 1, 2, 3, 4, 5 and 6, the latter independently between the different groups R1, R2 and R3, and R and R' independently of one another representing a hydrogen atom or a -(CH2),0-NH 2 group, q' being able to vary between i, 2, 3, 4, 5 and 6 and independently between the different groups R and R', m, n and p represent, independently of one another, an integer which can vary between 0 and 6 where, when n is greater than i, m can take different values and R3 different meanings in the general formula and -R4 represents a group of general formula R6

/R

X- CH) Y r U \R7 in which R6 and R7 independently of one another represent a hydrogen atom or a saturated or unsaturated, C10 to C22 aliphatic radical with at least one of the two groups being other than hydrogen, u is an integer chosen between 0 and 10 where, when u is an integer greater than 1, R5, X, Y and r can have different meanings in different motifs [X-(CHR5)r-Y], -X represents an oxygen or sulphur atom or an optionally monoalkylated amine group, Y represents a carbonyl group or a methylene group, R5 represents a hydrogen atom or a natural amino acid side chain, substituted if necessary, and -r represents an integer varying between 1 and 10 where, when r is equal to 1, R5 represents a substituted or unsubstituted natural amino acid side chain and when r is greater than 1, R5 represents a hydrogen atom.

As representative cases of these lipopolyamines, it is more particularly possible to mention those which follow: (H2N(CH2)3 2N(CH2)4N {(CH2)3NH2} (CH2)3NHCH2COGIyN[(CH2)17- CH312 (RP120525)

H

2

N(CH

2 3

NH(CH

2 4 NH(CH2)3NHCH2COGlyN((CH2)1812 RP120535)

H

2

N(CH

2 3

NH(CH

2 4 NH(CH2)3NHCH2COArgN[(CH2) 18] (RP 120531) In another particular embodiment of the invention, the non-viral vector is represented by at least one cationic polymer and more preferentially by a compound of general formula

SN-(CH

2 in which R can be a hydrogen atom or a group of formula

(CH

2 )n-N- J q where n is an integer between 2 and 10, p and q are integers, it being understood that the sum p+q is such that the average molecular weight of the polymer is between 100 and 107 Da. Such compounds are especially documented in the Patent Application W096/02655.

In particular, polyethylenimine (PEI) and polypropylenimine (PPI) polymers have wholly advantageous properties. The preferred polymers for carrying out the present invention are those whose molecular weight is between 103 and 5.106. By way of example, it is possible to mention polyethylenimine of average molecular weight 50000 Da (PEI50K) or polyethylenimine of average molecular weight 800000 Da (PEI800K). PEI50K or PEI800K are available commercially. As for other polymers represented by the general formula above, they can be prepared according to the process described in the Patent Application FR 94 08735.

Particularly advantageously, it is possible to use in the context of the invention lipofectamine, dioctadecylamidoglycylspermine (DOGS), palmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES), 2-5-bis(3-aminopropylamino)pentyl (dioctadecylcarbamoylmethoxy)acetate, 2-[1,3-bis(3-aminopropylamino)]propyl (dioctadecylcarbamoylmethoxy)acetate,

{H

2 N (CH 2 3 2

N(CH

2 4 N{ (CH 2 3 NH2} (CH 2 3

NHCH

2 COGlyN[ (CH 2 17-

CH

3 1 2

H

2

N(CH

2 3

NH(CH

2 4

NH(CH

2 3

NHC

H 2 COGlyN[(CH 2 18 2'

H

2

N(CH

2 3

NH(CH

2 4

NH(CH

2 3 NHCH2COArgN[ (CH 2 )18 2 and/or 17

H

2 N (CH 2 3 NH (CH 2 4 NH (CH 2 3

NHCH

2 COGlyN[ (CH 2 17CH3] 2.

The recombinant viruses employed according to the present invention are defective, that is to say incapable of replicating in an autonomous manner in the target cell. Generally, the genome of the defective viruses used in the context of the present invention is thus at least deprived of the sequences necessary for the replication of the said virus in the infected cell.

These regions can be either eliminated (in all or in o part), or rendered non-functional, or substituted by other sequences and especially by the nucleic acid inserted. Preferentially, the defective virus nevertheless conserves the sequences of its genome which are necessary for the encapsidation of the viral particles.

The recombinant virus employed in the context of the present invention are derived from a nonenveloped virus. As representative and non-limiting .20 cases of this type of virus, it is more particularly possible to mention the adenoviruses and the adenoassociated viruses (AAV). According to a preferred embodiment, the virus is an adenovirus.

Different serotypes of adenovirus exist, whose structure and properties vary somewhat. Among these serotypes, it is preferred to use in the context of the present invention the human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or the adenoviruses of animal origin (see the Application W094/26914). Among the *3 tL

LO

adenoviruses of animal origin which can be used in the context of the present invention, it is possible to mention the adenoviruses of canine, bovine, murine (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or alternatively simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferentially a CAV2 adenovirus [Manhattan or A26/61 (ATCC VR-800) strain, for example]. Preferably, in the context of the invention adenoviruses of human or canine or mixed origin are used.

Preferentially, in the genome of the adenoviruses of the invention, the El region at least is non-functional. The viral gene considered can be rendered non-functional by any technique known to the person skilled in the art, and especially by total suppression, substitution, partial deletion, or addition of one or more bases in the gene(s) considered. Other regions can likewise be modified, and especially the E3 (W095/02697), E2 (W094/28938), E4 (W094/28152, W094/12649, W095/02697) and (W095/02697) regions.

According to a preferred mode of employment, the adenovirus comprises a deletion in the El and E4 regions. According to another preferred embodiment, it comprises a deletion in the El region at the level of which are inserted the E4 region and the coding sequence. In the virus of the invention, the deletion in the El region preferentially extends from the nucleotides 455 to 3329 on the sequence of the adenovirus. According to another preferred embodiment, the exogenous nucleic acid sequence is inserted at the level of the deletion in the El region.

The defective recombinant adenoviruses according to 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, among other things, the exogenous nucleic acid. Homologous recombination occurs after co-transfection of the said adenoviruses and plasmid in an appropriate cell line. The cell line used must preferably be transformable by the said elements, and (ii) contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. By way of example of line, it is possible to mention the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which especially contains, integrated in its genome, the left part of the genome of an Ad5 adenovirus or lines capable of complementing the El and E4 functions such as especially described in the Applications Nos. W094/26914 and W095/02697. Next, the adenoviruses which have multiplied are recovered and purified according to the classical techniques of molecular biology, as illustrated in the examples.

The adenoviruses employed according to the invention can likewise be obtained according to an original process described in the Patent Application W096/25506 which employs as shuttle plasmid a procaryotic plasmid comprising a recombinant adenovirus genome bordered by one or more restriction sites not present in the said genome.

As an illustrative and non-limiting case of the compositions claimed it is especially possible to propose that associating with a recombinant adenovirus comprising in its genome at least one exogenous nucleic acid and a lipofectamine in sufficient quantity to improve its cellular transduction.

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

More particularly concerning the deoxyribonucleic acids, they can be single- or doublestranded 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, bonding regions to other cellular components, etc.

In the sense of the invention, therapeutic gene is especially understood as meaning any gene coding for a protein product having a therapeutic effect. The protein product coded in this way 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 this does not show any pathology). In this case, the expression of a protein allows, for example, insufficient expression in the cell or the expression of a protein which is inactive or weakly active because of a modification to be overcome, or alternatively the said protein to be overexpressed. The therapeutic gene can also code for a mutant of a cellular protein, having an increased stability, a modified activity, etc. The protein product can likewise be heterologous with respect to the target cell. In this case, an expressed protein can, for example, make up or contribute to a deficient activity in the cell, permitting it to combat a pathology, or to stimulate an immune response.

Among the therapeutic products in the sense of the present invention, it is more particularly possible to mention enzymes, blood derivatives, hormones, lymphokines, interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, HARP/pleiotrophin, etc., dystrophin or a minidystrophin (FR 9111947), CFTR protein associated with mucoviscidosis, genes associated with halting cell division and especially the gax gene, tumour suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), the genes coding for factors involved in coagulation: factors VII, VIII, IX, genes intervening in the repair of DNA, suicide genes (thymidine kinase, cytosine deaminase), haemoglobin or other protein transporter genes, genes corresponding to the proteins involved in the metabolism of lipids, of apolipoprotein type chosen from the apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and apo(a), metabolic enzymes such as, for example, lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase, 7-alpha cholesterol hydroxylase, phosphatidic acid phosphatase, or alternatively lipid-transfer proteins such as cholesterol ester-transfer protein and phospholipidtransfer protein, an HDL-bonding protein or alternatively a receptor chosen, for example, from the LDL receptors, chylomicron-remnant receptors and scavenger receptors, etc.

The therapeutic nucleic acid can likewise be a gene or an antisense sequence whose expression in the target cell allows the expression of genes or the transcription of cellular mRNA to be controlled. Such sequences can, for example, be transcribed in the target cell into complementary RNA of cellular mRNA and thus block their transduction into protein, according to the technique described in the Patent EP 140 308.

The therapeutic genes likewise comprise the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321 201).

As indicated further above, the nucleic acid can likewise contain one or more genes coding for an antigenic peptide capable of generating an immune response in man or animals. In this particular mode of employment, the invention thus allows the production either of vaccines or of immunotherapeutic treatments administered to man or to animals, especially against microorganisms, viruses or cancers. It can especially be specific antigenic peptides of the Epstein-Barr virus, of the HIV virus, of the hepatitis B virus (EP 185 573), of the pseudo-rabies virus, of the "syncytia-forming" virus, of other viruses or iTi. alternatively specifically of tumours (EP 259 212) Preferentially, the nucleic acid likewise comprises sequences allowing the expression of the therapeutic gene and/or of the gene coding for the antigenic peptide in the desired cell or organ. They can be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. They can likewise be sequences of different origin (responsible for the expression of other proteins, or even synthetic). Especially, they can be promoter sequences of eucaryotic or viral genes. For example, they can be promoter sequences arising from the genome of the cell which it is desired to infect.

In the same way, they can be promoter sequences arising from the genome of a virus. In this respect, it is possible to mention, for example, the promoters of the genes ElA, MLP, CMV, RSV, etc. In addition, these expression sequences can be modified by addition of activation or regulation sequences, etc. They can also be promoter, inducible or repressible sequences.

In addition, the nucleic acid can likewise contain, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the routes of secretion of the target cell. This signal sequence can be the natural signal sequence of the therapeutic product, but it can likewise be any other functional signal sequence, or an artificial signal sequence. The nucleic acid can likewise contain a signal sequence directing the therapeutic product synthesized towards a particular compartment of the cell.

In another mode of employment, the claimed compositions can additionally comprise an adjuvant of dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl or -myristoylphosphatidylethanolamines type as well as their derivatives which are N-methylated 1 to 3 times; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (especially such as galactocerebrosides), sphingolipids (especially such as sphingomyelins) or alternatively asialogangliosides (especially such as asialoGMl and GM2).

Very recently, the Applicant has demonstrated that it was likewise particularly advantageous to employ as adjuvant in the transfectant compositions a compound intervening, directly or indirectly, at the level of the condensation of nucleic acids. This compound is formed, in all or part, of peptide motifs (KTPKKAKKP) and/or (ATPAKKAA), the number of motifs being able to vary between 2 and 10. Such an agent can likewise derive in all or part from a histone, a nucleoline, a protamine and/or one of their derivatives (W096/25508). Such adjuvants can be profited from in order to improve the intracellular traffic of the recombinant virus in the claimed compositions.

The compositions according to the invention can likewise employ one or more targeting element allowing the viral and non-viral vectors to be directed towards receptors or ligands on the surface of the cells. By way of example, the composition of the present invention can comprise one or more antibodies directed against molecules of the cell surface, or alternatively one or more ligands of membrane receptors such as insulin, transferrin, folic acid or any other growth factor, cytokines or vitamins. Advantageously, the composition can use modified or unmodified lectins in order to target particular polysaccharides on the surface of the cell or on the neighbouring extracellular matrix. Proteins with the RGD motif, cyclic or acyclic peptides containing a pair of RGD motifs, as well as polylysine peptides can thus be used. These targeting agents can be conjugated either with the recombinant virus and/or with the non-viral transfection vector.

The doses of virus used for administration can be adjusted as a function of different parameters, and especially as a function of the mode of administration used, of the pathology concerned or alternatively of the duration of the treatment sought.

Generally speaking, the recombinant viruses according to the invention are formulated and administered in the form of doses of between 104 and 10 4 pfu/ml. The term pfu ("plaque-forming unit") corresponds to the infectious power of a suspension of virions, and is determined by infection of an appropriate cell culture, and measurement, generally after 48 hours, of the number of infected cell plaques. The techniques of determination of the pfu titre of a viral solution are well documented in the literature.

As far as the quantities of associated nonviral transfection vector are concerned, they vary, of course, according to its nature, the nature of the viral vector which is associated with it as well as the cell type aimed at. Generally speaking, a quantity varying between 50 ng and 1 ig is used in vitro. In exvivo applications, the quantity of associated non-viral transfection vector varies according to the quantity and type of tissue cells which it is desired to transfect. This quantity can vary between 60 ng/ml and 500 gg/ml.

The combinations according to the invention can be formulated with a view to administrations by the topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal route, etc. Preferably, the pharmaceutical combinations of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, especially for a direct injection at the level of the desired organ, or for administration by the topical route (on the skin and/or mucous membranes). In particular, they can be sterile, isotonic solutions, or dry, especially lyophilized, compositions which, by addition according to the case of sterilized water or of physiological serum, allow the formation of injectable solutions. The doses of nucleic acid used for the injection as well as the number of administrations can be adjusted as a function of different parameters, and especially as a function of the mode of administration used, of the pathology concerned, of the gene to be expressed, or alternatively of the duration of the treatment sought.

More particularly, as far as the mode of administration is concerned, it can be either a direct injection into the tissues or the circulatory passages, or a treatment of cells in culture followed by their reimplantation in vivo, by injection or graft. The present invention thus also relates to cells treated with the composition of the invention for ex-vivo use.

The present invention will be more completely described with the aid of the examples and figures which follow, which must be considered as illustrative and non-limiting.

FIGURES

Figure 1: Effect of lipofectamine on the efficiency of transduction of an Ad-pGal at the level of VSMC cells.

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

Figure 3: Detection with Crystal Violet of CMV cells transducted by Ad-Tk and treated with GCV, in the presence and in the absence of 0.125 gg of lipofectamine.

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

Figure 5: Inhibition of the proliferation of smooth muscle cells by overexpression of the Gax protein with the aid of a single adenovirus or using CLAAT (II).

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

MATERIALS AND METHODS The different methods used to show the synergism between recombinant adenoviruses and cationic liposomes in the transduction of vascular smooth muscle cells in primary culture are described below.

A- Primary culture of vascular smooth muscle cells

(VSMC)

The VSMC are obtained by enzymatic digestion of rabbit aorta according to an adapted method of Chamley et al. (Cell Tissue Res. 197,177,503-522) and then put into primary culture. To do this, the rabbit aorta is removed and then incubated for 45 minutes in the presence of collagenase (collagenase II, Cooper Biomedical) at 37 0 C. A second digestion in the presence of collagenase and elastase (Biosys) is carried out for 2 hours at 37 0 C. These two digestions allow a cellular suspension formed essentially of VSMC to be obtained.

The VSMC are maintained in culture in DMEM medium (Gibco) containing 20% of foetal calf serum (FCS, Gibco) and used for all the experiments before the tenth passage. On each passage, phenotyping of the VSMC is carried out by immunofluorescence with the aid of an antibody directed against the specific alpha-actin of the smooth muscle cells (Sigma).

B- Recombinant adenoviruses (Ad) Different recombinant adenoviruses are employed for the different examples of transduction of VSMC. All the recombinant Ad employed are of first generation and deficient in replication by deletion of the El region.

The Ad-Luc carries an expression cassette containing the luciferase gene under the control of the CMV promoter. This expression cassette comes from a commercial plasmid pUT650 (Cayla, Toulouse France) and contains the luciferase gene fused to the zeo resistance gene under the control of the CMV promoter.

The expression cassette has been inserted in the El region of the In340 adenovirus (Feldman et al., Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors Gene Ther, 1997. 4: 189-98; Robert, JJ et al Gene Neurochemistry., 1997, Vol 68, pp 2152-2160; Hearing et al 1983, Cell 33 p 695-703).

The Ad-3Gal carries a cassette coding for the P-galactosidase gene of Echerichia coli, fused in its N-terminal part to a nuclear localization sequence (NLS) identical to that of the T antigen of the virus, under the control of the LTR-RSV promoter (Stratford-Perricaudet et al., Widespread long-term gene transfer to mouse skeletal muscles and heart., J.

Clin. Invest., 1992, 90:626-630).

The Ad-tk codes for the gene of thymidine kinase, of the virus Herpes simplex, under the control of the 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 the VSMC by the recombinant Ad alone or complexed with cationic liposomes The VSMC are inoculated into 24-well culture plates (Falcon) at a density of 50000 cells per well in 1 ml of DMEM containing 10% FCS (DMEM-FCS). After 20 to 24 hours, the DMEM-FCS of the VSMC is replaced by 300 pl of DMEM without serum. The cells are then infected by the recombinant viruses, alone or complexed with liposomes, at multiplicities of infection (MOI) variable according to the experiments. This adenoviral preparation is added, after an incubation of 30 minutes at ambient temperature, to a suspension of 100 1l of water or of phosphate buffer (PBS) containing either Ad all on its own, at the desired MOI, or Ad with variable quantities of cationic liposomes (Lipofectamine, Gibco). The 100 p1 of Ad or of Ad-lipofectamine complex are directly added to VSMC in the 300 1l of DMEM. For the experiments intended to study the effect of neutralizing human serum (AHS), comparatively to FCS, on the efficiency of transduction after a period of 1 hour at 37 0 C, the transduction mixture is replaced by DMEM 0.5% FCS. 24 h after infection, cell proliferation is induced by addition of serum-rich medium (DMEM FCS). The cell viability is estimated 72 h postinfection with the aid of the Alamar Blue test (Biosource).

In the case of VSMC transducted by Ad-tk, the cells are brought together with 25 M of ganciclovir (GCV) for the 48 hours of culture. In parallel, noninfected cells or cells transducted by Ad-IGal serve as controls treated or untreated by GCV.

D- Transfer of the adenovirus coding for the luciferase gene in isolated arteries New Zealand White rabbit abdominal arteries are taken after euthanasia by overdosage of pentobarbital sodium. The abrasion of the arteries is carried out with the aid of the fogarty 3F probe inflated with 0.15 ml of air. The arteries are cut up into fragments of 5 mm and then opened longitudinally.

Each fragment is placed in the well of a 12-well plate in 1 ml of medium without serum.

Infection takes place on the very day of taking. The volume of the adenovirus mixture (AVI.oCMVluc or AVI.0CMV3Gal, 108 pfu) and lipofectamine is adjusted to 80 il/well with PBS. The solution is then incubated for 30 min at ambient temperature and then placed on artery fragments contained in 1 ml of medium without serum in one well of a 12-well plate.

The fragments are then incubated for 1 h at 37 0 C and then they are transferred to a new 12-well plate and incubated in 2 ml of DMEM 20% FCS medium. The evaluation of the transfer is made either by measurement of the luciferase activity (AVI.oCMVluc) or by histochemistry (AVI.

0 CMV3Gal) 3 days post infection.

Several doses of lipofectamine were tested for a constant MOI of AV 1 .0CMVluc.

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

SDetermination of the luciferase activity The VSMC transducted by Ad-Luc are collected in 200 il of lysis buffer for the luciferase test (Promega). After a 15-minute lysis period, 10 gl of each sample are used for the determination of the luciferase activity with the aid of a kit (Luciferase assay kit, Promega) and a luminometer (Berthold). The luciferase activity is expressed in RLU (Relative Right Units).

The measurement of the luciferase activity on the isolated arteries necessitates the artery fragments being ground in 500 il of lysis buffer containing antiproteases and then incubated for 20 min at ambient temperature with stirring. They are then centrifuged for 5 min at 10,000 rpm. Reading is carried out on 1l of extract in the presence of 50 p1 of substrate for 10 seconds.

SDetermination of the P-galctosidase activity in the cell extracts The P-galactosidase activity in the VSMC \transducted by Ad-Gal is determined by i chemiluminescence according to the Galacto-Light plus kit from Tropix, Inc. Briefly, the cells are washed in PBS and then collected in 250 .l of a lysis solution comprising 100 mM potassium phosphate pH 7.8 and 0.2% of Triton X-100. The lysates are centrifuged for 2 minutes at 10000 g, and then 5 gl of each sample are mixed with 50 .1 of Galacton-Plus T reaction buffer and incubated for 1 hour at ambient temperature. For luminescence measurement, the mixtures are placed in a luminometer (Berthold) and then receive 50 .1 of accelerator (light emission accelerator) and the luminescence is measured and expressed in RLU.

SHistochemical detection of the P-galctosidase activity in the cells The VSMC, on the culture support or detached by treatment with trypsin, are washed with PBS and then fixed in PBS containing 4% of formaldehyde. The fixing solution is withdrawn at the end of 10 min, and then the cells are stained for 1 to 4 hours at 370C in PBS containing 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 200 mM magnesium chloride and 0.4 mg/ml of X-Gal substrate. The percentage of blue cells is calculated from VSMC preparations in suspension while the coloured VSMC on the culture support are photographed.

Histochemical detection of the P-galctosidase activity on a tissue section 3 days post-infection, the tissue fragments (arteries) are fixed in 4% PBS-Formol for 3 h. They are then rinsed and treated in the following manner: PBS 3 x 20 min PBS-MgCl 2 30 min PBS-MgCl 2 30 min at 4 0

C

Permeabilization solution 30 min at (PBS-MgCl 2 2 mM, sodium deoxycholate 0.01% and 0.02%) Staining solution 4 to 22 h at 37 0

C

(permeabilization solution plus K 4 Fe(CN) 6 3H20 35 mM and

K

3 Fe(CN) 6 35 mM) They are then washed with PBS and then placed in a cassettes and dehydrated as follows: ethanol 1 h ethanol 1 h 100% ethanol 1 h 100% ethanol 1 h 100% ethanol 1 h xylene 1 h xylene 1 h xylene 1 h xylene 2 h paraffin 2 h paraffin 3 h They are then included in paraffin and sectioned on a microtome.

SMeasurement of the cell viability by staining with Crystal Violet To evaluate the efficiency of transduction by Ad-tk, the VSMC are submitted to treatment with GCV as described further above. The viable cells, derived from this treatment, are washed in PBS and then fixed in PBS containing 4% of formaldehyde. The fixing solution is withdrawn at the end of 15 min, and then the cells are stained for 20 minutes in a solution of Crystal Violet at 0.1% in water. After staining, the cells are washed three times in water and then photographed.

EXAMPLE 1 This example shows the increase in the transduction of VSMC when Ad-PGal is associated with a cationic liposome such as lipofectamine.

The VSMC are transducted according to the protocol described in MATERIAL AND METHODS, by Ad-pGal at an MOI of 10 in the presence of increasing doses of lipofectamine ranging from 0 to 0.5 pg. The histochemical detection of the 3 -galactosidase activity clearly shows that from the dose of 0.125 .g lipofectamine increases in a very significant manner the efficiency of transduction of the VSMC by Ad-pGal.

To quantify this increase in the efficiency of transduction, the percentage of cells positive for 3 -galactosidase activity is determined after histochemical staining of cells in suspension according to the protocol described in MATERIAL AND METHOD.

Figure 1 illustrates these results.

When Ad-pGal is used alone at an MOI of only 1 to 2% of VSMC are positive but as soon as lipofectamine is added, from the dose of 0.125 g, the number of positive cells reaches more than 40%. This efficiency of transduction is in perfect correlation with the 3-galactosidase activity measured by chemiluminescence (Figure A slight decrease in this activity is, however, observed when the quantity of lipofectamine is increased. This decrease is probably due to a slight toxicity as cell counting shows (Figure 1, dotted curve). This toxicity, however, and only at the highest dose of lipofectamine of 0.5 gg, does not affect more than 25% of the cells.

These results clearly show the gain in transduction due to the combination of an Ad-pGal with a cationic lipofectant.

EXAMPLE 2 Example 2 allowed a dose of lipofectamine to be determined allowing the transduction of the VSMC by Ad-pGal to be increased to an MOI of 10. To do this, Ad-Luc is employed at variable MOIs for the transduction of the VSMC and a determination of the luciferase activity is then carried out as described in MATERIAL AND METHOD. The luciferase activity increases almost linearly as a function of the MOIs of Ad-Luc used to transducte the VSMC (Figure 2, empty circles).

Interestingly, and whatever the MOI used, the addition of 0.125 gg of lipofectamine to the Ad-Luc (Figure 2, full diamonds) induces an increase of a factor of 100 in the luciferase activity recorded. This shows that, up to an MOI of 100 of Ad-Luc, a small quantity of lipofectamine, i.e. 0.125 gg, is sufficient to very significantly potentiate the transduction of the VSMCs.

EXAMPLE 3 The adenovirus employed in this example is the adenovirus Ad-TK, used for the cytotoxic property of the thymidine kinase gene of Herpes simplex in the presence of ganciclovir (GCV). The VSMCs were transducted with MOIs of 1.10 and 100 of Ad-TK and treated with GCV as described in MATERIAL AND METHOD.

The AD-PGal at an MOI of 100 is used as a control.

The Ad-TK and Ad-pGal are used either on their own or complexed with 0.125 gg of lipofectamine.

Figure 3 shows a staining of the VSMC with Crystal Violet after transduction and treatment with GCV.

The GCV, as expected, does not have any toxicity with respect to cells transducted by Ad-pGal in the presence or in the absence of lipofectamine. The transduction of the VSMC by Ad-TK, followed by treatment with GCV, only shows visible toxicity (30 to viable) at the highest MOI, i.e. MOI 100.

Interestingly, when Ad-TK is complexed with 0.125 ig of lipofectamine the treatment with GCV at an efficiency at MOI 1 comparable with that obtained with Ad-TK on its own at MOI 100. At MOI 10 or 100, the Ad-TK associated with lipofectamine is highly toxic in the presence of GCV. This example gives evidence of the interest of the present formulation. At the same time, it allows the quantity of Ad-TK recombinant viruses to be administered to be reduced significantly and this to be done for an equal or higher therapeutic efficiency by comparison with that observed with Ad-TK on its own.

EXAMPLE 4 Beyond the efficiency of transduction which can be improved as the preceding examples show, one of the major obstacles to an optimum efficiency of a recombinant adenovirus is its neutralization by the immune system. In fact, following a cell lysis after a first transfer in animals or following a first contact with an adenovirus in man, the immune system can effectively neutralize the virus in the circulation thus decreasing the possibilities of reinjection of virus.

This example reproduces in vitro the neutralization of the recombinant adenovirus by a pool ^0W C of human serums, and advantageously shows that the association of a recombinant adenovirus with liposomes allows this neutralization to be abrogated and, according to the dose of liposomes, the transduction can be higher than that observed with the virus on its own.

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

Although 10% FCS does not significantly modify the efficiency of transduction by Ad-Luc on its own with respect to DMEM, the luciferase activity is severely reduced in the presence of 10% of AHS.

Increasing doses of lipofectamine, added to the Ad-Luc allow a luciferase activity which is equivalent and even distinctly higher than that observed with the virus on its own to be restored at the highest doses of lipofectamine (Figure 4B).

This example thus shows that the claimed composition allows, on the one hand, the neutralization of a recombinant adenovirus by antibodies or a neutralizing serum to be totally abrogated and, on the other hand, the efficiency of transduction to be increased in the presence of neutralizing serum, as already shown in the preceding examples.

EXAMPLE This example shows the increase in the transduction of a therapeutic gene (gax) in rabbit smooth muscle cells by combination of the AV,.oCMVrGax adenovirus and a cationic lipid such as lipofectine

(CLAAT).

The rabbit smooth muscle cells are inoculated at a rate of 5 104 cells per well of an MW 48 in DMEM FCS medium. The infection is carried out 24 h later in DMEM medium without serum. Different dilutions of adenovirus are mixed with 60 ng of lipofectamine in a final volume of 25 li adjusted with PBS. This solution is incubated for 30 min at ambient temperature. The culture medium is replaced by 175 il of medium without serum per well and the mixture of the adenovirus and of the lipofectamine is added to the cells. The infection lasts 1 h at 37 0 C. The medium containing virus is replaced by DMEM 0.5% FCS. 24 h after infection, the cell proliferation is induced by addition of serum-rich medium (DMEM 10% FCS). The cell viability is estimated 72 h post-infection with the aid of the Alamar Blue test (Biosource).

The results show that lipofectamine, on its own and at the dose necessary for CLAAT, has no effect on the viability of the cells. However, the MOI necessary to obtain 50% inhibition of growth of the SMC is 10 times weaker when an AV 1 .0CMVrGax and lipofectamine mixture compared with the AVI.0CMVrGax alone is used. Figure 5 compares the inhibition of the .RA proliferation of smooth muscle cells by overexpression of the Gax protein with the aid of a single adenovirus or using CLAAT The cells were infected at MOIs varying between 0 and 105 PV/cell with AVIoCMV (a) or with AVi.

0 CMVrGax alone On using CLAAT, the MOIs vary from 0 to 3 104 PV/cell with the same viruses (II-a and After infection, the cells are incubated in DMEM 0.5% FCS medium for 24 h. The medium is then changed for serum-rich medium. The cell viability is estimated 72 h post infection.

This example thus shows the increase in the transduction of the therapeutic gax gene in the rabbit smooth muscle cells by combination of the AV, 0 CMVrGax adenovirus and the lipofectamine. This example likewise demonstrates that the combination of a cationic lipid and of an adenovirus allows weaker virus doses to be used.

EXAMPLE 6: This example describes the transfer of genes on isolated artery fragments using the combination of an adenovirus coding for luciferase or P galactosidase and lipofectamine (CLAAT).

The artery fragments are obtained and infected according to the protocol defined in MATERIAL AND METHOD For the preparation of cellular extracts and the measurement of the luciferase activity, the artery fragments are ground in 500 pR of lysis buffer containing antiproteases and then incubated for 20 min at ambient temperature with stirring. They are then centrifuged for 5 min at 10,000 rpm. Reading is carried out on 10 .l of extract in the presence of 50 gl of substrate for 10 seconds with the aid of a kit (Luciferase assay Kit, Promega) and of a luminometer (Berthold).

The results presented in Figure 6 allow it to be concluded that the luciferase activity is clearly increased on the one hand and on the other hand that it is dependent on the dose of lipofectamine used when the transfer of the adenovirus on the isolated arteries is carried out by means of CLAAT.

The detection of the p galactosidase activity is carried out as described in MATERIAL AND METHOD The histochemical results obtained indicate that the cells, which express P galactosidase after transfer using CLAAT on isolated arteries, are situated in the adventitia and are of fibroblastic type. In addition, the number of cells transferred appears greater than that obtained with the adenovirus alone at the same

MOI.

This example demonstrates that the transfer of genes is possible on isolated arteries using CLAAT. In addition, ex-vivo, this technique on the one hand allows the gene transfer to be improved and on the other hand the cells of the adventitia to be obtained. It is possible to 4ALi envisage the use of this technique for the treatment of vein grafts with the aid of genes of interest, as, for example, the FGF factor, carried by an adenoviral vector.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of ego.

15 integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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Claims (28)

1. Transfectant composition useful in gene therapy, which comprises one or more non-enveloped recombinant viruses which comprise in their genome at least one exogenous nucleic acid and at least one non- viral and non-plasmid transfection agent chosen from a lipofectant and polyalkylenimine.

2. Composition according to claim 1, wherein the non-viral transfection agent is a lipofectant.

3. Composition according to claim 2, wherein the lipofectant is a lipid capable of forming liposomes, stealth liposomes, immunoliposomes or targeted liposomes.

4. Composition according to claim 2, wherein the lipofectant comprises at least one polyamine region of general formula in which m is an integer greater than or equal to 2 and n is an integer greater than or equal to 1, m being able to vary among the different carbon groups contained, between 2 amines associated covalently with a saturated or unsaturated hydrocarbon chain type lipophilic region, of cholesterol, or a natural or synthetic lipid capable of forming lamellar or hexagonal phases.

5. Composition according to claim 4, wherein the polyamine region is represented by spermine, thermine or one of their analogues having conserved its bonding properties to the nucleic acid.

6. Composition according to claim 2, wherein the lipofectant is of general formula *i H2N-(-(pH)m-NH-)n-H in which the lipophilic region R is represented by the general formula SR3 -NR- with R' representing a C to C alkyl group, Y' indpch nd ently of one another represent a e 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 to C 4 alkyl radical, with p being able to vary between 0 and R 6 represents a cholesterol derivative or an 48 alkylamino group -NRiR 2 with RI and R 2 independently of one another representing a saturated or unsaturated, linear or branched C12 to C22 aliphatic radical.

7. Composition according to claim 2, wherein the lipofectant is of general formula R3 O R N -R4 S-(C (CH,) in which R1, R2 and R3 independently of one another represent a hydrogen atom or a -(CH2)q-NRR' group with q being able to vary between 1, 2, 3, 4, 5 and 6, the latter independently between the different groups R1, R2 and R3, and R and R' independently of one another representing a hydrogen atom or a -(CH2)q'-NH2 group, q' being able to vary between 1, 2, 3, 4, 5 and 6, the latter independently between the different groups R and m, n and p represent, independently of one another, an integer which can vary between 0 and 6 where, when n is greater than 1, m can take different values and R3 different meanings in the general formula III and R4 represents a group of general formula R 6 x CH) -y N in which R6 and R7 independently of one another represent a hydrogen atom or a saturated or unsaturated to C22 aliphatic radical with at least one of the two groups being other than hydrogen, u is an integer between 0 and 10 where, when u is an integer greater than 1, R5, X, Y and r can have different meanings in different motifs [X-(CHR5)r-Y], X represents an oxygen or sulphur atom or an optionally monoalkylated amine group, Y represents a carbonyl group or a methylene group, R5 represents a hydrogen atom or a natural amino acid side chain, substituted if necessary, and r represents an integer varying between 1 and 10 where, when r is equal to 1, R5 represents a substituted or unsubstituted natural amino acid side chain and when r *0ooo 20 is greater than 1, R5 represents a hydrogen atom.

8. Composition according to claim 2, wherein the polyalkylenimine is a polyethylenimine (PEI) or a polypropylenimine (PPI).

9. Composition according to claim 1, wherein the non-viral transfection agent is chosen from lipofectamine, polyethylenimine of average molecular weight 50000 (PEI50K), polyethylenimine of average molecular weight 800000 (PEI800K), dioctadecylamidoglycylspermine (DOGS), palmitoylphosphatidylethanolamine (DPPES), 2-5-bis(3-aminopropylamino)pentyl (dioctadecylcarbamoyl-methoxy)acetate, 2-[1,3-bis(3- aminopropylamino)]propyl (dioctadecylcarbamoylmethoxy)acetate, {H 2 N (CH 2 3 2 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 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 1 7 CH 3 2. 10. Composition according to any one of the preceding claims, wherein the exogenous nucleic acid contained in the recombinant virus is a deoxyribonucleic acid. S11. Composition according to any one of claims 1 to 9, wherein the nucleic acid contained in the recombinant virus is an exogenous ribonucleic acid. 12. Composition according to claim 10 or 11, 20 wherein the nucleic acid is chemically modified. 13. Composition according to any one of claims 10 to 14, wherein the nucleic acid is an antisense nucleic acid. 14. Composition according to any one of the preceding claims, wherein the nucleic acid contains a therapeutic gene. Composition according to claim 14, wherein the therapeutic gene codes for a product T\ selected from an enzyme, a blood derivative, a hormone, a lymphokine or a precursor thereof or synthesis enzyme, a trophic factor, dystrophin or a minidystrophin and CFTR protein. 16. A composition according to claim wherein the lymphokine is an interleukin, an interferon, TNF, a growth factor, or a neurotransmitter. 17. A composition according to claim 15 or 16, wherein the trophic factor is BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5 or HARP/pleiotrophin.

18. Composition according to claim wherein the therapeutic gene is selected from a gene associated with halting cell division, a tumour suppressor gene, a gene coding for a factor involved in coagulation, a gene intervening in the repair of DNA, a ee: suicide gene, haemoglobin or another protein transporter gene, a gene corresponding to a protein involved in the metabolism of lipids of apolipoprotein Stype chosen from the apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and apo(a), a metabolic enzyme, a lipid-transfer protein, an HDL- bonding protein or a receptor.

19. Composition according to claim 18, wherein the gene is the gax gene, p53, Rb, RaplA, DCC or K-rev, or encodes factors VII, VIII, or IX, thymidine kinase, cytosine deaminase, lipoprotein \T lipase, hepatic lipase, lecithin cholesterol 7 acyltransferase, 7-alpha cholesterol hydroxylase, phosphatidic acid phosphatase, cholesterol ester- transfer protein, phospholipid-transfer protein, a LDL receptor, a chylomicron-remnant receptor or a scavenger receptor. Composition according to any one of the preceding claims, wherein the non-enveloped recombinant virus is deprived at least of the regions of its genome which are necessary for its replication in the infected cell.

21. Composition according to any one of the preceding claims, wherein the non-enveloped recombinant virus derives from an adenovirus or an AAV.

22. Composition according to claim 21, wherein the non-enveloped recombinant virus derives from an adenovirus of animal or human origin.

23. Composition according to claim 28, 20 wherein the recombinant virus derives from an adenovirus of Ad 5 or Ad 2 type.

24. Composition according to any one of claims 1 to 3 and 10 to 23, which comprises a recombinant adenovirus comprising in its genome at least one exogenous nucleic acid and a lipofectamine in sufficient quantity to improve its cellular transduction. Composition according to any one of the preceding claims, which additionally comprises an f -y Pz AF~ adjuvant of dioleoylphosphatidylethanol-amine (DOPE) or oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl or -mirystoyl- phosphatidylethanolamines type as well as their derivatives N-methylated 1 to 3 times; a phosphatidylglycerol, a diacylglycerol, a glycosyldiacylglycerol, a cerebroside, a sphingolipid or a asialoganglioside.

26. Composition according to claim wherein the cerebroside is a galactocerebroside.

27. Composition according to claim wherein the sphingolipid is a sphingomyelin.

28. Composition according to any one of the **0000 Spreceding claims, which additionally comprises a compound intervening, directly or indirectly, at the level of the condensation of nucleic acids. S0*

29. Composition according to claim 28, S 20 wherein the compound is formed, in all or part, of peptide motifs (KTPKKAKKP) and/or (ATPAKKAA), the number of motifs being able to vary between 2 and Composition according to any one of the preceding claims, which additionally comprises a targeting element.

31. Composition according to claim wherein the targeting element is chosen from an antibody directed against a cell-surface molecule, a ligand of a \sT> membrane receptor, a modified or unmodified lectin, a protein with an RGD motif, a cyclic or acyclic peptide containing a pair of RGD motifs, and a polylysine peptide.

32. Use of one or more non-enveloped recombinant viruses which comprises in their genome at least one exogenous nucleic acid and at least one non- viral and non-plasmid transfection agent chosen from a lipofectant and polyalkylenimine for the manufacture of a composition for the transfer of nucleic acids to cells wherein the composition is as defined in any one of the preceding claims.

33. Recombinant cell which is obtained by transfection with a composition as defined in any one of claims 1 to 31. S

34. Method of transfer of a nucleic acid to the interior of a cell wherein the cell is contacted with a composition as defined in any one of claims 1 to 20 31. Composition according to claim 31, wherein the ligand of the membrance receptor is insulin, transferrin, folic acid or any other growth factor, cytokine or vitamin.

36. Composition according to claim 1, substantially as hereinbefore described in any one of the examples.

37. Use according to claim 32, substantially T? as hereinbefore described in any one of the examples.

38. Recombinant cell according to claim 33, substantially as hereinbefore described in any one of the examples.

39. Method according to claim 34, substantially as hereinbefore described in any one of the examples. DATED this 3 rdday of July, 2001 RHONE-POULENC RORER S. A. Its Patent Attorneys DAVIES COLLISON CAVE