CZ188299A3 - Transfection preparation suitable for gene therapy and containing recombinant virus and transfection 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

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

Chromosomal deficits and / or anomalies (mutations, aberrant expression, etc.) are the source of numerous diseases of hereditary or non-hereditary nature. For a long time medicine remained powerless to them. Today, with the development of gene therapy, it can be hoped that this type of chromosomal aberration can be repaired or prevented in the future. This new method of treatment consists in introducing genetic information into the affected cell or organ in order to correct this deficiency or anomaly or alternatively express the desired protein there.

A 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, which prevents it from passing through the cell membrane.

In order to overcome this obstacle, various techniques are now proposed, including, in particular, transfection of naked DNA alone across the plasma membrane in vivo (WO 90/11092) and transfection of DNA via a transfection vector.

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With regard specifically to this second technique, basically two strategies are proposed:

The first uses natural transfection vectors, which are viruses, and the second consists in the use of chemical or biochemical vectors.

As regards vectors of viral origin, they are widely used today for the cloning, transfer and expression of genes in vitro (for recombinant protein production, screening assays, regulatory protein studies, etc.) ex vivo or in vivo (for animal modeling or Of these viruses, adenoviruses, adeno-associated viruses (AAVs), retroviruses, herpesviruses or even vaccinia virus may be mentioned.

The Adenoviridae viral 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 capsule with icosahedric symmetry (Horwi'tz, In: Fields BN, Knipe DM, Howley PM , ed. Virology, Third edition ed. Philadelphia: Raven Publishers, 1996: 2149-2171). Their genome mainly comprises an inverted terminal repeat (ITR) at each end, an encapsidation sequence (Psi), early and late genes. The major early genes are contained in the E1, E2, E3 and E4 regions. Of these genes, those contained in the E1 region are essential for virus propagation. The major late genes are contained in regions L1 to L5. 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 retrovirus, whose life cycle is dependent on cell division, adenovirus can advantageously infect

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440 4 »40 actively dividing cells, but also resting cells, and its genome is maintained in an episomic form. In addition, it can be produced at a very high titer (10 ¹¹ pfu / ml). These major properties make it a vector of choice for cloning and expression of heterologous genes. Group C adenoviruses, especially types 2 and 5, as well as canine adenovirus type CAV-2, whose molecular biology is best known, are sources of 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 City 11724, p 187), for creation animals (WO95 / 22616), for transmission genes to cells ex vi vo (WO95 / 14785; WO95 / 06120) and pro transmission genes to cells in in vivo (see in particular

WO93 / 19191, WO94 / 24297 and WO94 / 08026).

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

These vectors, particularly adenoviruses, have been shown to be impressive in terms of transfection levels. Conventionally, infection of cells with a viral transfection vector is generally performed in two. · In the first stage · the target is recognized.

cell surface receptors that allow the attachment of recombinant virus. The nature of these receptors as well as their cell-type distribution is poorly documented, but in the particular case of adenoviruses, it appears to be a fiber, a protein on the outer surface of adenoviruses that may be involved in this attachment. The second step then consists in the interaction of the viral penton protein at the root of the strand via the RGD peptide motif with the avb3 and / or avb5 cellular integrins leading to virion internalization.

The efficacy with which a recombinant virus can infect a cell (transduction efficiency) is very variable and depends on the type of cells studied in the tissue culture or tissues being tested. important for in vivo treatment. In all cases, this is the first attachment step that determines the efficiency of virion internalization. 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 making this attachment step easier will remove this barrier.

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

A further object directed and achieved by the present invention relates to the reduction or even suppression of the immune response usually exerted by the treated cells against the 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 roles of the immune system consists in actually destroying elements that are not intrinsic or intrinsic, but which are intrinsic or intrinsic. ··· · · · · ·························

I changed. Administration of a gene treatment vector of viral origin introduces motifs that are not intrinsic to the organism. Similarly, cells infected with such a vector, and as a consequence expressing the exogenous gene, will become intrinsic altered elements. The immune response developed against these infected cells so. represents a major impediment to the development of these viral vectors because i) inducing and destroying infected cells limits the duration of gene expression and hence therapeutic effect, ii) induces a concurrent and significant inflammatory response, and iii) rapidly eliminates infected cells after repeated injections. ’

The present invention makes it possible to achieve advantageously these two objectives by combining the desired recombinant virus. with a chemical or biochemical compound that effectively assists in its transduction and protects it from immune system reactions manifested when encountered with the virus. '

As mentioned earlier, non-viral transfection vectors have also been developed in parallel with viral vectors at the level of gene therapy. 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. two nuclear membranes.

_: Although these chemicals are not as impressive vectors such as viral vectors, on the other hand advantageous in immune plane. They are not pathogenic, the risk of DNA multiplication, is zero in these vectors and has virtually no theoretical limit as to the size of the DNA to be transfected.

Of these developed synthetic vectors, the most preferred are cationic polymers of the polylysine and DEAEdextran type or even lipofectants. They are capable of condensing ι · · · φ · φ φ φ φ φ φ

DNA and promote its connection to the cell membrane. Recently, a method of targeted receptor-mediated transfection has been developed. This technique benefits from the principle of DNA condensation due to 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 be attached. 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 comprising at least one exogenous nucleic sequence in its genome with a view to more efficiently promoting cellular transduction of the recombinant virus.

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

The present invention advantageously benefits simultaneously from the properties of vehicles of liposome or lipofectant type chemical vectors, with a variety of cell types, of recombinant viruses, such as, in particular, adenovirus. This is followed by a synergy between the viral, which is the transduction agent of the gene of the desired therapeutic effect, and the lipofectant as an auxiliary transfection agent. This synergism is preferably manifested at 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 poorly expressed, transfection efficiency is 50 to 100 times higher for recombinant adenoviruses capable of fusion and internalization efficiency

9

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9 9 * associated with the liposome than the transfection observed with recombinant adenovirus alone. Moreover, this observation has been confirmed in different recombinant adenoviruses. It has been shown that in the presence of a chemical or biochemical vector, reduced amounts of recombinant viruses can be used with the same, but not higher, efficacy.

Finally, it has been unexpectedly observed that the association of a chemical vector with a recombinant adenovirus allows the phenomenon of neutralization, usually induced by the immune system upon encountering recombinant viruses, to be effectively attenuated. When adenovirus in isolated form is contacted with human serum containing anti-Ad neutralizing antibodies, a strong decrease in transduction efficiency is observed. This transduction is unexpectedly significantly increased in the presence of a lipofectant-type chemical vector. Depending on the dose of lipofectant used, the transduction efficiency is either restored or increased by 10 to 20 fold. comparison with adenovirus transduction under favorable conditions.

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

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

More specifically, as far as lipofectants are concerned, it is understood that within the meaning of the present invention this term covers each lipidic compound and already presented as an active agent with respect to cell transfection with nucleic acids are amphiphilic lipophilic acid molecules containing unrelated

Generally speaking, at least one with a hydrophilic portion. As a section joined or representative first ······································· 999999

9 9 9 9 9

In particular, lipids capable of forming liposomes, such as e.g. POPC, phosphatidylserine, phosphatidylcholine, cholesterol, lipophectamine, maleimidophenylbutyrylphosphatidylethanolamine, lactosylceramide in the presence or absence of polyethylene glycol for formation. fake liposomes or the formation of immunoliposomes or targeted liposomes, with or without antibodies or ligands.

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

as illustrative of this type of cationic lipids constructed on the following structural model: the lipophilic group associated with the amino group via a spacer branch, it is appropriate to mention specifically DOTMA, as well as those lipids which contain as lipophilic group two fatty acids or cholesterol derivatives, if necessary, as an amino group, a quaternary ammonium group. . In particular, DOTAP, DO.BT or - ChOTB may be mentioned as representative examples 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 quaternary ammonium group.

Advantageously, the lipofectants suitable for the invention may also be selected from lopopolyamines whose polyamine region corresponds to the general formula

H ₂ N- (- (CH) _m -NH-) _r -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 be

0000 • 0

0 »0000 00

0 0 0 0 · 0000 0 00 000 00 ·· between different carbon groups contained between two amines, this polyamine region is covalently linked to a lipophilic portion of a hydrocarbon chain that is saturated or unsaturated, cholesterol, or a natural or synthetic hexagonal phase lipid. This is represented by a spermine capable of forming a lamellar or polyamine stretch is most preferably thermin or one of its analogues, which retains the nucleic acid binding properties.

Patent application EP 394 111 describes lipopolyamines of this family which can be used in the sense of the present invention. As an example of these lipopolyamines, mention may be made in particular of dioctadecylamidoglycyl-spermine (DOGS) and palmitoylphosphatidylethanolamine-5-carboxyspermylamide (DPPES).

The lipopolyamines described in patent application WO 96/17823 can also be advantageously used according to the invention, represented by the general formula

H ₂ N; CH _{2 m} -NH are in which R represents

wherein X and X 'are, independently of each other, 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 group containing 1 to 4 carbon atoms (Ci-C ₄ alkyl group) ·· 44 4444 4444

4 4 · 4 · 444 · 4 4 · 4 4 '· 4

4444 44 444

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4 4 4

4 4 4

444 444

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44

Y and Υ 'independently of one another are methylene, carbonyl or. group C = S,

R 1, R ₄ and R ₅ , independently of one another, represent a hydrogen atom or a substituted or unsubstituted C ₂ to C ₄ alkyl group, wherein p may range from 0 to 5,

R 5 represents a cholesterol derivative or an aminoalkyl group -NR ₁ R ₂ , wherein R ₁ and R ₂ independently of each other

- j represents a saturated or unsaturated, linear or branched aliphatic group containing 12 to 22 carbon atoms (C ₁₂ to C ₂₂ aliphatic),

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

Finally, new lipopolyamines have been developed recently, as described in patent application FR 95/13490, and may also be useful in the sense of the present invention. They are compounds having the following general formula:

R3 O \ and _{R 4} (CH.) P η R2 wherein Rl, R2 and R3 independently of one another are hydrogen or - (CH _{2) q} -NRR ', with the proviso that q can range between 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 be between 1, 2, 3, 4, 5 and 6 independently 'between different R groups and R ','

Rl (CH.)

AA AAAA • A

AA AAAA

A A A A

A A A A

A A A · A

A A · A

AAAA AND AA

AA AA I A A A A A A

AAA AAA

A A

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

R 4 represents a group of the general formula. R 5 —f- X H CH-Y 1 ^-N

L ^r

R 6 • R 7 wherein R 6 and R 7 independently of one another represent a hydrogen atom or a saturated or unsaturated C ₁₀ to C ₂₂ aliphatic group containing, from 10 to 22 carbon atoms (C ₁₀ to C ₂₂ ) with at least one of two non-hydrogen groups; an integer between 0 and 10 where, when u is an integer greater than 1, R5, X, Y and _r may have different meanings in different themes [X- (CHR5) _r -Y].,

X represents an oxygen or sulfur atom or optionally a monoalkylene. amino.

Y represents a carbonyl group or a methylene group,

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

As a representative example of these lipopolyamines, the following are particularly useful:

{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 ) _n -CH ₃ j (RP120525)

HN (CH.) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₈ ] RP120535) • 9 9999 • 9 9999 • 9 9999

99 * · 9 • 9,999

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Η Ν ₂ (CH _{2) 3} {ΝΗ CΗ ₂₎ 4ΝΗ (CΗ _{2) 3} NHCH ₂ COAr g N _{[(CH2) I8] 2} (RP12 5 O 31)

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

Xn - ( ^{CH 2} ) - I j ~ ⁿ | in the formula wherein R can be a hydrogen atom or a group according to (CH ₂ ) _n ^--N - wherein n is an integer between 2 and 10, p and q are integers, it being understood that the sum of p + q is such that the molecular weight of the polymer is between, 100 and 10 ⁷ D. Such compounds are exemplified in patent application WO96 / 02655.

Particularly preferred properties are polymers of polyethyleneimine (PEI) and polypropyleneimine (PPI). Polymers preferred for carrying out the present invention are those whose molecular weight is between 10 ³ and 5 10 10 ⁶ . An example is polyethyleneimine of an average molecular weight of 50000 D (PEI50K) or polyethyleneimine of an average molecular weight of 800000 D (PEI800K). PEI50K or PEI800K are available commercially. As for the other polymers represented by the general formula above, they can be prepared according to the procedure described in patent application FR 94 08735.

It is particularly advantageous to use lipofectamine, dioctadecylamidoglycylspermine (DOGS), within the meaning of the invention, as defined in the claims 0, 0, 0, 0, 0, 0 and 0. • paimitoylf osphatidylethan lamin- 5 - car boxes of the company amide 1 (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 NH ₂ } (CH ₂ ) ₃ NHCH ₂ COGlyN [ ^: (CH ₂ ) _n -CH ₃ ] ₂ ,

H ₂ N (CH ₂ ) ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) is b,

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 ) ₁₇ CH ₃ b.

The recombinant viruses used according to the present invention are defective, i. incapable of replication autonomously in the target cell. In general, the genome of the defective viruses used according to the invention is thus at least devoid of sequences necessary for the replication of said virus in an infected cell. These regions can either be eliminated (in whole or in part) or rendered non-functional or substituted by other sequences, in particular the inserted nucleic acid. Preferably, however, the defective virus retains its genome sequences that are necessary for encapsidation (packaging) of viral particles.

The recombinant virus used in the sense of the present invention is an example of such a virus. According to a preferred type of virus, adenoviruses and adeno-associated viruses (AAVs) are non-limiting. The virus is an adenovirus.

There are various serotypes of adenoviruses whose structure and properties differ somewhat. Among these serotypes, preference is given; to use human adenoviruses type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO94 / 26914) for the purposes of the present invention. Among the adenoviruses of animal origin that may be used in the present invention, mention may be made of canine, bovine, murine adenoviruses (example: Mávl, Beard et al., Virology, 75, 81, 1990), sheep, porcine, or bird monkey (example: SAV). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [e.g., Manhattan strain or A26 / 61 (ATCC VR-800)]. Preferably, adenoviruses of human or canine or mixed origin are used in the present invention.

Preferably, at least one E1 region in the genome of the adenoviruses of the invention is rendered non-functional. Contemplated viral. the gene may be rendered inoperative by any technique known to those skilled in the art, in particular by complete suppression, substitution, partial deletion or addition of one or more bases to the genes under consideration. Other regions may also be modified, especially the E3 region (WO95 / 02697). E2 (WO94 / 28938), E4 (WO94 / 28152, WO94 / 12649, WO95 / 02697) and L5 (WO95 / 02697).

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 the virus according to the invention, the deletion in the E1 region preferably occupies the nucleotide region 455 to 3329 according to the sequence of the Ad5 adenoviruses. According to another preferred embodiment, the exogenous nucleic acid sequence is inserted at the deletion level of the E1 region.

Defective recombinant adenoviruses of the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 EMBO J. 3 (1984) 2917)

1991) 195, EP 185,573; Graham, especially

Homologous recombination between an adenovirus and a plasmid carrying inter alia an exogenous nucleic acid can be prepared. Homologous recombination occurs after 'co-transfection' (cotransfection) with said adenoviruses and plasmid into the respective cell line. The cell line used must. preferably be (i) transformable by said elements and (ii). it must comprise sequences capable of complementing a portion of the genome of the defective adenovirus, preferably

9

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¹⁵ in an integrated form to avoid the risk of recombination. As an example of the line, the human embryonic kidney line 293 may be mentioned (Graham et al., J. Gen. Virol. 36 t).

(1977) 59), which specifically includes, in its genome, the left part of the Ad5 genome of Ad5 (12%) or a line capable of complementing E1 and E4 functions, as specifically described in WO94 / 26914 and WO95 / 02697 . Further, the adenoviruses that have multiplied are obtained and purified by conventional molecular biology techniques, as shown in the examples.

Adenoviruses used according to the invention can likewise 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 associating 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, the nucleic acid introduced at the genome level of the recombinant virus may be a deoxyribonucleic acid as well as a ribonucleic acid. They may be sequences of natural or artificial origin, in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid. sequences or synthetic or semisynthetic sequences of modified or unmodified oligonucleotides. These nucleic acids may be of human, animal, plant, bacterial, viral, etc. origin. They may be obtained by any technique known to those skilled in the art, in particular by screening the banks, by chemical synthesis or · T 9 9 9 9 9 9 9 9 999 9 999 9 9 999 999

9 9 9 9 9 9 • 9999 99 999 99 9 · alternatively by mixed methods including chemical or enzyme modification of the sequences obtained by screening the banks.

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 is essentially any gene encoding a protein product that has a therapeutic effect. The protein product encoded by this method may be a protein, a peptide or the like. This protein product may be homologous to the target. a cell (i.e., a product that is normally expressed in a target cell that does not exhibit any pathology). In this case, the expression of the protein allows to overcome, for example, under-expression in the cell or the 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 increased stability, modified activity, and the like. The protein product may be equally heterologous to the target cell.

In this case, the expressed protein can, for example, generate 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, interfereons, TNF, and the like.

(FR 9203120), growth factors, neurotransmitters or their i

precursors or enzymes of synthesis, trophic factors: BDNF, CNTF, ·· ···· · · · ·

NGF, IGF, GMF, αFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc., dystrophin or minidystrophin (FR 9111947),. mucoviscidosis-associated CFTR protein, genes associated with arrest of cell division, in particular the gax gene, tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc. (FR 93 04745), genes encoding factors involved in coagulation: factors VII , VIII, IX-, interfering genes. DNA repair. suicide genes (thymidine kinase, cytosine deaminase), hemoglobin or other protein carrier gene, genes corresponding to proteins involved in the lipid metabolism of apolipoprotein type selected from apolipoproteins AI, A-II, A-IV, B, CI, C-II, C-III, D , E, F, G, H, J and apo (a) metabolic enzymes such as. for example, lipoprotein lipase, liver lipase, lecithincholesterolacyltransferase, 7-alpha-cholesterolsterol hydroxylase, phosphatidic acid phosphatase, or alternatively lipid transfer proteins, such as cholesterol ester transfer protein and phospholipid transfer protein, HDL binding protein, or alternatively a LDL receptor selected from, for example residual chylomicron receptors, scavenger receptors, etc.

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 the expression of the genes or allows the transcription of the cell mRNA to be suppressed. Such sequences may. For example, dd RNA complementary to cellular mRNA can be transcribed in a target cell to block its translation into a protein according to the method described in EP 140 '308. Therapeutic genes also contain ribozyme coding sequences that are capable of selectively destroying target RNA. 201).

As mentioned above, the nucleic acid may also contain one or more genes encoding antigenic peptides.

9 · · * ··· ···

9

999 ·· ·· responsible for expressing a sequence capable of producing an immune response in humans or animals. 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. They may be especially antigenic peptides specific virus · Epstein-Barr virus, HIV, hepatitis B virus (EP 185 ^573): pseudorabies virus, forming syncytia virus, or other viruses, or alternatively tumor-specific peptides (EP 259 212). ;

Preferably, the nucleic acid also comprises sequences allowing expression of the therapeutic gene and / or the gene encoding the antigenic peptide in the desired cell or organ. These may be sequences that are naturally of the gene of interest when these are 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 a cell that it is desired to infect. Likewise, they may be promoter sequences derived from the vi.ru genome. In this regard, it is appropriate to mention, for example, the promoters of the E1A, MLP, CMV, RSV, etc. In addition, these expression sequences may be modified by the addition of activation or regulatory sequences, etc. They may also be promoter, inducible or repressible sequences.

In addition, the nucleic acid may also 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 may also be any other functional signal sequence or diacylglycerols (especially galactosphingomyelins) or an artificial signal sequence. The nucleic acid may also comprise a signal sequence directing the synthesized therapeutic product toward a particular compartment of the cell.

In another method of the invention, the formulations of the invention may additionally comprise an adjuvant of dioleoylphosphatidylethanolamine (DOPE), oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl 'or -myristoylphosphatidylethanolamine type, as well as derivatives thereof which are 1 to 3x N-methylated; phosphatidylglycerols, glycosyldiacylglycerols, cerebrosides cerebrosides), sphingolipids (especially alternatively asialogangliosides (especially asialoGM1 and GM2).

Recently, the inventor has shown that it was also particularly advantageous to use as an adjuvant in transfection preparations a compound which interferes directly or indirectly at the level of nucleic acid condensation. The compound consists wholly or partially of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA) and the number of motifs can vary between 2 and 10. Such agent may also originate in whole or in part from histone, nucleoline, protamine and / or one derivatives thereof (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 may also be used in the composition of the invention to allow viral and non-viral vectors to be directed against cell surface receptors or ligands. 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 of these. Other growth factor, cytokines, or other growth factor, cytokines, or other vitamins. Modified or unmodified lectins may advantageously be used in the formulation to target particular polysaccharides on the cell surface or adjacent extracellular matrix. Thus, RGB motif proteins, 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 for administration can be adjusted as a function of various parameters, in particular as a function of the mode of administration used, relating to the pathology or, alternatively, to the length of treatment desired. In general, the recombinant viruses of the invention are formulated and administered in the form of doses between 10 ⁴ and 10 ¹⁴ pfu / ml. The term pfu (plaqueforming 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 infected cells. Methods for determining the pfu titre 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 targeting. Generally speaking, in vitro amounts varying between 50 ng and 1 pg are used. In ex vivo applications, the amount of associated non-viral transfection vector varies according to the number and type of cells of the tissue to be transfected. This amount can vary between 60 ng / ml and 500 pg / ml.

The compositions of the invention may be formulated for topical, cutaneous, oral, rectal, vaginal, parenteral, intranasal,

9999 99

9 9 9 99 9 99 9 • * · 9 9 9 9 9 99 9

9,999,999,999,999

9 9 9 9 9 9 »999 9« 9999 99 99 intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like. The pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable vehicle for an injectable preparation, particularly for direct injection at the desired organ level or for administration. topically (on the skin and / or mucous membranes). In particular, they may be sterile, isotonic solutions, or dry, in particular, lyophilized preparations 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, and the number of administrations can be adjusted as a function of various parameters, in particular as a function of the mode of administration used, relating to the pathology of the gene it has. be expressed or alternatively to the length of treatment desired. More specifically, as regards the mode of administration, it may be either a direct injection into tissues or circulation, or treatment of cells in culture followed by their reimplantation in vivo, injection or graft. The present invention is. also relates to cells treated with the preparation of the invention for use ex vivo.

The present invention will be described more fully by the examples and figures that follow, which are to be construed as illustrative and non-limiting.

Description of the picture

Figure 1: Effect of lipofectamine on the efficacy of Ad-pGal transduction at VSMC level. '

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

·· ··········· · · · · · ·

Figure 3: crystal violet detection of Ad-Tk transduced CMV cells and treated with GCV, in the presence and absence of 0.125 µg lipofectamine.

Figure ^4: Evaluating the effect of lipofectamine on neutralization of Ad-luc-FCS in the medium or AHS for doses ranging from 0 to 0.5 ug Lipofectamine (Figure 4A) and from 1 to 10 ug 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 lipophectamine.

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 a modified method of Chamley et al. (Cell Tissue Res. 197,177,503-522). and then seeded into the primary culture. For this purpose, the rabbit aorta is removed and then incubated for 45 minutes in the presence of collagenase (Collagenase II, Cooper Biomedical) at 37 ° C. A second cleavage in the presence of collagenase and elastase (Biosys) is carried out for two hours at 37 ° C. These two cleavages make it possible to obtain a cell suspension consisting of:

basically VSMC. VSMCs were maintained in culture in DMEM medium (Gibco containing 20% fetal calf serum (FCS, Gibco) and used for all subsequent experiments before passage 10. The VSMC phenotyping was performed by immunofluorescence using antibodies directed against specific alpha-actin at each passage. smooth muscle cells (Sigma).

B. Recombinant Adenoviruses (Ad) ·

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

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 (Cayla, Toulouse France) and contains a luciferase gene fused with 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., Improved Efficiency of Arterial Gene Transfer by 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).

Ad-pGal carries a cassette encoding the Echerichia coli β-galactosidase gene fused on its N-terminal part to a nuclear localization sequence (NLS) identical to the antigen sequence. SV40 T virus, under the control of the LTR-RSV promoter (Stratford-Perricaudet et al., Widespread long-term gene transfer to skeletal muscles and heart., J. Clin. Invest., 1992, 90: 626-630).

.Ad-tk encodes a 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.). P · · sim sim sim sim • sim sim sim sim sim sim sim sim sim p sim sim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p resonance imaging (Gene Ther, 1996, 3: 315-22).

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

VSMCs are inoculated into 24-well culture dishes (Falcon) at a density of 50,000 cells per well in 1 ml DMEM containing 10% FCS (DMEM-FCS). After 20-24 hours, the DMEMFCS of VSMCs is replaced with 300 μΐ serum-free DMEM. The cells are then infected with recombinant viruses, alone or in complex with liposomes, at multiple infections (MOIs) varying according to experiments. This adenoviral preparation was added after incubation for 30 minutes at ambient temperature to a suspension of 100 μΐ water or phosphate buffered saline (PBS) containing either Ad alone in 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 is replaced with DMEM 0.5% after 1 hour at 37 ° C.

FCS. 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 with the Alamar Blue assay (Biosource).

For VSMCs transduced with Ad-tk, cells are contacted with 25 µg of ganciclovir (GCV) for 48 hours of culture. Similarly, cells uninfected or cells transduced with Ad-pGal serve as controls untreated or treated with GCV.

9999 999 99999

D. Transfer of adenovirus encoding luciferase gene in isolated arteries

Abdominal arteries of rabbits of New Zealand White are collected after euthanasia with an overdose of sodium pentobarbital. Artery abrasion is performed with a 3F Fogarty catheter inflated with 0.15 ml of 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. V mixture of adenoviruses (AVI. ₀ CMVluc AVx.oCMVPGal or 10 ⁸ pfu) and Lipofectamine was adjusted to 80'μΐ / well with PBS. The solution is then incubated for 30 minutes at ambient temperature and then placed on the artery fragments contained 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. Transmission evaluations are made either by measuring luciferase activity (AVx.oCMVluc) or by histochemistry (AVi. ^ CMVpGal) 3 days after infection. Several doses of lipofectamine were tested. for constant MOI AVi.oGMVluc.

E. Analysis of VSMC transduction efficiency. recombinant Ad associated or not associated with liposomes

Determination of luciferase activity

AdMC-transduced VSMCs were collected in 200 µL luciferase assay lysis buffer (Promega). After a 15 minute lysis time, 10 μΐ of each sample was used to determine luciferase activity using a Luciferase assay kit (Promega) and a luminometer (Berthold). Luciferase activity

9999 • 9,999,999,999 • 9,999,999

9 9 9 999 9 99

9,999,999,999,999

9 9 9 9 9 9 • 999 «·· ··· ·· ·· is expressed in RLU (Relative Light 'Units _; relative light units).

Measuring luciferase activity on isolated arteries requires that the artery fragments be crushed in 500 μ 500 lysis buffer containing anti-proteases and then incubated with stirring at room temperature for 20 minutes. They are then centrifuged for 5 min at 10,000 rpm. Read on 10 μ na of extract in the presence of 50 μΐ of substrate for 10 s.

Determination of β-galactosidase activity in cell extracts β-galactosidase activity in VSMC transduced

Ad ~ 3Gal is determined by chemiluminescence using the kit. Galacto-Light plus kit (from Tropix, Inc.). Briefly, cells are washed in PBS, and then transferred to a 250 µL lysis solution containing 100 mM 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 Galacton-Plus ™ reaction buffer and incubated for 1 hour at ambient temperature. For luminescence measurements, the mixtures are placed in a luminometer (Berthold), and then a 50 μaccelerator (light emission accelerator) is added and the luminescence is measured, which is expressed in RLU.

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 to 4 hours at 37 ° C in PBS containing 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, 200 mM magnesium chloride and 0.4 mg / ml. substrate X-Gal. The percentage of blue cells is calculated from VSMC preparations

ΒΒΒΒ ΒΒΒΒ «· · · ···

ΒΒΒ Β

Β Β Β • Β ΒΒΒ

* * * · · · · ·

Β Β

ΒΒΒΒ Β

ΒΒ ΒΒ ΒΒΒ • • • • • •

ΒΒ ΒΒ in suspension while stained VSMC on the culture medium are photographed.

Histochemical detection of β-galactosidase activity in tissue sections days after infection, tissue fragments (arteries) were fixed for 3 hours in 4% PBS-formol. They were then rinsed and treated as follows;

PBS 3X20 minutes

_PBS-MgCl2 30 minutes _PBS-MgCl2 30 minutes at 4 ° C Permeabilization solution 30 minutes at 4 ° C (2 mM PBS-MgCl _2.0 ) .01% sodium deoxycholate and 0.02% NP40) Coloring solution 4 to 22 hours at 37 ° C permeabilization) solution plus 35 mM K ₄ Fe (CN) ₆

and 35mM K ₃ Fe (CN) _s )

They were then washed in PBS and placed in cuvettes and dehydrated as follows;

60% ethanol 1 80% ethanol 1 100% ethanol 1 100% ethanol 1

100% ethanol 1 xylene 1 xylene 1 xylene 1 xylene '2 paraffin 2 paraffin in' 3

Thereafter, hour-hour hour-hour-hour-hour-hour-hour-hour-hour-hours were cut on a microtome.

9 · 9

999 • · · 9

Evaluation of cell viability by crystal violet staining

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

Example 1

This example demonstrates an increase in VSMC transduction when Ad-3Ga1 is associated with a cationic liposome such as lipofectamine.

VSMCs are transduced according to the protocol described in Material and Methods, using Ad-PGal in an amount corresponding to an MOI of 10 in the presence of increasing doses of lipofectamine ranging from 0 to 0.5 gg. Histochemical detection of β-galactosidase activity clearly showed that from a dose of 0.125 gg lipofectamine has been shown to greatly increase the efficiency of VSMC transduction with the Ad-3Ga1 vector.

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

illustrated on giant . 1. If Yippee used Ad? Gal alone with MOI 1.0 is positive only 1 to 2% VSMC, but once added lipofectamine, so from batch 0, , 125 gg number of positive cells 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. it is probably 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 combination of Ad-PGal with a cationic lipofectant.

Example 2

In this example, the lipofectamine dose was determined by transducing the VSMC with an Ad-pGal vector with an MOI increasing to 10. AdLuc was used for transduction of VSMC at 'different' MOI rates and luciferase activity was determined as described in Material 'and methods. Luciferase activity grew almost linearly as a function of the Ad-Luc MOI used for VSMC transduction (Figure 2, open circles). Interestingly, at any of the MOIs used, the addition of 0.125 μο of lipofectamine to Ad-Luc (Figure 2, full diamonds) caused a 100-fold increase in the luciferase activity recorded. This shows that up to an MOI of 100 Ad-Luc, a small amount of lipofectamine, i. 0.125 μ9 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 gene. thymidine kinase from herpes simplex virus in the presence of ganciclovir (GCV). VSMCs were transduced with Ad-TK with MOIs of 1, 10, and 1000 and then treated.

GCV as described in Material and Methods. Ad-OGal 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 crystal violet staining after transduction and GCV treatment.

As expected, GCV showed no toxicity to Ad-PGal transduced cells either in the presence or absence of lipofectamine. VSMC Ad-TK transduction, followed by GCV treatment, showed toxicity (viability 30-50%) only at the highest MOI, i.e. MOI 100. Interestingly, when Ad-TK was in the p 0.125 µg lipofectamine complex, efficacy was after GCV treatment at an MOI of 1 comparable to that of Ad-TK alone with an MOI of 100. At MOI values of 10 or 100, the lipofectamine-associated Ad-TK 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 compared to that achieved with Ad-TK alone.

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

9 .9 ·

This example reproduces in vitro neutralization of recombinant adenoviruses by a collection of human sera and preferably shows that the association of recombinant adenoviruses with liposomes makes it possible to abolish neutralization, so that transduction, depending on the dose of liposomes, may be higher than that achieved by the use of 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 identical or even at the highest doses of lipofectamine significantly higher than when virus alone is used (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 adenoviruses by antibodies and, on the other hand, makes it possible to increase transduction efficiency in the presence of neutralizing serum.

Example 5

This example shows an increase in the transduction of the therapeutic gene (gax) into rabbit smooth muscle cells by way of example of adenoviruses. AV ₁₀ CMVrGAX and a cationic lipid such as lipofectin (CLAAT).

Rabbit smooth muscle cells in DMEM with 10% FCS were seeded on the MW48 microplate at 5 x 10 ⁴ cells per well. The infection was performed 24 hours later in serum-free DMEM medium. Different dilutions of adenoviruses were

mixed with 60 ng lipofectamine and supplemented with PBS to a total volume of 25 μΐ. 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 a mixture of adenoviruses and lipofectamine was added to the cells. The actual infection was carried out for 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 rich media. serum (DMEM with 10% FCS). Cell viability was evaluated 72 hours after infection with 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 required to obtain 50% inhibition of SMC growth is 10x less when using a mixture of AVi, ₀ CMVrGAX and lipofectamine as compared to AVi, ₀ CMVrGAX alone. Figure 5 shows a comparison of inhibition of smooth muscle cell proliferation by overexpressing GAX protein via adenovirus (I) alone or using CLAAT (II). Cells were infected at an MOI of 0-10 ⁵ PV / cell, either AVI., ₀ CMV (a) alone or AVI ^ CMVrGAX (b). using CLAAT, the MOI was from 0 to 3 x 10 ⁴ PV / cell of the same viruses (II-a, II-b). After infection, cells were incubated for 24 hours in DMEM with 0.5% FCS. The medium was then replaced with serum-rich medium. Cell viability was assessed 72 hours after infection.

This example showed an increase in the transduction of the therapeutic gene into rabbit smooth muscle cells using, for example, a combination of the _AV10 CMVrGAX adenoviruses and lipofectamine. The example has shown that a lower dose of virus can be used for the combination of cationic lipid and adenoviruses. '• 4,444 «

4,444 44 4 4 »4 4 4 4 4 4

I 4444 4 44 4> «44 444 444» 4 4 4 4 • 4,444 4 · ··

Example 6

This example shows gene transfer to isolated artery fragments using a combination of adenoviruses encoding luciferase or β-galactosidase and lipofectamine (CLAAT).

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

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

Based on the results shown 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 adenoviruses are transferred to isolated arteries by CLAAT.

substrate for 10 seconds (Promega) performed

Detection of β-galactosidase activity was as described in Material and Methods (E; histochemical results show that cells, β-galactosidase after CLAAT transmission, in adventitia (outer wall envelope) and are fibroblasts. higher than when adenovirus alone with the same MOI is transmitted.

This example demonstrates that gene transfer into an isolated artery by CLAAT is possible. Furthermore, this ex vivo method makes it possible, on the one hand, to improve gene transfer and, on the other hand, to obtain cells from Advent. Thus, it can be envisaged to use this method for the treatment of vascular grafts. · · · · P · · · · ·

4 genes such as FGF carried by adenoviral vectors.

Claims (28)

PATENTOVÉ Claims 9 · · «9 9 9 9 lil A transfection preparation useful for gene therapy, characterized in that it associates one or more packaging-free recombinant viruses which contain at least one exogenous nucleic acid in their genome with at least one transfection agent other than a virus or a plasmid. 2. A composition according to claim 1 wherein the non-viral transfecting agent is selected from cationic polymers and lipofectants. 3. A composition according to claim 1 or 2 wherein the non-viral transfecting agent is a lipofectant. A composition according to claim 3, characterized in that it 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, A1 wherein m is an integer equal to or greater than 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 stretch of a hydrocarbon chain that is saturated or unsaturated, cholesterol or natural to this to this to this or to a synthetic lipid capable of forming lamellas! or hexagonal phase. 6. The composition of claim 5, wherein the polyamine region is spermine, thermin, or one of the analogs thereof having retained nucleic acid binding properties. 7. The composition of claim 3, wherein the lipofectant has the general formula H ₂ N- (- (CH) _m -NH-) _n -H wherein R represents the lipophilic region of the general formula R 6 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' 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 each other, represent a hydrogen atom or a substituted or a. an 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, • 9,999 • 9 · 9 · 9 9 9 9 9 9 9 9 • 9 9 9 • 9 9 • 9 9 99 9 9 999 999 9 9 9 9 9 9 represent a saturated or unsaturated, linear or branched aliphatic group containing 12 to 22 carbon atoms. 8. The composition of claim 3 wherein the lipofectant has the general formula R.3 0 -to R2 (CH3) (CH3) η P R4 wherein Rl, R2 and R3 represent, independently of one another are hydrogen or - _{(CH2) q-NRR} 'group, with the proviso that q can take the values "1, 2, 3, 4, 5 and 6 independently for different R1, R2 and R3, R and R 'independently of one another represent a hydrogen atom or a - (CH ₂ ) _q > -NH _{2 group} , wherein q may be 1, 2, 3, 4, 5 and 6, independently for different R and R' groups , m, n and p represent, independently of one another, an integer which may be between 0 and 6, and wherein, when 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 R 5 / R 6 ', wherein R 6 and R 7 independently of one another represent a hydrogen atom or a saturated or unsaturated aliphatic group containing 10 to 22 carbon atoms, at least one of the two groups not being hydrogen, AA ···· A · AAA AAAA · · · · · · · · · · · · AA is an integer between 0 and 10, and when u is an integer greater than 1, R5, X / Y and _r can have different meanings in different themes [X- (CHR5) _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 side chain of a natural amino acid, if necessary so substituted, ar is an integer ranging between 1 and 10, and when r is 1, R 5 represents a substituted or unsubstituted side chain of a natural amino acid, and when r is greater than 1, R 5 represents a hydrogen atom. 9. A composition according to claim 1 or 2 wherein the non-viral transfecting agent is. a cationic polymer selected from polyalkyleneimines. A composition according to claim 9 characterized by. by being polyethyleneimine. (PEI) or polypropyleneimine (PPI). The agent selected from the group consisting of lipofectamine, polyethyleneimine weight 50,000 (PEI50K), molecular weight 800,000 glycylspermine (DOGS), palmitoylphosphatidylethanolamine-5-carboxyspermylamide (DPPES) is as claimed in claim 1. preferably with an average polyethyleneimine or 2 non-viral transfection groups containing a molecular with an average (PEI800K), dioctadecylamido, this to this, to this, to <RTIgt; · 2-5-bis (3-aminopropylamino) pentyl (dioctadecylcarbamoylmethoxy) acetate, 2-1,3-bis (3-aminopropylamino) propyl (di-octadecylcarbamoylmethoxy) acetate, {H ₂ N (CH ₂ ) ₃ j 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 NHGH ₂ COArgN [(CH ₂ ) ₁₈ ] ₂ and H ₂ N (CH ₂ t ₃ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NHCH ₂ COGlyN [(CH ₂ ) ₁₇ CH ₃ ] ₂ . Composition according to any one of the preceding claims, characterized in that the nucleic acid contained in the recombinant virus is deoxyribonucleic acid. The composition of any one of claims 1 to 11, wherein the nucleic acid contained in the recombinant virus is a ribonucleic acid. • 14. The composition of claim 12 or 13 characterized by that nuclear acid Yippee chemically modified. A composition according to claim 12, 13 or 14 characterized by that nuclear acid Yippee antisense nucleic acid. 16. A composition according to any preceding claim, wherein the nucleic acid comprises a therapeutic gene. 17. A composition according to claim 16, wherein the therapeutic gene encodes a product selected from the group consisting of enzymes, blood derivatives, hormones, lymphokines: interleukins, interfereons, TNF, growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin and others, dystrophin or minidystrophin and CFTR protein . '' The composition according to claim 16, characterized in that the therapeutic gene is selected from genes associated with arrest of cell division, in particular from the group comprising the gax gene, tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, genes coding factors involved in coagulation: factors VII,. VIII, IX, genes involved in DNA repair deriving, suicide genes (thymidine kinase, cytosine deaminase), hemoglobin or other protein transporter genes, genes corresponding to proteins involved in ^; 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, HDL binding protein, or alternatively a receptor selected from, for example, LDL receptors, receptor receptors selected from, or scavenger receptors. 19. A composition according to any one of the preceding claims wherein the packaging-free recombinant virus is free of at least those regions of its genome that are necessary for its replication in the infected cell. 44 4444 44 4444 4444 4 • 44 4 4 4 4 4 4 4 4 • 4 · 4 4 4 44 4 4 4 44 444 444 4 4 4 4 4 44 444 20. The composition of any preceding claim, wherein the packaging-free recombinant virus is derived from adenovirus or AAV. 21. A composition according to any one of the preceding claims, characterized in that the packaging is derived preferably 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 Ad 5 or Ad 2 adenovirus. A composition according to any one of claims 1 to 4 and 12 to 22, which links a recombinant adenovirus containing at least one in its genome. exogenous nucleic acid, and lipofectamine in an amount sufficient to improve cellular transduction. Composition according to any one of the preceding claims, characterized in that it additionally comprises a dioleoylphosphatidylethanolamine (DOPE) or oleoylpalmitoyl · phosphatidylethanolamine (POPE) adjuvant, a di-stearoyl, -palmitoyl or -myristoylphosphatidylethanolamine-type adjuvant, and 1- to 3x-N-methyl derivatives thereof ; phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (especially galactocerebrosides), sphingolipids (especially sf ingomyelines) or alternatively asialogangliosides. ·· ··· • · · 99 9999 • · • 9 9 ·· 99 999 A composition according to any one of the preceding claims, further comprising a compound affecting; directly or indirectly, the level of nucleic acid condensation. 26. The composition of claim 25, wherein the compound is comprised, in whole or in part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), wherein the number of these 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 connected to a targeting element. and 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, RGD motif protein, cyclic or acyclic peptides containing 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. A recombinant cell which is obtained by contacting a composition according to any one of claims 1 to 28. · ** ** ** ** ** ** ** ** **................ · · · · · · • · • • • • • • • • • » 4.3 31. A method of transferring nucleic acids into a cell interior comprising contacting the cell with a composition of any one of claims 1 to 28.