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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• the present invention relates to recombinant vectors of viral origin and their therapeutic use. More particularly, it relates to recombinant adenoviruses comprising a cassette capable of integrating into the genome of infected cells.
• the invention also relates to the preparation of these vectors, the pharmaceutical compositions containing them and their use for the transfer of genes in vitro, ex vivo and in vivo, in particular in the context of gene and cell therapies.
• Gene and cell therapy consists of correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) or ensuring the expression of a protein of therapeutic interest by introducing genetic information into the affected cell or organ.
• This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue.
• Different techniques have been described for the transfer of this genetic information, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al, J. Virol.
• viruses as vectors for gene transfer has appeared as a promising alternative to these physicochemical transfection techniques.
• different viruses have been tested for their ability to infect certain cell populations.
• retroviruses RSV, HMS, MMS, etc.
• the HSV virus adeno-associated viruses
• adenoviruses the viral vectors developed so far do not make it possible to satisfactorily resolve all the difficulties linked to the transfer of genes into cells and / or the organism.
• the adenovirus which has attractive properties for gene transfer (possibility of producing high titers, low pathogenicity) is an extrachromosomal virus.
• retroviral vectors or derivatives of adeno-associated viruses are capable of integrating into the genome of the cells they infect, they cannot be produced in high quantities, nor for example incorporate large transgenes.
• the present invention provides an advantageous solution to these problems.
• the present invention resides in fact in the development of recombinant vectors usable in gene therapy, having the properties of infection of a recombinant adenovirus vector and allowing the integration of a heterologous sequence in the genome of the cell or the infected organ.
• a first object of the invention relates more particularly to a defective recombinant adenovirus comprising a cassette capable of integrating into the genome of infected cells.
• the cassette comprises a desired DNA sequence, most often heterologous with respect to the adenovirus, and elements allowing its integration into the genome of the infected cells.
• the elements allowing integration are of viral origin.
• the vectors of the invention combine the properties of two types of virus: adenoviruses and integrative viruses.
• the vectors of the invention are particularly advantageous since they can be produced with high titers, are not pathogenic, have a broad host spectrum, are capable of incorporating large heterologous DNA sequences, and d '' integrate said sequences into the genome of infected cells.
• the vectors of the invention make it possible to limit the risks of dissemination of the DNA sequence which it is desired to transfer to the cell or the organism. Once it has been integrated into the genome, it can no longer be excised and incorporated into an infectious viral particle.
• the vectors of the invention advantageously make it possible to transfer to the cells a DNA sequence completely devoid of viral genes. Indeed, the cassette allowing integration into the genome can be totally devoid of any coding sequence of viral origin. The only viral regions that can be introduced are the elements of integration.
• the vectors of the invention allow integration of the heterogeneous DNA sequence at a precise site in the genome of the infected cell, and without cytotoxicity.
• the elements allowing the integration of the cassette consist of one or more repeated-inverted terminal sequences. Even more preferably, the elements allowing the integration of the cassette consist of one or more repeated-inverted terminal sequences of an adeno-associated virus (AAV).
• AAV adeno-associated virus
• a preferred object of the invention therefore relates to a defective recombinant adenovirus comprising a cassette containing at least one repeat-inverted terminal sequence of AAV and a heterologous DNA sequence.
• AAV adeno-associated virus
• AAVs like adenoviruses, are mild pathogenic viruses of the airways. In the absence of a helper virus, AAVs have the property of integrating in a stable manner, in the form of a provirus, at a preferential site of the genome of human cells (region of chromosome 19).
• the AAV genome has been cloned, sequenced and characterized. It comprises approximately 4700 bases, and contains at each end a terminal inverted repeat region (ITR) of approximately 145 bases.
• ITR terminal inverted repeat region
• the rest of the genome is divided into 2 essential regions carrying the packaging functions: the left part of the genome, which contains the reg gene involved in viral replication and the expression of viral genes; the right part of the genome, which contains the cap gene . encoding the capsid proteins of the virus.
• the inverted terminal repeat regions (ITRs) of AAVs serve as the origin of replication for the virus, and are also responsible for the integration of the virus into the genome of infected cells. It has now been shown that it is possible to introduce one or more of these ITRs on a recombinant adenovirus to target the integration of a heterologous DNA sequence on the chromosome of a target cell, and thus improve the stability. of expression over time.
• the Applicant has indeed shown that these sequences can be introduced into the genome of an adenovirus, that they retain their functionality in an adeno-viral context, and that they can be used in gene or cell therapy to integrate into human cells. stably a sequence introduced via an adenovirus.
• the integration cassette according to the invention comprises only a single AAV ITR, linked to the heterologous DNA sequence. It has in fact been shown that a single AAV ITR is sufficient to induce the integration of the heterologous sequence.
• the ITR can be located downstream or upstream of the heterologous DNA sequence (FIGS. 1a and 1b).
• the adenovirus according to the invention comprises a cassette containing at least one heterologous DNA sequence bordered by two ITRs of AAV (FIG. Le).
• This embodiment is particularly advantageous since the integration efficiency is very important.
• the AAV genome comprises 2 ITRs, located at its 2 ends: a 5 'ITR located at the left end, and a 3' ITR located at the right end.
• the sequence of these ITRs is identical, and they are oriented in opposite directions to each other.
• the structure of the ITRs can be modified by rearrangements, but the basic composition is not altered.
• the cassette comprises a heterologous DNA sequence bordered by an ITR 5 'and an ITR 3' from AAV.
• AAV ITRs can be obtained in different ways. They can first of all be isolated from the genome of an AAV, by conventional techniques of molecular biology (see in particular WO91 / 18088, WO93 / 09239). As indicated above, these sequences have approximately 145 bp, are located at the ends of the AAV genome, and can be excised using appropriate restriction enzymes. In this regard, they can be isolated from the various AAV serotypes, such as in particular the AAV1, AAV2, AAV3 and AAV4. Furthermore, the sequence of ITRs being known, they can also be synthesized artificially, using nucleic acid synthesizers, or obtained by mixed techniques (isolation from the genome, then elongation by synthesis techniques).
• these ITRs can also be modified by any technique known to those skilled in the art (molecular biology, chemistry, enzymology, etc.), in the aim of improving their functionality, reducing their size, increasing their stability after integration or their specificity of integration, etc. In particular, they can be modified by mutation, deletion and / or addition of base pairs, according to conventional techniques of molecular biology.
• the ITR (s) used are ITRs of AAV-2.
• the ITRs used in the present invention can comprise only the sequences necessary and sufficient for integration into the genome of the cells (strict ITRs).
• the strict ITRs correspond to 145 bp located on either side of the genome (SEQ ID No. 4).
• the ITRs used can also include additional sequences, for example adjacent sequences of the AAV genome, and / or deletions, insofar as these sequences and / or deletions do not suppress the integration capacity of The tape.
• they can be isolated in the form of fragments of greater length (for example up to 1000 bp), which can be used as they are, if they do not remove the integration capacity of the cassette, or previously digested for reduce their size (see for example SEQ ID n ° 1-3 + 5).
• the chosen sequence or sequences can allow integration into the chromosome, it is for example possible to transfect a human cell line (for example Hela or 293) on the one hand with a plasmid carrying a selection gene of the neoR type between the sequences to be tested or next to the sequence to be tested and on the other hand with a control plamide carrying the same selection gene without said sequences, to select clones by adding the antibiotic G418, then to compare between the two types of transfections the number of clones obtained.
• the integration frequency obtained with the test sequence (s) must be higher than that obtained by simple selection, for said sequences to be usable.
• Another way of testing the integration capacity of the sequences is to transfect the same line on the one hand with a plasmid carrying a marker gene (eg ⁇ gal) between the sequences to be tested and on the other hand with a control plamide carrying the same marker gene without said sequences, and to compare as the enzyme activity passes between the two transfections; the activity must decrease exponentially in the control transfections and remain more stable in the other.
• the "integrating" cells expressing ⁇ gal can be identified, after fixation, by Xgal staining.
• the heterologous DNA sequence and the ITR (s) must be arranged so as to allow integration of the cassette into the genome of the infected cell. They can either be directly joined or spaced by sequences which do not alter the integration property of the cassette. In particular, it may be one or more restriction sites, regions originating from a plasmid used during construction (example: bacterial plasmid), or any neutral region, etc.).
• the adenoviruses of the invention contain a cassette allowing the integration of a heterologous DNA sequence into the genome of the infected cell.
• the heterologous DNA sequence can be any sequence whose transfer to a cell or an organism is desired.
• the heterologous DNA sequence contains a therapeutic gene.
• the term “therapeutic gene” in particular means any gene coding for a protein product having a therapeutic effect.
• the protein product thus coded can be a protein, a peptide, etc. This protein product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter presents no pathology).
• the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein.
• the therapeutic gene can also code for a mutant of a cellular protein, having increased stability, modified activity, etc.
• the protein product can also be heterologous towards the target cell.
• an expressed protein can, for example, supplement or bring about a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.
• the therapeutic products within the meaning of the present invention there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc.
• FR 9203120 growth factors (erythropoietin, G- CSF, M-CSF, GM-CSF, etc.), neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF. aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc; apolipoproteins: ApoAI, ApoAIV, ApoE, etc. (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), the CFTR protein associated with cystic fibrosis, tumor suppressor genes: p53, Rb, RaplA, DCC, k- rev, etc.
• trophic factors BDNF, CNTF, NGF, IGF, GMF.
• apolipoproteins Apo
• FR 93 04745 the genes coding for factors involved in coagulation: Factors VII, VIII, IX, the genes involved in DNA repair, the suicide genes (thymidine kinase, cytosine deaminase), etc. .
• the therapeutic gene can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.
• Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.
• the antisenses also include the sequences coding for ribozymes , which are capable of selectively destroying target RNAs (EP 321,201).
• the therapeutic gene can also include one or more sequences coding for an antigenic peptide capable of generating an immune response in humans or animals.
• the recombinant adenoviruses can be used for the production of either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers . These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, or even specific for tumors (EP 259 212).
• the heterologous DNA sequence may comprise, in addition to the therapeutic gene, a marker gene, such as for example the ⁇ -galactosidase gene or the neo gene.
• a marker gene such as for example the ⁇ -galactosidase gene or the neo gene.
• the adenoviruses of the invention in which the cassette has two genes (a therapeutic gene and a marker gene in particular) are particularly interesting because their construction is greatly facilitated.
• the heterologous DNA sequence also comprises sequences allowing the expression of the therapeutic gene in the desired cell or organ.
• These may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic).
• they may be promoter sequences of eukaryotic or viral genes.
• they may be promoter sequences originating from the genome of the cell which it is desired to infect.
• they may be promoter sequences originating from the genome of a virus.
• these expression sequences can be modified by adding activation, regulation sequences, or conferring specificity of tissue expression on the gene of interest.
• heterologous DNA sequence may also include a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell.
• This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.
• the defective recombinant adenoviruses according to the invention are adenoviruses incapable of replicating autonomously in the target cell.
• the genome of the defective adenoviruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the cassette.
• the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
• serotypes of adenoviruses the structure and properties of which vary somewhat.
• adenoviruses of type 2 or 5 Ad 2 or Ad 5
• Ad 2 or Ad 5 adenoviruses of animal origin
• adenoviruses of animal origin mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or even simian (example: SAV).
• the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example].
• adenoviruses of human or canine or mixed origin are used.
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the cassette. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used must preferably (i) be transformable by 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.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%).
• Strategies for the construction of vectors derived from adenoviruses have also been described in applications No. WO94 / 26914 and FR 2,707,664 which are incorporated into the present application by reference.
• the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• the recombinant adenoviruses prepared according to the present invention can be used for the transfer of genes of interest in vitro, ex vivo or in vivo.
• in vitro they can make it possible to transfer a gene to a cell line, for example in order to produce a recombinant protein.
• Ex vivo they can be used to transfer a gene onto a population of cells taken from an organism, optionally selected and amplified, in order to confer on these cells desired properties with a view to their re-administration to an organism.
• they can be used for gene transfer by direct administration of a purified solution, optionally combined with one or more pharmaceutical vehicles.
• the present invention also relates to any pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above.
• These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
• the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes.
• the doses of defective recombinant adenovirus used for injection can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought.
• the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ⁇ and 101 pfu / ml, and preferably 10 ⁇ to 1010 pfu / ml.
• the term pfu (“plaque forming unit”) corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
• the present invention thus provides a very efficient means for the transfer of genes into cells.
• the vectors of the invention can be used in many applications, such as genetic diseases (myopathy, cystic fibrosis, SCID, etc.), pathologies of the central nervous system (Alzheimer, Parkinson, etc.), cardiovascular diseases (hemophilia, atherosclerosis ), AIDS, cancers, etc.
• the vectors of the invention are very particularly advantageous for the transfer of genes in dividing cells. Indeed, thanks to the infection capacities of the adenoviral vector and to the integration properties of the casette, the vectors of the invention make it possible to confer stable properties over generations on cells which divide. Preferred examples of these cell types include hematopoietic cells (stem cells, progenitors, etc.), and cancer cells.
• the vectors of the invention can be used for the transfer of genes into CD34 cells.
• the invention also relates to any mammalian cell modified by an adenovirus as defined above. More preferably, it is a human cell, and even more preferably, chosen from hematopoietic cells, in particular CD34, or tumor cells.
• vectors of the invention can be used both for modifying human and animal cells (sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.).
• the invention thus provides a particularly effective method for the administration of genes in vivo, comprising the administration of a vector as defined above comprising a cassette composed of said gene and of elements allowing its integration into the genome of any or part of the infected cells.
• the adenoviruses of the invention comprising a cassette consisting of a heterologous DNA sequence bordered by 2 AAV ITRs can also be used for the production of recombinant AAVs.
• AAVs indeed need to replicate the presence of a helper virus. It may in particular be an adenovirus of a herpes virus or a vaccinia virus. In the absence of such a helper virus, AAVs remain in latent form in the genome of infected cells, but cannot replicate.
• recombinant AAVs are generally produced by co-transfection in a cell line infected with the helper virus (in particular adenovirus) of a plasmid carrying the AAV cassette (gene bordered by ITR) and of a plasmid carrying the packaging genes (rep / cap plasmid).
• helper virus in particular adenovirus
• a plasmid carrying the AAV cassette gene bordered by ITR
• a plasmid carrying the packaging genes (rep / cap plasmid).
• another application of the invention resides in a process for the preparation of recombinant AAVs according to which the producer cells are infected with an adenovirus as described above and transfected with a plasmid carrying the rep and cap genes.
• the rep and cap genes are also carried by a virus (in particular an adenovirus) used to co-infect the producer cells.
• a virus in particular an adenovirus
• the method of the invention uses a production line containing, integrated into its genome, the rep and cap genes.
• a single step of infection with the virus of the invention is sufficient.
• a virus according to the invention containing, in addition to the heterologous DNA / ITR AAV cassette, an expression cassette for the rep and cap genes.
• the rep and cap genes can be placed under the control of a strong and / or inducible promoter.
• the adenovirus is deleted from all of the viral genes except for the E4 region, and it is co-infected with another adenovirus carrying the entire genome except for the E4 region, in the 293 cells. This embodiment is particularly advantageous.
• the E4 virus used is the same for all heterologous DNAs.
• the construction of the virus takes place via a plasmid carrying E4, the PSI sequence and the ITRs of the adenovirus (plasmid ⁇ E4ITR, cf. FR 2,707,664), in which the rep and cap genes are introduced. and heterologous DNA framed by AAV ITRs.
• This strategy thus makes it possible to propagate the 2 viruses to all cells, in contrast to the transfection of a repapid plasmid which affects only a fraction of the cell population and therefore gives only low AAV titers.
• Figure 1 Schematic representation of cassettes according to the invention.
• Figure 2 Representation of vector pXL2373
• Figure 3 Construction of vector pXL2373
• Figure 4 Representation of vector pXL2384
• Figure 5 Construction of vector M 13 ITR FL
• Figure 6 Representation of vector pITRFL
• Figure 7 Representation of vector pXL2388
• Figure 8 Representation of vector pXL2389
• Figure 9 Representation of the vector papoAI ITRAAV
• the pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).
• the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.
• the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
• the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
• the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 12 (1985) 8749-8764] using the kit distributed by Amersham.
• Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5477] using the kit distributed by Amersham.
• Example 1 Construction of a plasmid carrying the ⁇ -galactosidase gene inserted between the ITRs of 1 ⁇ AV2.
• This example describes the construction of a vector, designated pXL2373, carrying 2 ITRs regions flanking a marker gene ( ⁇ gal) and a polyadenylation site, serving as an intermediary for the preparation, by recombination, of a recombinant adenovirus.
• the plasmid pXL2373 (FIG. 2) contains in particular a fragment comprising
• Rous sarcoma virus RSV
• the vector pXL2373 also contains an adenovirus region allowing homologous recombination.
• the vector pXL2373 was constructed in the following manner (FIG. 3): The DNA fragment carrying the LTR ("long terminal repeat") of the Rous sarcoma virus (RSV), the lacZ gene of Escherichia coli with the sequences localization and polyadenylation signals from the SV40 early region was isolated as an Xbal-Kpnl fragment from the plasmid pRSV- ⁇ gal (Stratford-Perricaudet et al., J. Clin.Invest.90 (1992 ) 626). This fragment was then inserted at the corresponding sites of the plasmid pAICMVIXCAT.3 (Philip et al., Molecular and Cellular Biology, in press). This step allows this fragment to be inserted between the first 650 and the last 191 base pairs (bp) of 1 ⁇ AV2. The resulting plasmid was called pXL2359 ( Figure 3a).
• the Xbal-BglII fragment of pXL2359 comprising the Rous sarcoma virus (RSV) LTR, the Escherichia coli lacZ gene with the nuclear localization sequences and polyadenylation signals from the SV40 early region, as well as the 191 last bp of AAV, was inserted at the corresponding sites of pBS KS +, to generate the plasmid pXL2360. This subcloning makes it possible to isolate this same fragment in the form of an Xbal-Smal fragment.
• the Xbal-Smal fragment of pXL2360 was then inserted at the compatible Xbal-EcoRV sites of pRSV- ⁇ gal to give the vector pXL2364 (FIG. 3a).
• the plasmid psub201 has been described in Samulski et al. (J. Virol 61 (1987) 3096).
• the Xbal-PvuII fragment of this plasmid carrying the sequences 4484 to 4675 of the AAV was inserted at the Xbal-EcoRV sites of pCRII (Invitrogen) to generate the plasmid pXL2362.
• the Spel-Xbal fragment of pXL2362 was introduced into the Xbal compatible site of pXL2364 (FIG. 3b).
• the plasmid obtained was designated pXL2373 ( Figure 2).
• the capacity of this vector to allow the integration of the cassette is verified by transfection in the Hela and 293 cell lines.
• Example 2 Construction of a plasmid carrying the ⁇ -galactosidase gene inserted between the ITRs of 1 ⁇ AV2.
• This example describes the construction of a vector, designated pXL2384, carrying 2 ITRs regions flanking a marker gene ( ⁇ gal) and a polyadenylation site, serving as an intermediary for the preparation, by recombination, of a recombinant adenovirus.
• the plasmid pXL2384 (FIG. 4) contains in particular a fragment comprising
• Rous sarcoma virus RSV
• the vector pXL2384 also contains an adenovirus region allowing homologous recombination.
• the vector pXL2384 was obtained by inserting the fragments
• Example 3 Construction of a plasmid carrying the ⁇ -galactosidase gene inserted between the strict ITRs of AAV2.
• the ITRs sequences used have a 46 bp extension downstream of the left ITR or upstream of the right ITR, and / or a 9 bp deletion 5 ′ of the left ITR (see SEQ ID n ° 1-3).
• This example describes the construction of a plasmid comprising a gene of interest inserted between the strict ITRs of AAV2. More particularly, this example describes the construction of a vector, designated pITRFL, carrying 2 strict ITR regions surrounding a marker gene ( ⁇ gal) and a polyadenylation site, serving as an intermediary for the preparation, by recombination, of a recombinant adenovirus. .
• the plasmid pITRFL (FIG. 6) contains in particular a fragment comprising - the LTR ("long terminal repeat") of the Rous sarcoma virus (RSV),
• the pITRFL vector also contains an adenovirus region allowing homologous recombination.
• the vector pITRFL was constructed as follows: The strict ITR sequence was constructed using the following oligodeoxynucleotides: seq 4259: (SEQ ID No. 6)
• M13ITR5 oligodeoxynucleotides
• seq 4560 and seq 4561 on the one hand and seq 4263 and seq 4264 on the other hand are hybrid two by two and introduced between the Hindi ⁇ and BamHI sites of M13mpl8, the bacteriophage obtained is called M13ITR3 '.
• the KpnI-Ahall fragment of M13ITR5 'comprising the 5' region of the AAV ITR to the Ahall site (region 1 to 63 on the AAV sequence) and the AhalI-BamHI fragment comprising the 3 'region of ITR (region 64 to 145) are introduced at the Kpnl-BamHI sites of M13mpl8 to give the vector M13 "ITRFL".
• the BamHI-Spel fragment of M13 "ITRFL” containing the entire sequence of the ITR of AAV (bases 1 to 145) is introduced between the unique BglII-Spel sites of pXL2384 to give the plasmid pRSV ⁇ gal ITRlss.
• the Kpnl-HincIIde fragments M13 "ITRFL” containing the entire sequence of the ITR of AAV (bases 1 to 145) and EcoRV-XhoI of pXL2384 carrying the protein IX of Ad5 are introduced between the Xhol-Kpnl compatible sites of pRSVBgaUTRlss to give pITRFL ( Figure 6).
• Example 4 Construction of a plasmid carrying the ⁇ -galactosidase gene and the neoR gene inserted between the ITRs of TAAV2.
• This example describes the construction of a vector, designated pXL2388, carrying 2 ITRs regions flanking 2 genes ( ⁇ gal and neoR) and a polyadenylation site, serving as an intermediary for the preparation, by recombination, of a recombinant adenovirus.
• the plasmid pXL2388 (FIG. 7) contains in particular a fragment comprising:
• neoR neomycin
• RSV Rous sarcoma virus
• the vector pXL2388 also contains an adenovirus region allowing homologous recombination.
• the vector pXL2388 was constructed as follows: The DNA fragment carrying the gene conferring resistance to neomycin (neoR) under the control of the SV40 promoter, as well as the polyadenylation signals of the SV40 virus was isolated in the form of 'a BamHI fragment from the plasmid pMAMneoluc (Clontech). This fragment was then introduced into the BamHI site of the plasmid pBS KS + (Stratagene) to introduce new restriction sites on either side of this fragment. The plasmid thus obtained is called pXL2363.
• the DNA fragment carrying the gene conferring resistance to neomycin (neoR) under the control of the SV40 promoter, as well as the polyadenylation signals of the SV40 virus was then isolated from pXL2363 in the form of an EcoRI-Xbal fragment. and introduced to the EcoRI-Xbal sites of pCRII (Invitrogen) to give rise to the plasmid pXL2372.
• the DNA fragment carrying the gene conferring resistance to neomycin (neoR) under the control of the SV40 promoter, as well as the polyadenylation signals of the SV40 virus was then isolated in the form of a Spel fragment from pXL2372, and introduced into the Spel site of pXL2384 (Example 2) to give rise to the plasmid pXL2388 ( Figure 7).
• the sequence of the neomycin resistance gene under the control of the SV40 promoter comes from the plasmid pXL2388 digested with Spel, and this fragment was inserted at the Spel site of pRSVGAIIX to give pXL2429.
• the capacity of this vector to allow the integration of the cassette is verified by transfection into the Hela and 293 cell lines.
• the 293 cells (2,106 cells cultured in 100 mm dishes) are transfected with the plasmids pXL2388, pXL2429 according to the technique. with calcium phosphate.
• the day after the transfection the medium is changed. After 72 hours, the cells are harvested and returned to culture after dilution 1/10 and 1/50 in a non-selective medium. 72 hours later, the medium is changed and a selective medium containing geneticin at 400 microg / ml is applied.
• Clones appear after approximately 2 weeks of culture in this medium and it is found that the frequency of appearance of the clones is 100 times higher with the plasmid pXL2388 than with the plasmid pXL2429 which testifies to the capacity of the ITRs to integrate the transgene in the genome of the cell.
• Example 5 Construction of a plasmid carrying a Sh ble :: lacZ fusion gene inserted between the ITRs of AAV2.
• This example describes the construction of a vector, designated pXL2389, carrying 2 ITRs regions framing 1 fusion gene (Sh ble :: lacZ), serving as an intermediary for the preparation, by recombination, of a recombinant adenovirus.
• the plasmid pXL2389 contains in particular a fragment carrying, under the control of the SV40 promoter, a fusion between the gene for resistance to Phleomycin (or zeomycin) and the reporter gene lacZ, followed by polyadenylation signals from the virus SV40, which fragment being inserted between the sequences of two ITRs of the AAV (sequences shown in SEQ ID No. 2 and 3).
• the vector pXL2389 also contains an adenovirus region allowing homologous recombination.
• the fusion carrying a dominant marker (Sh ble) and the reporter gene lacZ makes it possible to obtain a dominant phenotype associated with a rapidly identifiable phenotype (blue coloration on X-gal) with a size which does not exceed 3.5 kb. Therefore the region inserted between the two ITRs of the AAV has a size which does not exceed 4.3 kb.
• the vector pXL2389 was constructed as follows: The Spel-NdeI fragment of the plasmid pUT593 (Cayla, Toulouse) carrying the promoter SV40 and the start of the Sh ble :: lacZ fusion was isolated, then inserted between the Spel- Ndel of pXL2384 (example 2). The plasmid thus obtained was designated pXL2389 ( Figure 8). The capacity of this vector to allow the integration of the cassette is verified by transfection in the Hela and 293 cell lines.
• Example 6 Construction of a plasmid carrying the apolipoprotein AI gene inserted between the ITRs of 1 ⁇ AV2.
• Apolipoprotein AI is a protein made up of 243 amino acids, synthesized in the form of a prepropeptide of 267 residues, having a molecular mass of 28,000 daltons. It is synthesized in humans specifically in the liver and the intestine and it constitutes the essential protein of HDL particles (70% of their mass in proteins). It is abundant in plasma (1.0-1.2 g / 1). Its activity which is best characterized biochemically is the activation of lecithin cholesterol acyl transferase (LCAT), but many other activities are attributed to it, such as in particular the stimulation of the efflux of cellular cholesterol.
• LCAT lecithin cholesterol acyl transferase
• Apolipoprotein AI plays a major role in resistance to atherosclerosis, probably linked to the reverse transport of cholesterol, since the mere expression of this apolipoprotein in transgenic mice makes it possible to reduce the surface of lipid deposits at the level of 40 times.
• aorta versus control mice (Rubin et al. 1993 Science, In Press). Its gene, 1863 bp long, was cloned and sequenced (Sharpe et al., Nucleic Acids Res. 12 (9) (1984) 3917).
• various natural variants of apoAI have been described in the prior art.
• the plasmids used to generate, by homologous recombination, the recombinant adenoviruses expressing the apoAI gene were constructed as follows: Construction of the papoAI ITR AAV plasmid (FIG. 9): The papoAI ITR AAV plasmid contains in particular the cDNA encoding the preproapoAI under the control of the RSV promoter, the polyadenylation signals of the SV40 virus, the whole inserted between the AAV ITRs, as well as an adenovirus region allowing homologous recombination.
• DNA fragment carrying in particular the left ITR and the packaging sequence of the adenovirus 5 as well as the LTR of the RSV virus was isolated in the form of an XmnI-ClaI fragment of the plasmid pXL2384 (Example 2) and the fragment of DNA carrying the cDNA encoding preproapoAI and the SV40 virus polyadenylation signals was isolated in the form of a ClaI-BamHI fragment from the plasmid pXL2244 (FR93 05125). These two fragments were then inserted at the XmnI-BamHI sites of the plasmid pXL2384, to generate the plasmid papoAI ITR AAV. During this last step, the lacZ region followed by the polyA of SV40 was eliminated.
• This example describes the construction of a vector pXL2629 carrying 2 ITRs regions flanking the lacZ marker gene and a polyadenylation site, serving as an intermediary for the preparation by recombination of a recombinant adenovirus.
• This plasmid differs from plasmid pXL2384 by the sequence of the left AAV ITR (SEQ ID No. 5).
• the plasmid pXL2359 (Example 1) was digested with Hinfl, the end of which was made blunt by treatment with the DNA polymerase of bacteriophage T4 and then digested with PstI, a fragment of approximately 200 bpd carrying the ITR of the AAV (sequence figure) was isolated and introduced into the plasmid pBSKS-i- at the PstI and Smal sites (blunt end).
• the plasmid thus constructed is pXL2580.
• the plasmid pXL2581 is derived from pXL2384 by insertion of 2 oligonucleotides seq 4674 and sep 4675 at the BglII-Spel sites of pXL2384, this plasmid therefore carries in place of the ITR of the left AAV of pXL2384 a unique BstBI site which could be used to construct recombinant AdITRsAAVRSVBgal adenoviruses by the technique of transfection of a ligation mixture of two linearized plasmids into cells 293.
• seq4674 5'GATCTTTCGAAT3 '(SEQ ID No. 12)
• seq 4675 5'CTAGATTCGAAA3' (SEQ ID n ° 13)
• the plasmid pXL2629 was constructed as follows: the plasmid pXL2580 was digested with BamHI-BglII and the fragment of approximately 170 bp containing the complete sequence of the ITR of AAV was introduced into the plasmid pXL2581 previously linearized by BglII.
• Example 8 Preparation of the Ad ITRsAAVRSV ⁇ Gal Adenovirus
• This example describes the construction of a defective recombinant adenovirus comprising a cassette allowing the integration of a gene into the genome of cells. More particularly, this adenovirus, designated Ad ITRsAAVRSV ⁇ Gal comprises a cassette composed of 2 ITRs of AAV surrounding the ⁇ gal gene.
• This adenovirus was obtained by cotransfection of the plasmid pXL2384 for recombination with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus.
• the Ad ITVsAAVRSV ⁇ gal adenovirus was prepared by homologous in vivo recombination between the AdRSV ⁇ Gal adenovirus (Stratford-Perricaudet et al., J. Clin.Invest. 90 (1992) 626) and the plasmid ⁇ XL2384 according to the following protocol:
• the plasmid pXL2384 linearized by the enzyme Xmnl and the adenovirus AdRSV ⁇ Gal linearized by Clal are cotransfected in line 293 in the presence of calcium phosphate to allow recombination.
• the recombinant adenoviruses thus generated are selected by plaque purification.
• the recombinant adenovirus is amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer of approximately l ⁇ 1 "pfu / ml.
• the viral particles are centrifuged on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
• Ad ITRsAAVRSV ⁇ gal is stored at -80 ° C in 20% glycerol.
• Example 9 Preparation of Ad ⁇ ITRsAAVRSV ⁇ gal adenovirus.
• This example describes the construction of a defective recombinant adenovirus comprising a cassette allowing the integration of a gene into the genome of cells. More particularly, this adenovirus, designated Ad ⁇ ITRsAAVRSV ⁇ Gal, comprises a cassette composed of 2 truncated AAV ITRs surrounding the ⁇ gal gene.
• This adenovirus was obtained by cotransfection of the plasmid pXL2373 for recombination with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus.
• the protocol used is the same as that described in Example 7 for the preparation of the adenovirus Ad ITRsAAVRSV ⁇ gal.
• the Ad ⁇ ITRsAAVRSV ⁇ gal adenovirus is stored at -80 ° C in 20% glycerol.
• Example 10 Preparation of Ad ITRFL adenovirus.
• This example describes the construction of a defective recombinant adenovirus comprising a cassette allowing the integration of a gene into the genome of cells. More particularly, this adenovirus, designated Ad ITRFL comprises a cassette composed of 2 strict AAV ITRs surrounding the ⁇ gal gene.
• This adenovirus was obtained by cotransfection of the plasmid pITRFL for recombination with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus.
• Adenovirus Ad ITRFL is stored at -80 ° C in 20% glycerol.
• Example 11 Preparation of the adenovirus Ad apoAI ITRAAV.
• This example describes the construction of a defective recombinant adenovirus comprising a cassette allowing the integration of a gene into the genome of cells.
• this adenovirus designated Ad apoAI ITRAAV, comprises a cassette composed of 2 truncated AAV ITRs surrounding the preproapoAI gene.
• This adenovirus was obtained by cotransfection of the papoAI ITR AAV plasmid for recombination with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions encoded by the El (El A and E1B) regions of adenovirus.
• Example 12 Preparation of the Ad2629 adenovirus.
• This example describes the construction of a recombinant adenovirus Ad2629 carrying a cassette composed of 2 ITRs of AAV surrounding the Bgal gene.
• This adenovirus was obtained by cotransfection of the plasmid pXL2629 with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the E1 (E1A and E1B) regions of adenovirus.
• Ad2629 adenoviruses were prepared by homologous in vivo recombination between the adenovirus dl324 and the plasmid pXL2629 according to the following protocol: the plasmid pXL2629 linearized by Xmnl and the adenovirus dl324 linearized by Clal are cotransfected in the line 293 in the presence of calcium phosphate to allow recombination. The recombinant adenoviruses thus generated are selected by plaque purification.
• the recombinant adenovirus is amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer of approximately 109-1010 pfu / ml.
• the viral particles are centrifuged on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
• NAME RHONE-POULENC RORER S.A.
• RUE 20, avenue Raymond ARON
• SEQ ID NO: 2 GCGCGCTCGC TCGCTCACTG AGGCCGCCCG GGCAAAGCCC GGGCGTCGGG CGACCTTTGG 60 TCGCCCGGCC TCAGTGAGCG AGCGAGCGCG CAGAGAGGGA GTGGCCAACT CCATCACTAG 120 GGGTTCCTTG TAGTTAATGA TTAACCCGCC ATGCTACTTA TCTACGTAGC CATG 174

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