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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
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  • A61L2/0023—Heat
• • A—HUMAN NECESSITIES
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
  • A61L2/04—Heat
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  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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  • C07—ORGANIC CHEMISTRY
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • 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
  • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

• the present invention relates to new viral vectors, their preparation and their use in gene therapy. It also relates to pharmaceutical compositions containing said viral vectors. More particularly, the present invention relates to recombinant adenoviruses as vectors for gene therapy.
• Gene therapy consists of correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) 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 exist, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol.
• adenoviruses have certain properties of interest for use in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated to date with significant pathologies in man. Adenoviruses have thus been used to transfer genes of interest into the muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al. Nature genetics 3 (1993) 224), etc.
• Adenoviruses are linear double-stranded DNA viruses approximately 36 kb in size. Their genome includes in particular a repeated reverse sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and genes late (Cf figure 1).
• the main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the El region (El a and Elb in particular) are necessary for viral replication.
• the E4 and L5 regions for example, are involved in viral propagation.
• the main late genes are contained in regions L1 to L5.
• the genome of the Ad5 adenovirus has been fully sequenced and is accessible on the database (see in particular Genebank M73260). Likewise, parts or even the entire genome of adenoviruses of different serotypes (Ad2, Ad7, Adl2, etc.) have also been sequenced.
• adenoviruses Given the properties of the adenoviruses mentioned above, these have already been used for gene transfer in vivo. To this end, different vectors derived from adenoviruses have been prepared, incorporating different genes ( ⁇ -gal, OTC, ⁇ -
• the adenovirus was modified so as to render it incapable of replication in the infected cell.
• the constructions described in the prior art are adenoviruses deleted from the El regions (El a and / or Elb) and possibly E3 into which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al.
• line 293 a complementation line in which a part of the adenovirus genome has been integrated. More specifically, line 293 contains the left end (approximately 11-12%) of the genome of the adenovirus serotype 5 (Ad5), comprising the left ITR, the packaging region and the El region, including El a, Elb and part of the region coding for the protein pIX.
• This line is capable of trans-complementing recombinant adenoviruses defective for the E1 region, that is to say devoid of all or part of the E1 region, necessary for replication.
• the recombinant El- adenoviruses can be prepared in 293 cells due to the good trans ⁇ complementation of the El region contained in this line.
• Contamination with replicating particles is a major drawback. Indeed, the presence of such particles in therapeutic compositions would induce in vivo viral propagation and uncontrolled dissemination, with risks of inflammatory reaction, recombination, etc. Contaminated batches cannot therefore be used in human therapy.
• the present invention overcomes these drawbacks.
• the present invention indeed describes new constructions allowing the production of defective recombinant adenoviruses devoid of any contamination by replicative particles.
• the present invention also describes a method for the production of these recombinant adenoviruses. It thus provides new defective recombinant vectors derived from adenoviruses, which are particularly suitable for use in gene therapy, in particular for the transfer and expression of genes in vivo.
• the present invention more particularly resides in the construction of defective recombinant adenoviruses comprising an adenovirus genome whose genetic organization is modified and whose possible recombination with the genome of the production line leads to the generation of non-replicative viral particles. and / or non-viable.
• the Applicant has now shown that it is possible to modify the genomic organization of the adenovirus to avoid the production of replicative particles during the production of stocks.
• a first object of the present invention therefore relates to a recombinant adenovirus comprising an adenovirus genome (i) whose E1 region is inactivated,
• the term “genetic or genomic organization” means the arrangement of the various genes or functional regions present in the genome of the wild adenovirus, as represented in FIG. 1.
• a modified genetic or genomic organization therefore corresponds to a genome in which certain genes or regions are not in their original position.
• certain genes or certain regions can be moved from the genome and inserted into another site. It is also possible to insert a given gene or region at a particular site, and to delete or inactivate the region of origin (by mutation, deletion, insertion, etc.)
• non-viable viral particle designates, within the meaning of the invention, an adenovirus incapable of replicating its DNA and / or of propagating autonomously in infected cells.
• a non-viable viral particle therefore has an adenovirus genome lacking at least the sequences necessary for its replication and / or its propagation in the infected cell. These regions can either be eliminated (in whole or in part), or made non-functional, or substituted by other sequences.
• the sequences necessary for replication and / or propagation are for example the E1 region, the E4 region or the L5 region. More particularly, with regard to the E4 region, the important genes are the ORF3 and ORF6 genes.
• the Applicant has more particularly shown that it is possible to displace a function essential for viral replication or propagation without affecting the properties of adenovirus as a vector for gene therapy, namely its high power of infection of cells, in particular human, and its ability to efficiently transfer a gene of interest into said cells.
• the present invention relates to recombinant adenoviruses, a region essential for viral replication and / or propagation is present in a genomic position other than its original position.
• this region is located at or near another genomic region rendered non-functional.
• the vectors of the invention are particularly advantageous since they allow the incorporation of large genes of interest and that they can be produced at high titers, without production of contaminating replicative viral particle.
• the E1 region or any other region can be inactivated or made non-functional by various techniques known to those skilled in the art, and in particular by deletion, substitution, deletion, and / or addition of one or more several bases.
• Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, using genetic engineering techniques, or alternatively by treatment with mutagenic agents.
• the said genetic modification (s) may be located in a coding part of the region, or outside of a coding region, and for example in the regions responsible for the expression and / or transcriptional regulation of said genes. Inactivation can therefore manifest itself by the production of inactive proteins due to structural or conformational modifications, by the absence of production, by the production of proteins having an impaired activity, or by the production of natural proteins at an attenuated level. or according to a desired regulation mode.
• physical agents such as energy radiation (X-rays, ⁇ rays, ultra violet rays, etc.
• chemical agents capable of reacting with different functional groups of the bases of DNA and for example alkylating agents [ethylmethane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroquinoline-1-oxide (NQO)], bialkylating agents, intercalating agents,
• Genetic modifications can also be obtained by gene disruption, for example according to the protocol initially described by Rothstein [Meth. Enzymol. 1PJ . (1983) 202]. In this case, all or part of the coding sequence is preferably disturbed to allow replacement, by homologous recombination, of the genomic sequence with a non-functional or mutant sequence.
• the region concerned is inactivated by mutation and / or deletion of one or more bases. Even more preferably, it is inactivated by total or partial deletion.
• deletion is understood to mean any deletion of all or part of the gene considered. It may especially be all or part of the coding region of said gene, and / or all or part of the region promoting the transcription of said gene. Removal can be accomplished by digestion using enzymes appropriate restriction, then ligation, according to conventional molecular biology techniques, as illustrated in the examples.
• the inactivation of the genes is carried out in such a way that it affects only the gene considered and not the other viral genes, in particular the neighboring genes. Furthermore, certain alterations such as point mutations being by nature capable of being corrected or attenuated by cellular mechanisms, it is particularly preferred that the inactivation is perfectly segregationally stable and / or non-reversible.
• the region essential for viral viability is preferably located at or near the site of deletion. It is however possible to use other insertion sites, such as for example restriction sites already present in the wild genome. In this regard, it is nevertheless preferred that the insertion be carried out at least near the deletion site, that is to say outside the deletion site, but sufficiently ready so that it cannot occur. recombination events in the interval between the deletion site and the insertion site. Preferably, the distance between the deletion site and the insertion site should not exceed 50 bp.
• the region essential for viral replication and / or propagation is displaced to be included at the level of the inactivated E1 region and / or of the E3 region.
• the E1 region is inactivated by deletion of a PvuII-BglII fragment going from nucleotide 454 to nucleotide 3328, on the sequence of the adenovirus Ad5. This sequence is accessible in the literature and also on the database (see in particular Genebank n ° M73260).
• the E1 region is inactivated by deletion of a HinfII-Sau3A fragment going from nucleotide 382 to nucleotide 3446.
• the region essential for replication and / or viral propagation according to the present invention is advantageously chosen from all or part of the E4 region and / or of the pIX-IVa2 region and / or of the L5 region, etc.
• the essential region consists of all or a functional part of the E4 region and it is inserted at or near the El deletion site.
• the E4 region essential for viral propagation, is inserted in a position other than its original position, so that this region is absent in any construction which would result from recombination with the genome of the production line (cf. FIG. 3).
• the present invention therefore also relates to a recombinant adenovirus whose genome is characterized by the presence of inactivated El and E4 regions, and in that all or a functional part of the E4 region is inserted at or near the El region .
• Such an adenoviral vector preferably comprises two ITRs, an encapsidation region, a deletion in the E1 region at the level of which all or a functional part of E4 is inserted, and an E4 region of inactivated origin.
• the E4 region is involved in the regulation of late gene expression, in the stability of late nuclear RNA, in the quenching of expression of host cell proteins and in the efficiency of DNA replication viral. Mutants lacking E4 are unable to spread. E4 thus constitutes an essential region for viral replication and / or propagation.
• This E4 region consists of 7 open reading phases, designated ORF1, ORF2, ORF3, ORF4, ORF3 / 4, ORF6 and ORF6 / 7 ( Figure 4).
• ORF3 and ORF6 are the two genes essential for viral spread. Each of these genes is capable of inducing viral spread.
• inactivation of the E4 region involves inactivation of ORF3 and ORF6.
• the entire E4 region is inserted at or near the El deletion site. It may in particular be a Maell-Mscl fragment corresponding to nucleotides 35835-32720.
• E4 only a functional part of E4, that is to say sufficient to allow viral propagation, is inserted.
• This part includes at least one functional ORF3 or ORF6 gene.
• the functional part of E4 consists essentially of ORF3 or ORF6.
• these coding phases can be isolated from the E4 region in the form of PvuII-AluI and BglII-PvuII fragments respectively, corresponding to nucleotides 34801-34329 and 341 15-33126 respectively.
• the E4 region or the functional part of this region also comprises a transcription promoter region II can be the promoter of the E4 region or any other functional promoter, such as the viral promoters (El a, SV40, LTR -RSV, etc), eukaryotes or mammal
• the promoter used is the promoter of the E4 region
• the functional part of E4 inserted at the level of El does not necessarily correspond to the part of E4 deleted in the original position.
• the initial region can be inactivated by point mutation (without deletion) and a region Functional E4 inserted at El level
• the initial E4 region can be entirely deleted and only a functional part inserted at El level
• the inactivation of the E4 region implies, within the meaning of the invention, the functional inactivation of at least the ORF3 and ORF6 regions. These regions of origin can be inactivated by any technique known to those skilled in the art. In particular, all of the methods given above can be applied to the inactivation of ORF3 and ORF6 or any additional region of E4. For example, the deletion of the E4 region of the virus Ad2 dl808 or of the viruses Ad5 dll004, Ad5 dll007, Ad5 dllQl l or Ad5 dll014 can be used in the context of the invention (cf. example 3)
• adenoviruses can be obtained for example by co-transfection in a line for producing a first plasmid carrying the left part of the genome of the virus which it is desired to produce (having a deletion in the El region at or near which at least one functional part of E4 is inserted) and a viral genomic DNA fragment bringing the right part of the virus genome (having an inactivated E4 region) After recombination, the viruses produced are amplified and isolated
• These adenoviruses can also be obtained by permutation of the ends of the genome, comprising the ITRs plus the contiguous region
• the invention also relates to a recombinant adenovirus characterized in that its genome has an inactivated E1 region and in that the left end, comprising the ITR and the packaging region, and the right end, comprising the ITR and all or a functional part of the E4 region, are permuted More particularly, the left end comprising the left ITR and the packaging region is contained in the first 382
• the right end comprising '' ITR right and all or a functional part of the E4 region, including the promoter of the E4 region is contained in the last 3215 nucleotides of the genome of the adenovirus Ad5 (for example from the Mscl site at position 32720).
• Ad5 for example from the Mscl site at position 32720.
• the genome of the recombinant adenovirus thus obtained is particularly advantageous - since the essential region E4 displaced to the left is maintained in its natural environment and therefore in conditions of optimal activity for a high-titer infectious cycle. In addition, this region now precedes the region whose presence in the genome of production line 293 was at the origin of the appearance of viable particles.
• the essential region consists of the region coding for the proteins pIX and IVa2 and it is inserted at the level of the E3 region, possibly as a replacement for deleted sequences (cf. FIG. 6). More particularly, the region coding for the proteins pIX and IVa2 is included in a BglII-NruI fragment corresponding to nucleotides 3328 to 6316 on the sequence of the wild-type Ad5 adenovirus.
• the possible recombination of the recombinant adenovirus with the region of the adenovirus integrated into the production line only generates viral particles which are not viable because they are deleted from the main late genes essential for viability.
• two essential regions of the genome of the edenovirus are displaced from their original positions. More preferably, these essential regions are represented by the region coding for the protein pIX and the region coding for all or only a functional part of the E4 region. According to a preferred mode, they will be displaced at the level of the E1 region, replacing deleted sequences and retaining or not the orientation of their reading frame.
• the region coding for the protein pIX is displaced in the deleted region E1, to the right of the left ITR which has become, right end of the recombinant virus.
• the region coding for the protein pIX is also placed there in reverse reading.
• the region essential of E4 it is represented there by the genes ORF3-ORF6 / 7, under the control of the promoter E4, and is also inserted there at the level of the deletion site of E 1, between the region coding for the protein pIX and the region coding for the IVa2 protein whose position has not been affected; in the case of the specific construction of FIG. 8, the two regions coding respectively for the pIX protein and the IVa2 protein have distinct polyadenylation sites.
• Such a construction is particularly advantageous in terms of reliability and safety. Indeed, any parasitic recombination between 2 viral molecules of this type, at the level of the E4 region for example, will lead to a recombinant virus deprived of its encapsidation sequence. Likewise, a recombination between such a viral molecule with the complementary region of dudit adenovirus, integrated into a production cell line, will only generate viral particles deleted from their main late genes, essential for their viability.
• the recombinant adenoviruses according to the invention advantageously comprise the ITR sequences and a region allowing the encapsidation.
• the inverted repeat sequences constitute the origin of replication of adenoviruses. They are located at the 3 ′ and 5 ′ ends of the viral genome (cf. FIG. 1), from which they can be easily isolated according to the conventional techniques of molecular biology known to those skilled in the art.
• the nucleotide sequence of the ITR sequences of human adenoviruses (in particular the Ad2 and Ad5 serotypes) is described in the literature, as well as canine adenoviruses (in particular CAV1 and CAV2).
• the left ITR sequence corresponds to the region comprising nucleotides 1 to 103 of the genome.
• the packaging sequence (also called Psi sequence) is necessary for the packaging of the viral genome. This region must therefore be present to allow the preparation of defective recombinant adenoviruses according to the invention.
• the packaging sequence is located in the genome of wild adenoviruses, between the left ITR and the E1 gene (see FIG. 1). In the adenoviruses of the invention, it can be located next to the left ITR as well as the right ITR (cf. FIG. 5). It can be isolated or artificially synthesized by conventional molecular biology techniques.
• nucleotide sequence of the packaging sequence of human adenoviruses (in particular of the serotypes Ad2 and Ad5) is described in the literature, thus than canine adenoviruses (notably CAV1 and CAV2).
• Ad5 adenovirus for example, a functional encapsidation sequence is between nucleotides 194 and 358 of the genome.
• the adenoviruses according to the invention may have other alterations in their genome.
• other regions can be deleted to increase the capacity of the virus and reduce these side effects linked to gene & viral expression.
• all or part of the E3 region in particular can be deleted.
• Recombinant adenoviruses according to the invention have properties which are particularly attractive for use in gene therapy. These vectors indeed combine very high infection, safety and gene transfer properties.
• the recombinant adenoviruses of the invention also comprise a heterologous nucleic acid sequence whose transfer and / or expression in a cell, an organ or an organism is sought.
• the heterologous DNA sequence may include one or more therapeutic genes.
• the therapeutic genes which can thus be transferred are any gene whose transcription and possibly translation into the target cell generate products having a therapeutic effect.
• the protein product thus coded can be a protein, a peptide, an amino acid, 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 provide a deficient activity in the cell allowing it to fight against a pathology.
• 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, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc; apolipoproteins: ApoAI, ApoAIV, ApoE, etc (FR 93 05125), dystrophin or a minidystrophin (FR 9111947), tumor suppressor genes: p53, Rb, RaplA, DCC, k-rev, etc (FR 93 04745) , the genes coding for factors involved in coagulation: Factors VII, VIII, IX, etc., the suicide genes: Thymidine kinase, cytosine desaminase, etc; or all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.), etc.
• trophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF
• 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 therapeutic gene may also be a gene coding for an antigenic peptide capable of generating an immune response in humans.
• the invention therefore makes it possible to produce vaccines making it possible to immunize humans, in particular against microorganisms or viruses.
• 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 nucleic acid sequence also comprises a promoter region for functional transcription in the infected cell, as well as a region located 3 ′ of the gene of interest, and which specifies a transcriptional end signal and a site for polyadenylation. All of these elements constitute the expression cassette.
• the promoter region it may be a promoter region naturally responsible for the expression of the gene considered when it is capable of functioning in the infected cell. They can also be regions of different origin (responsible for the expression of other proteins, or even synthetic).
• they may be gene promoter sequences eukaryotic or viral. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect.
• heterologous nucleic acid sequences may also comprise, in particular upstream of the therapeutic gene, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.
• the therapeutic gene expression cassette can be inserted at different sites in the genome of the recombinant adenovirus according to the invention. It can first of all be inserted at the level of the El deletion. In this case, it is located next to (in 5 ′ or 3 ′) the region or the functional part of E4. It can also be inserted at the E3 region, in addition to or in substitution for sequences. It can also be located in the inactivated E4 region
• the vectors of the invention also have a functional E3 gene under the control of a heterologous promoter. More preferably, the vectors have a part of the E3 gene allowing the expression of the protein gpl9K. This protein in fact makes it possible to prevent the adenovirus vector from undergoing an immune reaction which (i) limits its action and (ii) could have undesirable side effects.
• the recombinant adenoviruses according to the invention can be of various origins. There are in fact different serotypes of adenoviruses, the structure and properties of which vary somewhat, but which have a comparable genetic organization. Therefore, the teachings described in the present application can be easily reproduced by a person skilled in the art for any type of adenovirus. ⁇ More particularly, the adenoviruses of the invention can be of human, animal, or mixed (human and animal) origin.
• adenoviruses of human origin it is preferred to use those classified in group C. More preferably, among the various serotypes of human adenovirus 5, it is preferred to use, within the framework of the present invention, adenoviruses of type 2 or 5 (Ad 2 or Ad 5).
• the adenoviruses of the invention can also be of animal origin, or contain sequences derived from adenoviruses of animal origin.
• the Applicant has indeed shown that adenoviruses of animal origin are capable of infecting human cells with great efficiency, and that they are unable to propagate in the human cells in which they have been tested (cf. request FR 93 05954).
• the Applicant has also shown that adenoviruses of animal origin are in no way trans-complemented by adenoviruses of human origin, which eliminates any risk of recombination and of propagation in vivo, in
• adenoviruses or adenovirus regions of animal origin is therefore particularly advantageous since the risks inherent in the use of viruses as vectors in gene therapy are even lower.
• the adenoviruses of animal origin which can be used in the context of the present invention can be of canine, bovine, mutine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or still simian (example: after-sales service). More particularly, among the avian adenoviruses, mention may be made of serotypes 1 to 10 accessible to ATCC, such as for example the strains Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR- 827), IBH-2A (ATCC VR-828), J2-A
• ATCC VR-829 25 (ATCC VR-829), T8-A (ATCC VR-830), Kl 1 (ATCC VR-921) or the strains referenced ATCC VR-831 to 835.
• the various known serotypes can be used , and in particular those available at ATCC (types 1 to 8) under the references ATCC VR-313, 314, 639-642, 768 and 769. Mention may also be made of the urine adenoviruses FL (ATCC VR-550) and E20308 ( ATCC VR-
• adenoviruses or regions of adenovirus of origin canine, and in particular all the strains of the CAV2 adenoviruses (Manhattan or A26 / 61 strain (ATCC VR-800) for example).
• Canine adenoviruses have been the subject of numerous structural studies. Thus, complete restriction maps of the CAV1 and CAV2 adenoviruses have been described in the prior art (Spibey et al., J. Gen. Virol. 70 (1989) 165), and the Ela, E3 genes as well as the ITR sequences were cloned and sequenced (see in particular Spibey et al., Virus Res. 14 (1989) 241; Linné, Virus Res. 23 (1992) 11-19, WO 91/1 1525).
• the defective recombinant adenoviruses according to the invention can be prepared in different ways.
• a first method consists in transfecting the DNA of the recombinant virus
• a second approach consists in co-transfecting into a suitable cell line the DNA of the defective recombinant virus prepared in vitro and the DNA of one or more viruses or helper plasmids.
• it is not necessary to have a competent cell line capable of complementing all the defective functions of the recombinant adenovirus. Part of these functions is in fact complemented by the helper virus or viruses. This or these helper viruses are themselves defective.
• the preparation of defective recombinant adenoviruses of the invention according to this method is also illustrated in the examples.
• the present application also describes the construction of plasmids carrying the modified left part of the genome of the Ad5 adenovirus (plasmids of the pCOl-E4 series for example). These plasmids are particularly useful for the construction of recombinant adenoviruses as vectors for gene therapy.
• the plasmids pCOl-E4 carry the left region of the adenovirus genome, from the left ITR to nucleotide 6316, with a deletion of the region between nucleotides 382-3446, corresponding to the El locus, at the level from which is inserted all or a functional part of E4.
• Plasmids pC01-E4 can be used to prepare the defective recombinant adenovirus by cotransfection with DNA, preferably of viral origin, corresponding to the right part of the genome of the adenovirus having an inactivated E4 region, in a competent cell line.
• Ad2 dl808 which is deleted from the E4 region (Weinberg et al., J. Virol. 57 (1986) 833),
• the invention also relates to a process for the preparation of recombinant adenoviruses devoid of replicating particles according to which a competent cell line is co-transfected with - a first DNA comprising the left part of the genome of said adenovirus, having a deletion in the El region at or near which at least one functional part of the E4 region is inserted, and
• a second DNA comprising at least the right part of the genome of said adenovirus, having an inactivated E4 region, and a part of adenovirus common with the first DNA, and the adenoviruses are recovered by homologous recombination between said
• the human embryonic kidney line 293 (Graham et al., J. Gen Virol. 36 (1977) 59). As indicated previously, this line contains in particular, integrated into its genome, the left part of the genome of the human adenovirus Ad5 (12%).
• the first DNA is chosen from plasmids of the pCOl-E4 type.
• the first or second DNA used in the method of the invention further carries a heterologous DNA sequence of interest.
• the plasmids pCOl-E4 thus allow the construction of recombinant adenoviruses carrying a deletion in the E1 region going from nucleotide 382 to nucleotide 3446, at the level of which all or a functional part of E4 is inserted, possibly as well as a therapeutic gene.
• the present invention also relates to any pharmaceutical composition comprising one or more defective recombinant adenoviruses as described previously.
• the pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
• the pharmaceutical composition contains pharmaceutically acceptable vehicles for an injectable formulation.
• pharmaceutically acceptable vehicles for an injectable formulation can be in particular saline solutions (monosodium phosphate, disodium, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes.
• the doses of virus used for the 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 4 and 10 14 pfu, and preferably 10 6 to 10 10 pfu.
• 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 15 days, 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 adenoviruses of the invention can be used for the treatment or prevention of many pathologies, including genetic diseases (dystrophy, cystic fibrosis, etc.), neurodegenerative diseases (alzheimer, parkinson, ALS , etc.), cancers, pathologies linked to coagulation disorders or dyslipoproteinemias, pathologies linked to viral infections (hepatitis, AIDS, etc.), etc.
• genetic diseases distrophy, cystic fibrosis, etc.
• neurodegenerative diseases alzheimer, parkinson, ALS , etc.
• cancers pathologies linked to coagulation disorders or dyslipoproteinemias
• pathologies linked to viral infections hepatitis, AIDS, etc.
• Figure 2 Recombination events between the adenovirus and the 293 line.
• Figure 3 Representation of a type of vector of the invention, and of its recombination with the line 293.
• Figure 5 Representation of an adenovirus of the invention with permuted ends.
• E4 + denotes a functional E4 region
• ⁇ E4 denotes a non-functional E4 region
• ⁇ denotes an encapsidation sequence.
• the expression cassette for the gene of interest is not shown but can be inserted as indicated in the text.
• Figure 6 Representation of a type of vector of the invention (pIX-IVa2)
• pIX + IVa2 contains at least one functional IVa2 region.
• ⁇ El ⁇ pIX designates a deletion of the adenovirus sequences between the region ⁇ and the end of the region coding for the protein 140kD (position 5200) of the region E2. This deletion also includes the IVa2 region and concerns the sequence present in the chromosome of line 293 downstream of the E & b region.
• Figure 7 Restriction map of the HindIII fragment contained in the plasmid pCOl.
• Figure 8 Representation of the expression plasmids pPY40 and pPY6
• Figure 9 Representation of the plasmid pGY10
• Figure 10 Representation of the plasmid pCOl- (ORF6 + ORF7)
• Figure 1 1 Representation of the plasmids pPY78 and pPY77
• Figure 12 Representation of plasmids pPY15 and pJYl
• Figure 13 Representation of a type of virus according to the invention
• Figure 14 Representation of the plasmids pXL2675 and pXL2757
• Figure 15 Representation of the restriction maps of plasmids pPY66, pPY82 and pPY75
• Figures 16 (A): Schematic representation of homologous recombination between the HincP / Ad5 replicon and the plasmid pPY66 and (B).
• Figure 17 Protocol for generating the HincP / Ad5 viral genome [delE4 (Y + ITR)] via homologous recombination of the two replicons pPY82 and HincP / Ad5 [delE4-
• Figure 18 Representation of HincP / Ad5 [ITRYdelEl (SacB + SpecR) delE4 (Y + ITR)] and
• 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 DNA ligase phage T4 (Biolabs) according to the supplier's recommendations
• the filling of the prominent 5 'ends can be carried out by the Klenow fragment of E coli DNA Polymerase I (Biolabs) according to the supplier's specifications
• Destruction of the 3' ends prominent is carried out in the presence of DNA Polymerase from phage T4 (Biolabs) used according to the manufacturer's recommendations
• Destruction of the prominent 5 ′ ends is carried out by a gentle treatment with nuclease S 1
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al [Nucleic Acids Res 1_3 (1985) 8749-8764] using the kit distributed by Amersham
• the enzymatic amplification of DNA fragments by the technique called PCR can be performed using a "DNA thermal cycler "(Perkin Elmer Cetus) according to the manufacturer's specifications
• Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al [Proc. Natl Acad Sci USA, 74 (1977) 5463-5467] using the kit distributed by Amersham
• the EcoRI-Xbal fragment corresponding to the left end of the genome of the Ad5 adenovirus was first cloned between the EcoRI and Xbal sites of the vector pIC19H. This generates the plasmid pCA.
• the plasmid pCA was then cut by Hinfl, its prominent 5 'ends were filled with the klenow fragment of DNA polymerase I from E. coli, then it was cut by EcoRI.
• the fragment thus generated of the plasmid pCA which contains the left end of the genome of the adenovirus Ad5 was then cloned between the EcoRI and Smal sites of the vector pIC20H (Marsh et al., Gene 32 (1984) 481).
• the plasmid pCB was then cut with EcoRI, its prominent 5 'ends were filled in with the klenow fragment of DNA polymerase I from E. coli, then it was cut with BamHI.
• the fragment thus generated of the plasmid pCB which contains the left end of the genome of the adenovirus Ad5 was then cloned between the NruI and BglII sites of the vector pIC20H. This generates the plasmid pCE, an interesting characteristic of which is that it has the first 382 base pairs of the adenovirus Ad5 followed by a cloning multisite.
• the Sau3A (3346) - SstI (3645) fragment and the SstI (3645) - Narl (5519) fragment of the Ad5 adenovirus genome were first ligated and cloned between the ClaI and BamHI sites of the vector pIC20H, this which generates the plasmid pPY53.
• the Sall-Taql fragment of the plasmid pPY53 prepared from a dam- context, containing the part of the genome of the adenovirus Ad5 between the Sau3A (3346) and Taql (5207) sites was then cloned between the SalI and ClaI of the vector pIC20H, which generates the plasmid pCA '.
• the Narl (5519) - NruI (6316) fragment of the genome of the Ad5 adenovirus prepared from a dam context and the Sall-Narl fragment of the plasmid pCC were then ligated and cloned between the SalI and NruI sites of the vector. pIC20R. This generates the plasmid pCD '.
• This example describes the construction of plasmids of the pCOl-E4 type, that is to say of plasmids obtained by incorporating all or a functional part of the E4 region of an adenovirus in the plasmid pCOl (Example 1).
• the plasmid pPY2 corresponds to the cloning of the Avr2-Sall fragment (approximately 1.3 kb including the promoter / LTR of the MMTV virus) of the plasmid pMSG (Pharmacia) between the Xbal and Sali sites of the plasmid pIC20H prepared from an E. coli context dam +.
• the plasmid pPY4 is derived from the plasmid pPY2 by deletion of a fragment of 35 bp after cleavage with BamHI and Bgl2 then religation.
• the plasmid pPY5 corresponds to the cloning of the Taql-Bgl2 fragment (position 35576- 32490) including the E4 region of Ad5 between the ClaI and BamHI sites of the plasmid pIC20H.
• the entire E4 region of Ad5 is therefore now bordered by EcoRV and Sphl sites originating from the cloning multisite. Partial digestion of the plasmid pPY5 with EcoRV and then digestion with Sphl makes it possible to purify an EcoRV-Sphl fragment of approximately 3.1 kb including the entire E4 region of Ad5.
• the plasmid pPY6 * is identical to the plasmid pPY6 with the exception of the Xbal site which was destroyed after cutting by filling with the Klenow fragment of DNA polymerase from E coli (Biolabs), then religation.
• the plasmid pPY6 * therefore contains the entire E4 region (from the Taql site at position 35576 and up to position 32490 located approximately 300 bp after the polyadenylation site) expressed under the control of the LTR promoter of the RSV virus.
• the plasmid pFG144 [FL Graham et al. EMBO J. (1989) 8 2077-2085] contains in particular a Sau3A fragment of 1162 bp including the right end of the Ad5 genome from position 34773 This fragment is then cloned into the BamHI site of the plasmid pIC20H which generates the plasmid pGY9 in which the Sau3A fragment is now included in a BamHI-EcoRI fragment of 1184 bp due to the orientation of the fragment relative to the vector This fragment is then purified by electroelution, cut by Avril, then subjected to partial hydrolysis by the enzyme Maell.
• One of the restriction products corresponds to the Maell (35835) -AvrII (35463) fragment which includes the promoter of the E4 region of AdS and up to the limit of the right ITR.
• This 372 bp fragmen is then purified by electroelution and ligated in the presence of the AvrII-SalI fragment of the plasmid pPY6 * between the ClaI and SalI sites of the plasmid pCOI which generates the plasmid pGYlO (FIG. 9) which is of the pCOI-E4 type ( Figure 7).
• the plasmid pPY6 is the source of an Xhol-Sall fragment of approximately 4.5 kb corresponding to the LTR expression cassette MMTV / E4.
• the cloning of this cassette at the SalI site of the plasmid pCOl generates the plasmid pCOl-MMTV / E4 (2 possible orientations of the E4 region with respect to the ITR and of the sequence ⁇ ) which is of the pCOl-E4 type.
• Cloning of the Xbal fragment (approximately 1 kb) of the plasmid pGRE5.1 included an expression cassette composed of a minimal promoter highly inducible to glucocorticoids and of a polyadenylation signal [Mader and White Proc. Natl. Acad. Sci. (1993) 90 5603-5607],
• the cloning of this fragment between the Xbal sites of the plasmid pIC20H prepared from a dam context generates the plasmid pPY21 in which the 5 binding sites for the glucocorticoid receptor are now located at immediate proximity to the Bgl2 site from the cloning multisite.
• the plasmid pPY21 is the source of a Bgl2-EcoRI fragment of approximately 0.8 kb corresponding to the minimal promoter highly inducible to glucocorticoids. The cloning of this fragment between the Bgl2 and EcoRI sites of the plasmid pIC20H generates the plasmid pPY26.
• the plasmid pPY5 is the source of an EcoRV-Hind3 fragment of approximately 0.65 kb which includes the Taql-Hind3 fragment from positions 35576 to 34930 on the genome of the Ad5.
• the cloning of this fragment between the EcoRV and Hind3 sites of the plasmid pIC20R generates the plasmid pPY24 in which an EcoRI site is now located close to the Taql site (position 35576).
• This plasmid is the source of an EcoRI -Sphl fragment of approximately 3.1 kb which comprises the entire E4 region of Ad5 at positions- 5576 to 32490.
• the expression cassette pGRE5 / E4 of the plasmid pPY40 is available in the form of a Bgl2-SalI fragment of approximately 3.9 kb, the cloning of which between the BamHI and SalI sites of the plasmid pCOl generates the plasmid pCOl-pGRE5 / E4 which is pCOl-E4 type ( Figure 7).
• 35835 to 35464 is purified by electroelution after partial hydrolysis by Mae2 and total hydrolysis by Avr2. This fragment is then hydrolyzed by Taql and the Mae2-Taql fragment (positions 35835 to 35576) is cloned in the presence of the ClaI-SalI fragment (1.65 kb) originating from the plasmid pGY47 'between the ClaI and SalI sites of the plasmid pCOl.
• One of the reaction products corresponds to the plasmid pCOl- (ORF6 + ORF7) ( Figure 10) which is of the pCOl-E4 type.
• the sequence located between the stop codon of ORF7 and the Bgl2 site (up to position 32490 which includes the polyadenylation site of E4) is deleted and replaced by a heterologous polyadenylation site.
• the Fragment Xbal -Sali (about 0.25 kb) corresponding to the signal SV40 virus late polyadenylation is isolated from the plasmid pGL3 and cloned between the corresponding sites of the plasmid pIC20H prepared from a dam + context, which generates the plasmid pPY76.
• This plasmid is the source of a Kpnl-Sall fragment ( about 0.7 kb including the sequences between positions 33598 to 32891), the cloning of which between the corresponding sites of the plasmid pC ⁇ l- (ORF6 + ORF7) generates the plasmid pPY78 ( Figure 11) which is of the pCOl-E4 type.
• This plasmid is the source of a Kpnl-Sall fragment (approximately 0.5 kb including the sequences between positions 33598 to 33126) whose cloning between the corresponding sites of the plasmid pCOl- (ORF6 + ORF7) generates the plasmid pCOl- (ORF ⁇ ) which is of the pCOl-E4 type.
• ORF3 sequence only A plasmid of the pC01-E4 type in which only the ORF3 reading phase of the E4 region is also present can be constructed in an analogous manner. Indeed, it is known that the mere expression of ORF3 is sufficient to complement viruses deleted for the E4 region.
• the Bgl2-Xbal fragment (approximately 1.65 kb) of the plasmid pPY6 includes the sequence of Ad5 between positions 34115 and 32490 (ORF6 + ORF7 from the E4 region). The cloning of this fragment between the Bgl2 and Xbal sites of the plasmid pIC20H generates the plasmid pPY13.
• This plasmid now includes the E4 subregion (ORF6 + ORF7) of Ad5 in an Xhol-Sphl fragment of approximately 1.65 kb. The cloning of this fragment between the SalI and Sphl sites of the plasmid pPY4 generates the plasmid pPY15 (FIG.
• the Bgl2-SalI fragment of the plasmid pPY13 (approximately 1.65 kb) includes the E4 subregion (ORF6 + ORF7) of Ad5.
• the cloning of this fragment between the BamHI and SalI sites of the plasmid pIC20H generates the plasmid pPY45 in which the subregion (ORF6 + ORF7) is now included in an EcoRl-Sphl fragment of about 1.65 kb.
• the cloning of this fragment between the EcoRI and Sphl sites of the plasmid pPY26 generates the plasmid pJYl (FIG.
• This example describes the construction of recombinant adenoviruses carrying a deletion in the E1 region going from nucleotide 382 to nucleotide 3446, an inactivated E4 region, and all or a functional part of E4 inserted in the E1 deletion.
• Plasmids of the pCOI-E4 type can for example be cotransfected in 293 cells in the presence of a genome viral carrying a modification / deletion in the E4 region so that this region is nonfunctional (mutation in ORF3 and ORF6 at least). Such viruses are therefore not viable in a line which does not functionally transcomplement the E4 region. viruses can be previously propagated in the line W162 [Weinberg and Ketner Proc. Natl. Acad. Sci. (1983) 80: 5383-5386], or in one of the lines 293 E4 + described in patent FR.
• viruses include the Ad2dl808 [Challberg and Ketner Virology (1981) 1_14: 196-209], Ad5dll004, Ad5dll007, or Ad5dll014 [Bridge and Ketner J. Virol. (1989) 63: 631-638] viruses, or again Ad5dll011 [Bridge et al. Virology (1993) 193: 794-801] etc.
• viruses are therefore characterized by the presence of the left ITR and a functional packaging sequence (sequence ⁇ , nucleotide 1-382 for example), followed by a functional E4 region (whole or at least ORF3 or ORF6) expressed from a functional promoter and for example the original promoter of the E4 region [included in the Taql (35576) - MaeII (35835) J fragment, or an inducible promoter, followed by the region located downstream from the Sau3A site located at position 3446 of the Ad5 genome, and continuing up to the right ITR and therefore including the deletion of the E4 function present in the starting E4 "virus (FIG. 13).
• E1" E4 + viruses characterized by an E4 deletion on the right of the genome originating from the virus Ad5dll01 1 can be propagated in line 293 at titers greater than 10 ⁇ PFU / ml.
• the Bcll-Avr2 fragment (approximately 0.5 kb) originating from the plasmid pFG144 corresponds to the right end of the AdS genome from the Avr2 site at position 35464.
• the cloning of this fragment between the sites Xbal and BamHI of the plasmid pIC19H prepared from an E. coli dam context generates the plasmid pPY23.
• This plasmid is the source of a Sall-Hae3 fragment of approximately 320 bp including the right end of the Ad5 genome up to the Hae3 site at position 35617.
• the cloning of this fragment between the Xhol and EcoRV sites of plasmid pIC20H generates the plasmid pPY29.
• the Bgl2-Smal fragment corresponding to the Ad5 genome between positions 32490 and 33093 is then cloned between the BamHI and Smal sites of the plasmid pPY29 which generates the plasmid pPY64.
• This plasmid is the source of an Xbal-Hind3 fragment, the cloning of which between the corresponding sites of the multi-cloning site of the plasmid pXL2675 (FIG. 14) generates the plasmid pPY65.
• Plasmid pXL2675 (2513 bp) is a ColEl-type replicon (included in the BsAl-Pvu2 fragment of approximately 1.15 kb and originating from the commercial plasmid pBKS +) having a gene conferring resistance to kanamycin (originating from Tn5, plasmid Pharmacia pUCKXXX ) at E. -coli and a multisite of synthetic cloning.
• Plasmid pXL2757 ( Figure 14) is the source of a Smal -EcoRV fragment of approximately 4 kb containing the sacB gene of B. subtilis and a gene conferring resistance to spectinomycin in E. coli.
• the cassette (SacB + SpecR) of this fragment cloned into the SmaI site of the plasmid pPY65 generates the plasmid pPY66 ( Figure 15).
• the plasmid pPY66 can be used to introduce the cassette (SacB + SpecR) by homologous recombination with a viral genome derived from Ad5 and included in a functional replicon in E. coli polA.
• the technology described in patent application FR 95 016323, is based on the use of certain properties of replication in E. coli. Indeed, the replicons of the HincP incompatibility class (and for example RK2) replicate in the absence of the enzyme coded by the polA gene. Conversely, replicons of the ColEl type (plasmids of the pUC, pIC, pBR, etc.) need this enzyme to replicate.
• the Ad5 genome derived from the plasmid pFG144 was first cloned on the plasmid RK2 (of the HincP class) and then introduced by electroporation into a strain of E. coli mutated in the polA gene.
• the Xbal fragment which contains the ColEl replicon (pBR) included in place of the E3 region in the plasmid pFG144 was then deleted from the genome (Patent Application FR 95 016323). This generates the strain of E. coli called E. coli polA / Ad5.
• An important point is that the adenoviral genome present in this strain is bordered by Pacl sites on either side of the ITRs and that such genomes are infectious after transfection into 293 cells.
• the introduction of the cassette (SacB + SpecR) by homologous recombination in E. coli polA / Ad5 is carried out as follows: a) The plasmid pPY66 is first introduced by electroporation in E. coli polA / Ad5. Selection in the presence of spectinomycin and kanamycin is carried out. The resistant clones then correspond to the formation of a cointegrate between the HincP / Ad5 replicon and the plasmid pPY66. In almost all cases, the plasmid pPY66 was inserted by a homologous recombination event between the two types of replicon.
• a clone corresponding to the result of a recombination at the E4 region between positions 32490 to 33093 (603 bp) is then isolated (FIG. 16 A).
• This bacterial clone has a "direct repetition" of the sequences (A and B) on either side of the cassette (SacB + SpecR): 603 bp on the one hand and 320 bp (right end of the genome) on the other hand.
• the presence of such sequences is a source of instability and gives rise to rare homologous recombination events on either side of the cassette (SacB + SpecR).
• the Sall-Smal fragment originating from the plasmid pPY6 * and corresponding to the genome of Ad5 of positions 32490 to 33093 is first cloned between the corresponding sites of the plasmid pCO7 which generates the plasmid pPY81
• the plasmid pCO7 obtained after total restriction of the plasmid pCOl by Pstl and religation, therefore contains the left end of l ⁇ d5 up to position 382, the multi-cloning site of plasmid pCOl, followed by the sequence of Ad5 from positions 3446 up to the Pstl site located at position 3788.
• the plasmid pPY81 is the source of an H3-Xbal fragment of about 1.4 kb, the cloning of which between the corresponding sites of the plasmid pXL2675 generates the plasmid pPY82, a restriction map of which is given in Figure 15. 4.2. 2 Construction of the viral genome in E. coli
• the introduction of the sequence ⁇ in the immediate vicinity of the right ITR is also done by homologous recombination after introduction of the plasmid pPY82 by electroporation in E. coli generates the viral genome HincP / Ad5 [delE4 ( ⁇ + ITR)]
• the event of replicon fusion is first selected in the presence of kanamycin.
• a clone corresponding to a homologous recombination event between sequences 32490 and 33093 common to the two replicons is then isolated ( Figure 16B).
• the ejection event of the ColEl replicon (originating from the plasmid pXL2675) is then amplified by lifting the selection with kanamycin for a sufficient number of generations.
• Glucose as carbon source is then replaced by sucrose and the bacterial clones are isolated for which the event of ejection of the ColEl replicon occurred by homologous recombination at the ITR level which generates the HincP viral genome. / Ad5 [delE4 ( ⁇ + ITR)] ( Figure 16B). A clone corresponding to this event is then isolated: E. coli polA / Ad5 [delE4 ( ⁇ + ITR)].
• the Hind3-EcoRV fragment (approximately 0.4 kb) of the plasmid pCOl includes the left end of the viral genome up to position 382.
• the cloning of this fragment between the corresponding sites of the plasmid pXL2675 generates the plasmid pPY83.
• the plasmid pCOlDSal is obtained after digestion of the plasmid pCOlDS (prepared in a dam + context) with the enzyme Xbal, treatment with the Klenow fragment of DNA polymerase I of E. coli, then religature.
• This plasmid is the source of a BamHI -Nsil fragment of approximately 1 kb which includes the sequences of Ad5 at positions 3446 to 4419 (Nsil site).
• the cloning of this fragment between the BamHI and Pst1 sites of the plasmid pIC20R generates the plasmid pPY86 wherein the sequences between the positions 3446 to 4419 ⁇ are now included in a BamHI-Bgl2 fragment cloning of this fragment into the BamHI site of the plasmid pPY83 generates the plasmid pPY87.
• the SmaI -EcoRV fragment corresponding to the cassette (SacB + SpecR) of the plasmid pXL2757 is then cloned into the EcoRV site of the plasmid pPY87, which generates the plasmid pPY88 (FIG. 17).
• This plasmid is used to replace, according to a procedure analogous to the procedure described in FIG. 16 A, the left part of the HincP / Ad5 genome [delE4 ( ⁇ + ITR)] by the corresponding part coming from the plasmid pPY88, which generates the viral genome noted
• HincP / Ad5 [ITR ⁇ delEl (SacB + SpecR) delE4 ( ⁇ + ITR)] whose structure is given in Figure 18. 5.2. Deletion of the ⁇ sequence and introduction of a functional E4 region.
• the right end of the adenovirus genome was amplified by PCR with the oligonucleotides 5'-
• the PCR amplification therefore generates a fragment of 418 bp corresponding to the right end of the AdS and into which a Pacl site and then an EcoRI site were introduced immediately upstream of the ITR, while a Pstl site is found located in the immediate vicinity of position 35517.
• the cloning of this fragment between the EcoRI and Pst1 sites of the plasmid pUC19 generates the plasmid pXL2624 (see patent application FR.95 016323).
• the EcoRI -Taql fragment of the plasmid pXL2624 corresponding to the right end of the viral genome from the Taql site at position 35576 is cloned between the EcoRI and ClaI sites of the plasmid pGY47 ', which generates the plasmid pPY89.
• This plasmid is the source of an EcoRI -Sali fragment of approximately 2 kb including the right end of the viral genome up to position 35576, then the E4 sub-region (ORF6 + ORF7) of positions 341 to 32490.
• the Kpnl-Sstl fragment (approximately 0.95 kb) of the plasmid pPY78 corresponds to the C-terminal part of the E4 region between positions 32891 to 33598, immediately followed by the "late polyadenylation signal of the SV40 virus.
• the cloning of this fragment between the corresponding sites of the plasmid pPY89 generates the plasmid pPY90
• This plasmid is the source of an EcoRI -Sali fragment of approximately 2 kb including the right end of the viral genome up to position 35576, then the E4 sub-region (ORF6 + ORF7) of positions 34115 to 32891.
• the plasmids pGY50 'or pPY90' are used to replace, according to a procedure analogous to the procedure described in FIG. 16B, the left part of the HincP / Ad5 genome [ITR ⁇ delEl (SacB + SpecR) [delE4 ( ⁇ + ITR)] by the corresponding part from said plasmids.
• the plasmid pPY32 is described in patent application Fr. 94 04590 (04/18/94).
• This plasmid is the source of a Bgl2-Hind3 fragment of approximately 0.65 kb corresponding to the right end of the Ad5 genome from the Bgl2 site (position 32490) and deleted between the Mae2 sites (position 3281 1) up to 'at the Hae3 site (position 35617).
• the cloning of this fragment between the BamHI and Hind3 sites of the plasmid pXL2675 generates the plasmid pPY70, a restriction map of which is given in Figure 19.
• This plasmid can be used to introduce the corresponding E4 deletion by homologous recombination, for example in E. coli polA / Ad5 [delE4 (SacB + SpecR)].
• the plasmid pGY12 corresponds to the cloning of the BssH2-Mscl fragment (positions 33249-32720) between the BssH2 and EcoRV sites of the commercial plasmid pSL1180 ( Figure 20).
• the cloning of EcoRI -Taq 1 fragments from the plasmid pXL2624 (this fragment corresponds to the right end of the Ad5 genome up to the Taql site at position 35576) and Taql-BssH2 (positions 35576 to 33249) between the EcoRI sites and BssH2 of the plasmid pGY12 generates the plasmid pGY13.
• This plasmid is therefore the source of an EcoRI -Hpal fragment (approximately 3.25 kb) including the right end of the Ad5 genome up to position 32720.
• the cloning of this fragment between the EcoRI and Smal sites of the plasmid pIC20H generates the plasmid pGY14 ( Figure 20).
• This plasmid is the source of an EcoRI -BspH1 fragment corresponding to the right end of the Ad5 genome up to position 34700.
• the Sphl-Taql fragment located between positions 31224 to 33055 on the Ad5 genome is purified from the plasmid pMC2 and then cloned between the Sphl and ClaI sites of the plasmid pIC20H, which generates the plasmid pYJ5.
• this plasmid is the source of a Bgl2-Xbal fragment which includes the viral genome sequence located between positions 32490 to 33055. This Bgl2-Xbal fragment is then cloned with the Avr2 fragment.
• This plasmid can be used to introduce the corresponding E4 deletion by homologous recombination, for example in E. coli polA Ad5 [delE4 (SacB + SpecR)].
• the plasmid pMC2DSmal is obtained after total digestion of the plasmid pMC2 with Smal then religation.
• This plasmid is the source of a Bgl2-Hind3 fragment of approximately 1.2 kb including the entire right end of the Ad5 genome from position 32490 and deleted in the E4 region between positions 33093 and 35356.
• the cloning of this fragment between the BamHI and Hind3 sites of the plasmid pXL2675 generates the plasmid pPY72, a restriction map of which is given in Figure 19.
• This plasmid can be used to introduce the corresponding E4 deletion by homologous recombination, for example in E. coli polA / Ad5 [delE4 (SacB + SpecR)].
• Smal-Smal deletion (33093-35356)
• the BamHI -EcoRI fragment of the plasmid pGY9 (approximately 1.2 kb) includes the right end of the Ad5 genome from the Sau3A site at position 34773.
• This restriction fragment is purified by electroelution, cut by SmaI, then subjected to a partial hydrolysis by the enzyme Mae2.
• One of the restriction products corresponds to the Maell (35835) -SmaI (35356) fragment.
• the cloning of this fragment between the SmaI and ClaI sites of the plasmid pPY82 generates the plasmid pPY75 ( Figure 15).
• This plasmid can be used to introduce the corresponding E4 deletion by homologous recombination, for example in E. coli polA / Ad5 [delE4 (SacB + specR) ( ⁇ + ITR)].
• the Taql fragment of the plasmid pPY82 which includes positions 32490 (SalI site of the cloning multisite) at 33055 is cloned into the ClaI site of the plasmid pIC20H which generates the plasmid pICTaq in which the Xhol site of the multisite is positioned in the immediate vicinity of the position 32490.
• the plasmid pICTaq is the source of an Xhol-Smal fragment including positions 32490 to 33055, the cloning between the SalI and Smal sites of the plasmid pPY75 generates the plasmid pPY91 ( Figure 21).
• Hpa2-Smal deletion (32980-35356)
• the Sall-Hpa2 fragment of the plasmid pPY82 which includes positions 32490 to 32980 is cloned between the SalI and ClaI sites of the plasmid pIC20H which generates the plasmid pPY93.
• This plasmid is the source of a Sali -EcoRV fragment including positions 32490 to 32980, the cloning of which between the Sali and. Smal of the plasmid pPY75 generates the plasmid pPY94 ( Figure 21).
• the Sau3A fragment of the plasmid pPY82 which includes positions 32490 to 32891 is cloned into the BamHI site of the plasmid pIC20H which generates the plasmid pICSau in which the SalI site of the multisite is positioned in the immediate vicinity of position 32490.
• the plasmid pICSau is the source of a SalI -Smal fragment including positions 32490 to 32891, the cloning of which between the corresponding sites of the plasmid pPY75 generates the plasmid pPY92 (FIG. 21).
• Example 7 Protocol for transfection of the 293 cells by the recombinant genomes constructed in E. Coli according to Examples 4 and 5.
• the construction of the plasmids described according to examples 4 and 5, or of analogous plasmids which, for example, are characterized by different E4 deletions, or by modified functional E4 regions, or for example corresponding to the introduction of an expression cassette of a given therapeutic gene ect ..., are used to generate the recombinant viral genomes by homologous recombination in E. coli polA After verification by restriction, a bacterial clone is then isolated and its plasmid content is extracted and purified.
• the DNA is then subjected to a total digestion with the enzyme Pacl and transfected into the 293 cells.
• This DNA is infectious (virus E1-E4 +) and a cytopathic effect (CPE) appears after approximately 2 weeks.
• the CPE is then gradually amplified in cells 293 and a viral stock is prepared (cf. Patent Application FR 016323).
• the plasmid pIC-pIX-pA was constructed by cloning of the EcoRV-Eagl fragment of pCR2-pIX and of the Eagl-BamHI fragment of the plasmid pCI (Promega) containing the SV40 polyA in the plasmid pIC20R digested with EcoRV and BamHI.
• the plasmid pCR2-IVa2 was constructed by cloning into the plasmid PCR2 (Invitrogen) of the product of amplification by PCR of the plasmid pCOl with the oligonucleotides 5'-AAGCTTATTGCCATCATTATGGAC-3 '(SEQ ID No. 6) and 5'- ACTAGTTATTTAGGGGTTTTGCGC-3' (SEQ ID No. 7).
• the plasmid pIC-IVa2-pA was constructed by cloning of the Hind3-Spel fragment of pCR2-IVa2 and of the Xbal -Sphl fragment of the plasmid pCDNA3 (Invitrogen) containing the signal for polyadenylation of the bovine growth hormone gene, in the plasmid pIC20R digested with Hind3 and Sphl.
• the BstXl-Sall fragment of the plasmid pIC-IVa2-pA was then cloned between the BstXl-Sall sites of the plasmid pCOl, thus creating the plasmid pCOl-_pIX.
• the plasmid pCOl-pIX was constructed by introducing the ClaI cassette of pIC-pIX-pA into the ClaI site of pCOl-_pIX, so that the gene coding for pIX is oriented in the same direction as that coding for IVa2, unlike its orientation in the adenoviral genome
• the plasmid pCOl-pIX therefore contains the ITR- ⁇ sequences of Ad5, a cassette for expression of the virus pIX (promoter and pIX gene followed by the SV40 polyA) oriented in the opposite direction from its natural orientation to the within Ad5, followed by sequences of IVa2 whose polyA has been replaced by that of bovine growth hormone
• the recombinant virus was constructed "in a conventional manner" by cotransfection in cells 293 of the plasmid pCOl-pIX linearized with Xhol and by the viral DNA of the AdRS VBGal virus digested by Cla 1
• NAME RHONE POULENC RORER S.A.

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