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  • 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 cell lines which can be used for the production of defective recombinant adenoviruses. It also relates to the purified viral preparations produced in these lines, as well as the plasmids allowing their construction. More particularly, the new cell lines according to the invention allow the transcomplementation of the E4 region and a clonal production with high titers of defective recombinant adenoviruses in particular for all or part of the E4 region.
• Adenoviruses have certain properties which are particularly advantageous for use as a vector for gene transfer 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 cell. infected, and have not been associated to date with significant pathologies in humans 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 (Akh et al., Nature genetics 3 (1993) 224) was
• Adenoviruses are linear double-stranded DNA viruses approximately 36 kb in size. Their genome includes in particular a repeated inverted sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes (see FIG. 1).
• the main early genes are contained in the regions E 1, E2, E3 and E4. Among these, the genes contained in the region E 1 in particular are necessary for viral propagation.
• the main late genes are contained in regions L 1 to L5
• the genome of the adenovirus Ad5 has been completely sequenced and is accessible on database (see in particular Genebank M73260) Likewise parts, or even all other adenoviral genomes (Ad2, Ad7, Ad 12, summer) have also been sequenced.
• adenovirus constructs described in the prior art are delete adenoviruses of the E1 region, essential for viral replication, at the level of which heterologous DNA sequences are inserted (Levrero et al ., Gene 101 (1991) 195, Gosh-Choudhury et al., Gene 50 (1986) 161).
• adenoviruses are produced in a complementation line (line 293) in which part of the adenovirus genome has been integrated More specifically line 293 contains 1 left end (approx iron I 1 - 12%) of the genome of the adenovirus serotvpe 5 (Ad5), comprising the left ITR the encapsidation region the E1 region including E1 a, E1b the region coding for the pIX protein and part of the region coding for the pIVd2 protein.
• This line is capable of transcomplementing recombinant adenoviruses defective for the E 1 region. that is to say devoid of all or part of the E1 region, and of producing viral stocks having high titers.
• vectors deficient for the E1 region have certain disadvantages for therapeutic use In particular, they may not be totally defective for replication in vivo, in particular due to the existence of certain transcomplementing cellular functions.
• a transcomplementation activity of E1 has been demonstrated in the cells of embryonic carcinoma F9. (Imperial et al., Mol Cell Biol 4, 1984 867-874).
• a similar activity, regulated by interleukin-6 has also been demonstrated (Spergel et al., J Virol. 66, 1992, 1021 - 1030)
• Other disadvantages linked to these vectors are the presence of numerous genes viral, capable of being expressed in vivo after gene transfer, and of inducing an immune and / or inflammatory response.
• E4 region is indeed 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 replication of viral DNA Adenoviral vectors in which the E 1 and E4 regions are deleted therefore have very reduced transcription background and expression of viral genes (see in particular application PCT / FR94 / 00851)
• the construction and the industrial and therapeutic exploitation of such vectors involves the provision of an efficient system for transcomplementing these two functions for the production of viral stocks.
• the present invention provides a solution to this problem.
• the present invention indeed provides cell lines allowing the transcomplementation of the E4 region and a clonal production and with high titers of recombinant adenoviruses defective for this region.
• the lines according to the invention are advantageously capable of transcomplementing the two functions E1 and E4, and therefore make it possible to produce viruses deficient for these two functions.
• the present invention also provides plasmids allowing the construction of these lines; a process for the preparation of defective recombinant adenoviruses and purified viral stocks More particularly, the Applicant has now shown that production lines capable of efficiently transcomplementing the E4 region are obtained by introducing only part of the E4 region. Thus, lines having particularly advantageous properties are obtained when only a reduced functional unit of the E4 region, corresponding to the reading phase ORF6 or to the reading phases ORF6 and ORF6 / 7, are present.
• a first object of the invention therefore resides in a cell which can be used for the production of defective recombinant adenoviruses comprising, inserted into its genome, part of the E4 region of an adenovirus genome comprising the reading phase ORF6 under the control of 'a functional promoter.
• the cells of the invention comprise a part of the E4 region of an adenovirus genome comprising the reading phases ORF6 and ORF6 / 7 under the control of a functional promoter.
• the cell lines according to the present invention have particularly advantageous properties. They allow first of all the transcomplementation of the functions E1 and E4. However, in a particularly advantageous manner, they are capable of inducing the formation of plaques of viruses deficient in these functions, which is essential for the cloning of the recombinant viruses, then for their amplification and their purification.
• the Applicant has indeed shown that lines possessing the entire E4 region or larger functional units, including for example the ORF4 reading phase, are incapable of forming ranges of viruses deficient for the E4 region .
• the identification of specific functional units of the E4 region allows the creation of a very efficient system of transcomplementation and production of defective viruses for the E1 and E4 functions.
• Other advantages of the lines according to the invention are in particular their aptitude for the amplification in a liquid medium of such deficient viruses for regions E 1 and E4 the high titers of such viruses which they produce, and the absence of production of contaminating replicative viral particles.
• the E4 region of the adenoviral genome consists of 7 open reading phases, designated ORF 1, ORF2, ORF3, ORF4 ORF3 / 4 ORF6 and ORF6 / 7 ( Figures 2 and 3)
• ORF 1 open reading phases
• ORF2 ORF3
• ORF6 ORF6 / 7
• Figures 2 and 3 the cells of the invention are characterized in particular by the presence of only part of this region, comprising the ORF6 reading phase possibly associated with the ORF6 / 7 reading phase. It is particularly important that the part of the E4 region present in the cells of the invention does not contain the functional ORF4 reading phase.
• the E4 region present in the cells of the invention does not contain the ORF 1 reading phases.
• the E4 region present in the cells of the invention is deleted from at least part of the reading phases ORF 1 -ORF4
• the cell lines of the invention comprise an inserted fragment containing less than 2 kb of the E4 region of an adenovirus genome containing all of the phases.
• the ORF6 reading phase can be isolated from the E4 region in the form of a fragmen t BglII-PvuII, corresponding to nucleotides 341 15-33126, and the reading phases ORF6-ORF6 / 7 can be isolated from the E4 region in the form of a BglII-BglII fragment corresponding to nucleotides 341 15-32490 of the genome of l 'Ad5
• the reading phase ORF6 is merged in the translational phase with the domain of a nuclear receptor which is responsible for the recognition of its specific ligand
• the domain for binding to the hormone (HBD Hormone Binding Domain) of the glucocorticoid receptor (GCR HoUenberg et al 1985, Nature, 318, 635-341) is advantageously chosen because it makes it possible to retain, in the cytoplasmic compartment of the cell, the fusion protein
• Another particularly preferred embodiment of the invention consists of a cell comprising, inserted into its genome, a BglII-PvuII fragment corresponding to the nucleotides 341 15-33126 of the Ad5 genome
• the ORF6 region at least, is coupled to the HBR of the GCR, either at its C-terminal end (fusion GCR-ORF6), or at its end N-terminal (ORF6-GCR fusion).
• the sequence of the virus coding for ORF6 and ORF7 is advantageously inserted in the translational phase downstream of the sequence specifying the HBD of the GCR.
• the expression of the chimeric gene then generates a primary RNA whose translation product is the protein GCR-ORF6 (SEQ ID No. 7).
• the alternative splicing of this transcript generates a messenger RNA which codes for the fusion protein GCR-ORF6 / 7 (cf. Example 1.5).
• the part of the E4 region present in the cells according to the invention can come from adenoviruses of different origins or serotypes. There are in fact different adenovirus serotypes, the structure and properties of which vary somewhat, but which have a comparable genetic organization. More particularly, the E4 region present in the cells according to the invention can be derived from an adenovirus of human or animal origin.
• adenoviruses of human origin there may be preferably mentioned those classified in group C. More preferably still, among the various serotypes of human adenovirus, it is preferred to use, within the framework of the present invention, adenoviruses of type 2 or 5 ( Ad 2 or Ad 5).
• Ad 2 or Ad 5 adenoviruses of type 2 or 5
• adenoviruses of animal origin it is preferred to use, within the framework of the invention, adenoviruses of canine origin, and in particular all the strains of adenoviruses CAV2 [Manhattan strain or A26 / 61 (ATCC NR-800) for example] .
• Other adenoviruses of animal origin are cited in particular in application WO94 / 26914 incorporated herein by reference.
• the part of the E4 region comes from a genome of human adenovirus of group C. More preferably, it comes from the genome of an adenovirus Ad2 or Ad5.
• the part of the E4 region present in the cells of the invention is placed under the control of a functional promoter in said cells.
• a functional promoter in said cells.
• it is an inducible promoter, making it possible to control the levels and / or the periods of expression of these genes.
• it is the promoter of the LTR of MMTV (Pharmacia), which is induced by dexamethasone or of a promoter regulated by tetracycline (WO94 / 29442, WO94 / 04672) It is understood that others promoters can be used, and in particular variants of the LTR of MMTY carrying, for example, heterologous regulatory regions (“enhancer” regions in particular).
• the expression of the hybrid genes is under the control of regulated promoters in order to avoid a constitutive accumulation of the fusion protein in the cytoplasm, or to minimize "leakage" towards the nuclear compartment and a certain cytotoxicity
• the chimeric gene is under the control of an inducible promoter which responds to glucocorticoids such as the GRE5 promoter (S. Mader and J White, 1993, PNAS, 90, 5603-5607. cf Example 1 .5).
• the cells according to the invention can be prepared from different pharmaceutically usable cells, that is to say cultivable under industrially acceptable conditions and not having a recognized pathogenic character II can be established cell lines or primary cultures and in particular of human embryonic retina cells. These are advantageously cells of human origin, which can be infected with an adenovirus. In this regard, mention may be made of KB, Hela, 293, Vero, gmDBP6, etc. cells.
• the cells of the KB line are derived from a human epidermal carcinoma. They are accessible to ATCC (ref. CCL17) as well as the conditions allowing their culture.
• the human cell line Hela comes from a carcinoma of the human epithelium. It is also accessible to ATCC (ref. CCL2) as well as the conditions allowing its cultivation.
• the cells of line 293 are human embryonic kidney cells (Graham et al, J Gen Virol 36 (1977) 59). This line contains in particular, integrated into its genome, the left part of the genome of the human adenovirus Ad5 (12%).
• the gm DBP6 cell line (Brough et al., Virology 190 (1992) 624) consists of Hela cells carrying the E2 adenovirus gene under the control of the LTR of MMTV. It can also be cells of canine origin (BHK, MDCK, ete) In this regard, the cells of the canine line MDCK are preferred.
• the culture conditions of MDCK cells have been described in particular by Macatney et al., Science 44 (1988) 9.
• the cell lines according to the invention can be constructed in different ways In general, they are prepared by transformation of a cell culture with a plasmid carrying the selected fragment of the E4 region under the control of a functional promoter
• the transfection of the cells can be carried out by any technique known to those skilled in the art, and in particular in the presence of calcium phosphate, by electroporation, ete
• the plasmid used also carries a marker gene making it possible to identify and select the cells transformed They may especially be any gene for resistance to an antibiotic (geneticin, hygromycin, etc.).
• the marker gene can also be carried by a separate construct, co-transfected with the plasmid After transfection and selection for the marker gene, the cells obtained can be selected for their capacity to transcomplement adenoviruses lacking the E4 region.
• different adenoviruses mutants defective for different parts of the E4 region can be used, such as in particular the adenoviruses Ad2dl808 (Weinberg and Ketner, J Virol 57 (1986) 833), Ad5dll 004, dl 1007 or dl 1014 (Bridge and Ketner, J Virol 63 ( 1989) 631), d1101 1 (Bridge et al., Virology 193 (1993) 794), as shown in the examples.
• the cells according to the invention are also capable of transcomplementing for the E1 region.
• These can be constructed as described above from cells which already transcomplement the E 1 region (example cells 293), or by sequential introduction of a construct providing the E1 region and of a construct providing the part of the E4 region according to the invention, for example in retinoblasts of human origin.
• the cells according to the invention derive from the cell line 293
• particularly advantageous results have been obtained with cells of the line 293 transformed by the plasmid pORF6Gen or the plasmid pGG0 which codes for the proteins HBD-ORF6 and HBD-ORF6 / 7.
• the present invention also describes the construction of plasmids comprising a part of the E4 region of an adenovirus genome carrying the reading phase ORF6 or ORF6 and ORF6 / 7 under the control of an inducible promoter (see in particular the plasmids pORF6Gen and pGG0 ). These plasmids can be used directly to transfect a selected cell population, then, by selection, for the identification of cells which have stably acquired the E4 function. Another object of the invention resides in the use of the cells described above for the production of defective recombinant adenoviruses at least for the E4 region.
• the invention indeed provides a method for producing defective recombinant adenoviruses at least for the particularly advantageous E4 region, using the above cells.
• a culture of cells as described above is transformed with one or more plasmids providing the different regions of the genome of said defective recombinant adenovirus and then harvesting the viruses produced
• This process is particularly advantageous for the production of adenoviruses having non-functional E1 and E4 regions.
• These are in particular vectors in which the E1 and E4 regions have been inactivated or made non-functional by total or partial deletion.
• Such modifications can be obtained in vitro (from isolated DNA) or in situ, for example, using in genetic engineering techniques, or even by treatment with mutagenic agents
• the said genetic modification (s) can be localized in a coding part of the region, or outside a coding region and for example in the regions responsible for the expression and / or transcriptional regulation of said genes
• the deletion can be carried out by digestion using appropriate restriction enzymes, then ligation, according to conventional techniques of molecular biology.
• the method of the invention is used for the production of recombinant adenoviruses in which the E1 region is inactivated by deletion of a PvuII-BglII fragment going from nucleotide 454 to nucleotide 3328, on the sequence of l adenovirus Ad 5
• the E1 region is inactivated by deletion of a HinfII-Sau3A fragment ranging from nucleotide 382 at nucleotide 3446
• the process allows the production of vectors comprising a deletion of the entire E4 region.
• the cell lines according to the invention are indeed capable of transcomplementing and amplifying adenoviruses carrying any type of deletion or inactivation of the reg ion E4
• a functional part of E4 is deleted.
• This part comprises at least the ORF3 and ORF6 phases.
• these coding phases can be deleted from the genome in the form of Pvull-AluI and BglII- fragments.
• the cells of the invention are particularly advantageous for the production of virus comprising an inactive E1 region and a deletion in the E4 region of the type present in the genome of Ad5 d11014. that is to say of virus E4- retaining the reading phase ORF4.
• the present invention therefore also describes defective recombinant adenoviruses the genome of which comprises a deletion in the E1 region and a deletion in the E4 region. More particularly, it describes defective recombinant adenoviruses the genome of which comprises a deletion in the E1 region and a deletion in the E4 region corresponding at least to the ORF3 and ORF6 reading phases.
• the adenoviruses according to the invention preferably comprise the following deletions affecting all or part of the E1 and E4 regions.
• This double strand sequence represents the right part of the adenoviral genome, including the E4 region and the right ITR of nucleotide 32749 (1 in SEQ ID No. 4) at 35935 (3186 in SEQ ID n ° 4). It is a purely illustrative sequence and other published sequences can also be used.
• the different reading phases of the E4 region are represented, in particular ORF7, ORF6, ORF4, ORF3, ORF2 and ORF1
• An adenovirus ⁇ E1, ⁇ E4, ORF1 + of the invention advantageously comprises a deletion in the E1 region and a deletion of a fragment whose 5 'end is included in the ORF7 reading phase and whose 3' end is located in the ORF2 Encore reading phase more preferably, the deletion relates to a fragment whose 5 'end is between nucleotides 32920 to 33190 of the adenovirus genome and whose 3' end is between nucleotides 34710 to 35090 of the adenoviral genome This deletion corresponds approximately at nucleotides 1 70 to 440 (5 'end) and 1960 to 2340 (3' end) on the sequence SEQ ID No. 4.
• ⁇ E1, ⁇ E4, ORF4 + adenovirus of the invention advantageously comprises a deletion in the E1 region, a deletion of a fragment whose end 5 is included in the phase of this ORF7 and whose end 3 'is located in the reading phase ORF6 (with the exception of the part overlapping the reading phase ORF4), and a deletion of 'a fragment whose I 5' end is included in the reading phase ORF3 and whose 3 'end is located in the reading phase ORF1 or in the promoter region of E4 More preferably, these adenoviruses according to the invention comprise:
• the corresponding positions of the deletion (n) on the sequence SEQ ID n ° 4 are 180 to 445 (5 'end) and 450 to 1250 (end 3')
• the corresponding positions of the deletion (iii) on the sequence SEQ ID n ° 4 are 1610 to 1950 (5 'end) and 2410 to 2870 (3' end).
• deletions in the E4 region preferentially cover nucleotides 33093 (SmaI) -33695 and 34634 (SspI) -35355 (SmaI) They can be obtained for example from the Ad5 mutant d11014.
• - Adenovirus ⁇ E 1, ⁇ E4 deletion of all or part of the E 1 region and of nucleotides 32720-35835, or 33466-35355 this deletion can be obtained for example from the mutant Ad5 d11007) or 33093-35355 (this deletion be obtained for example from the mutant Ad5 d1101 1).
• These 3 deletions cover the entire E4 region.
• the deletion in the E1 region advantageously covers all or part of the E1 A and E1B regions. This deletion must be sufficient to render the virus incapable of autonomous replication in a cell.
• the part of the E region is deleted in the adenoviruses.
• nucleotides 454-3328 or 382-3446 The positions given above refer to the sequence of the wild-type Ad5 adenovirus as published and accessible on the database. Although minor variations may exist between the different adenovirus serotypes, these positions are generally applicable to the construction of recombinant adenoviruses according to the invention from any serotype, and in particular from adenoviruses Ad2 and Ad7.
• the adenoviruses of 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 the expression of viral genes.
• all or part of the E3 or IVa2 region in particular can be deleted.
• the E3 region it may however be particularly advantageous to keep the part coding for the gp 19K protein. This protein in fact makes it possible to prevent the adenov irus vector from undergoing an immune reaction which (i) would limit its action and (ii) could have undesirable side effects
• the E3 region is deleted and the sequence coding for the protein gp19k is reintroduced under the control of a heterologous promoter.
• the recombinant adenoviruses according to the invention have properties which are particularly attractive for use in gene therapy. These vectors indeed combine infection, safety (the risks of immune and / or inflammatory reaction are greatly reduced) and transfer capacity. very high genes
• the lines of the invention allow the production of viral stocks totally devoid of contaminating replicative particles (RCA).
• RCA contaminating replicative particles
• viruses according to the invention lies in their increased cloning capacity, allowing the insertion of large transgenes (greater than 10 kb). This allows in particular the use of transcription regulatory sequences making it possible to improve the efficiency, the regulation and the duration of expression. This also makes it possible to use lower doses of virus and to obtain a comparable therapeutic effect with very reduced cytopathic side effects.
• adenoviruses constitute very efficient gene transfer vectors for gene and cell therapy applications. For this, a heterologous nucleic acid sequence whose transfer and / or expression in a cell, an organ or a organism is research can be inserted into their genome This sequence can include!
• one or more therapeutic genes such as a gene whose transcription and possibly translation into the target cell generate products before a therapeutic effect
• therapeutic products mention may be made more particularly of enzymes, blood derivatives, hormones, interleukin lymphokines, interferons, TNF, ete (FR 9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors BDNF.
• CNTF NGF, IGF. GMF, aFGF, bFGF, NT3 NT 5, 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 blocks; thus 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 in humans an immune response, with a view to producing vaccines II 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. II may also be regions of different origin (responsible for the expression of other proteins, or even synthetic).
• they may be promoter sequences of eukaryotic or viral genes or any promoter sequence or derivative, stimulating or expressing the transcription of a gene in a specific way or not and in an inducible way or not
• they may be promoter sequences originating from the genome of the cell which it is desired to infect, or of the genome of a virus, and in particular, the promoters of the E1 A, MLP genes of adenovirus, the CMV promoter, LTR-RSV, ete
• ubiquitous promoters HPRT vimentin ⁇ -actin tubulin ete
• promoters of intermediate filaments desmin neurofilaments, keratin, GFAP, ete
• promoters of therapeutic genes type MDR, CFTR, factor VIII, ete
• tissue-specific promoters pyruvate kinase, villin.
• intestinal protein promoter of fatty acids promoter of actin ⁇ of smooth muscle cells, specific promoters for the liver
• Apo AI Apo AII, human albumin ete
• promoters responding to a stimulus hormone receptor s teroids, retinoic acid receptor, ete,
• these expression sequences can be modified by addition of activation sequences, regulation, or allowing tissue-specific or majority expiession
• the acid inserted nucleic acid does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence.
• heterologous nucleic acid sequence 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 product. therapeutic, but it can 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 techniques described in the prior art. It can first of all be inserted at the E1 deletion. It can also be inserted at the E3 region, in addition to or in substitution for sequences. It can also be located at the deleted E4 region.
• the cells according to the invention can also be used for the production of recombinant adeno-associated viruses (AAV).
• AAV adeno-associated viruses
• the present invention also relates to the use of a cell comprising, inserted into its genome, all or part of the E4 region of the genome of an adenovirus comprising at least the ORF6 reading phase for the production of recombinant AAVs
• the cells are advantageously as defined above
• AAV is a DNA virus from the family of human parvoviruses, of relatively small size, which integrates into the genome of the cells which it infects, in a stable and site-specific manner.
• AAVs are capable of infecting a large spectrum of cells, without inducing an effect on cell growth, morphology or differentiation Moreover, they do not seem to be involved in pathologies in humans
• the genome of AAVs has been cloned, sequenced and characterized It comprises approximately 4700 bases , and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as the origin of replication for the virus
• ITR 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 rep gene involved in viral replication and expression of viral genes, the right part of the genome, which contains the cap gene encoding the capsid proteins of the virus
• ITR inverted repeat region
• AAV adenovirus or l virus.
• a helper virus for example an adenovirus
• a plasmid containing a nucleic sequence of interest bordered of two inverted repeat regions (ITR) of AAV and a plasmid carrying the packaging genes (rep and cap genes) of AAV.
• the major drawback of this system is that it uses a helpei virus. It is generally replicative and present in mixture with the AAVr products. The viral stocks are therefore potentially contaminated by a helper virus, which makes these stocks incompatible with therapeutic use. In addition, due to the high number of components involved in this process, the virus titers obtained are quite low, of the order of 10 8 .
• the present invention makes it possible to remedy these drawbacks.
• the present invention in fact provides an effective method for producing AAVr, making it possible to obtain stocks of virus with very high titers (greater than 10), not contaminated by a replicative virus.
• AAV E1 A, E1B, E2A, VA and E4 Five genes of the adenovirus are necessary for the replication of the AAV E1 A, E1B, E2A, VA and E4 These genes must be present and expressed in the producer cell for optimal production of infectious particles
• the method of l he invention now uses a production cell line already containing in its genome some of these genes, and in particular all or part of the E4 region, preferably combined with the E1 region.
• the advantage of using this type of cell line is that it makes it possible to use a defective adenovirus helper, that is to say not capable of autonomously generating infectious particles.
• the inventories of AAVi produced are not contaminated
• the functions integrated into the line can be deleted from the genome of the adenovirus helper. This is particularly advantageous for the use of an adenov irus helper of human origin.
• helper of human origin (ad5 or ad2 for example) defective for these functions
• Such an helper adenovirus could not be used effectively in the procedures of the prior art because the absence of regions essential for the production of AAVr (E 1 and E4) considerably limited the effectiveness of the process.
• such an adenovirus is totally incapable of autonomously generating infectious particles. Therefore, its possible presence in a stock of AAVr does not affect the pharmaceutical quality of this preparation.
• adenovirus helper of animal origin preferably canine
• the method according to the invention makes it possible to obtain particularly high virus titers.
• the titles produced which can exceed 10 viral genomes per ml, are read up to 1000 times greater than the titles observed in the prior art.
• These results are completely unexpected and of capital importance in terms of industrial exploitation.
• the results are particularly remarkable with the cell lines derived from the line 293 and which express ORF6, possibly associated with ORF6 / 7, or all of E4, under the control of the promoter MMTV (they therefore contain E1 constitutively expressed and E4 or a part of E4 comprising at least ORF6 conditionally expressed in the presence of dexamethasone) .
• a cell line which is particularly advantageous for implementing the method according to the invention is represented by a cell of the line 293 comprising. inserted into its genome, a BglII-BglII fragment corresponding to nucleotides 341 15- 32490 of the Ad5 genome. It is, for example, a cell of line 293 transformed by the plasmid pORF6Gen (see examples).
• adenovirus helper for example human with wild phenotype, or deleted for E1, or a double deletion for E1 and E4, or canine adenovirus
• a plasmid- AAV carrying the AAV ITRs framing a nucleic acid of interest
• a plasmid carrying the rep and cap functions it is possible to produce, in the presence of dexamethasone, particles of infectious AAV virus at high titers.
• genome titers of 10 11 genomes / ml can be obtained when operating on small quantities of cells.
• Another object of the invention resides in a process for producing recombinant AAVs characterized in that a part of the E4 region of the genome of a genome is introduced into a culture of cells comprising, inserted into their genome adenovirus comprising at least the ORF6 reading phase:
• AAV plasmid carrying a nucleic acid of interest bordered with AAV dTTRs, - an adenovirus helper.
• the producer cell is a cell comprising the entire E4 region
• the producer cell is a cell comprising part of the E4 region comprising at least the reading phase ORF6 and possibly the reading phase ORF6 / 7.
• It is particularly preferably a cell as defined above for the production of adenovirus.
• it is advantageously a cell capable of transcomplementing the E1 and E4 functions of the adenovirus.
• a preferred example is represented by a cell of line 293 containing inserted into its genome, the whole E4 region or a part of the E4 region comprising at least the reading phase ORF6 and optionally the reading phase ORF6 / 7.
• the adenovirus helper can be a human adenovirus with a wild phenotype, or defective for the E1 region, or a double deletion agent for E1 and E4, or also canine adenovirus
• the advantage of the method according to the invention resides on the one hand in the titles very high in AAVr, and also in the safety character of the stocks which it allows to produce
• a defective adenovirus helper that is to say incapable of generating autonomously infectious particles
• the adenovirus helper according to the method of the invention is advantageously a human adenovirus having a deletion in the E4 region More preferably still, it is a human adenovirus defective for the E1 and E4 regions
• it is a canine adenovirus, preferably chosen from the CAV2 strains
• the rep and cap functions of the AAV are preferably provided by co-transfection of the cells with a plasmid carrying the rep and cap regions of the AAV These regions can be under the control of the homologous P5 promoter or a constitutive promoter such as LTR-RSV These functions can also be provided directly by the helper virus used II is indeed possible to insert into the adenovirus helper a cassette containing the rep and cap regions of the AAV
• the transfection of the plasmid (s) (plasmid-AA V and plasmid-RepCap if necessary) can be carried out by any technique known to those skilled in the art.
• the Applicant has now developed a particularly effective method for transfection of plasmids into production cells
• This method is based on the use of a polycationic lipid and an acid-compacting agent
• One of the advantages of this method lies in the fact that it does not seem to alter the morphology or physiological state of the cells.
• Different types of cationic lipids can be used, such as hpofectamine, Transfectam, ete
• DNA compacting agents peptides derived from nuclear proteins such as histones, nucleolin, and the like can advantageously be cited.
• the different plasmids and helper viruses can be introduced into the productive cell concomitantly or separately. In the case of a separate introduction, the order in which the different components are introduced does not seem to be essential for obtaining high titers. As illustrated in the examples, important titles have been obtained when, in the first stage, the plasmids are co-transfected in the cells then, in the second stage, the cells are infected with the helper virus
• a specific embodiment of the invention resides in a process for producing recombinant AAV characterized in that, in a culture of cells transcomplementing the E1 and E4 functions of the adenov irus, one cotransfects, in the presence of a lipid canonical polv and of an agent compacting an AAV plasmid carrying a nucleic acid of interest bordered by AAV ITRs and a plasmid carrying the rep and cap regions of AAV, said culture is co-infected with a selected helper adenovirus among human adenoviruses of Ad2 or Ad5 origin defective for regions E1 and E4 and canine adenoviruses of CAV2 origin, then the viruses produced are harvested
• the present invention also relates to the purified viral preparations (adenovirus and AAV) obtained according to the process of the invention, as well as any pharmaceutical composition comprising one or more defective recombinant adenovirus or AAV prepared according to this process
• the pharmaceutical compositions of the invention can be formulated for administration by parenteral oral topical route intranasal intravenous intramuscular, subcutaneous, transdermal intraocular, ete
• the pharmaceutical composition contains pharmaceutically acceptable vehicles for an injectable formulation. It can be in particular saline solutions (monosodium phosphate, disodium, sodium chloride potassium, calcium or magnesium, ete, or mixtures of such salts), sterile.
• a hydrogel can be prepared from any biocompatible and non-cytotoxic (homo or hetero) polymer
• biocompatible and non-cytotoxic (homo or hetero) polymer Such polymers have for example been described in application WO93 / 08845 Some of them, such as in particular those obtained from ethylene oxide and / or propylene are commercial
• the doses of virus used for injection can be adapted according to different ts parameters, and in particular depending on 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 under in the form of doses between 10 4 and 10 14 pfu, and preferably 10 6 to 10 10 pfu and the AAV, between 10 6 and 10 11 particles.
• pfu plaque forming unit
• plaque forming unit corresponds to the infectious power of an adenovirus 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.
• viruses thus produced can be used for the treatment or prevention of many pathologies, including genetic diseases
• ALS ete
• cancers pathologies linked to coagulation disorders or dyslipoproteinemias
• pathologies linked to viral infections hepatitis, AIDS, summer
• the present invention will be described more fully with the aid of the following examples, which should be considered as illustrative and not limiting.
• FIG. 1 Genetic organization of the Ad5 adenovirus The complete sequence of Ad5 is available on the database and allows those skilled in the art to select or create any restriction site, and thus to isolate any region of the genome .
• Figure 2 Genetic organization of the E4 region
• Figure 3 Genetic organization of the E4 region in wild and defective E4 adenoviruses. The size of the deletion is represented by a thick bar.
• FIG. 4 (A) Schematic representation of the MMTV LTR / (ORF6 +
• AAA Polyadenylation site.
• Figure 5 Analysis of the production of adenoviral fiber by immunological detection using a polyclonal serum against the fiber (Boulanger et al.).
• Cell extracts are prepared after 72 hours of viral infection, dexamethazone is added at the same time as the virus (final concentration 600 nM)
• A) Infection with the d1010 virus (MOI 10)
• (wt) Infection with the Ad5 virus (ME 10).
• 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, extracts with phenol or with a phenol / chloroform mixture, precipitated with ethanol then incubations in the presence of DNA ligase from phage T4 (Biolabs) according to the recommendations of the supplier
• the filling of the prominent 5 ′ ends can be carried out with the fragment of Klenow of E. DNA Polymerase I. 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 a gentle treatment with nuclease S1.
• Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al [Nucleic Acids Res 13 (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
• Sequence verification nucleotides can be performed by the method developed by Sanger et al. [Proc Natl Acad Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
• Example 1 Construction of plasmids carrying different functional units of the E4 region under the control of a promoter 1.1. Construction of the plasmid pE4Gen
• the plasmid pPY2 corresponds to the cloning of the Avr2-Sall fragment (approximately 13 kb including the MMTV promoter) of the plasmid pMSG (Pharmacia) between the XbaI and Sali sites of the plasmid pIC20H prepared from an E. coli dam + context.
• 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 plasmid pIC20H in which the TaqI -Bgl2 fragment including the E4 region of the adenovirus type 5 located between positions 35576 (Taq 1) and 32490 (Bgl2), has been cloned between the ClaI and BamHI sites
• the E4 region of the plasmid pPY5 is therefore included in an EcoRV-Sphl fragment which can be cloned after partial digestion between the sites Smal and Sphl of the plasmid pPY4, which generates the plasmid pPY6.
• the main donor 5 ′ site for splicing located upstream of the reading phase ORF1 (towards position 35548) is therefore preserved to ensure correct alternative splicing, making it possible to generate the various expression products of all the coding phases of the E4 region and in a comparable manner to the alternative splicing observed during the viral cycle
• the plasmid pPY13 corresponds to the cloning of the Bgl2-Xbal fragment of the plasmid pPY6 between the corresponding sites of the plasmid pIC20H.
• This 1.6 kb fragment therefore includes the adenovirus type 5 sequence from position 341 (Bgl2) to position 32490 (Bgl2, followed by the Xbal site from the cloning multisite of the plasmid pIC20H)
• the plasmid pPY13 therefore contains the all of the open reading phases ORF6 and ORF7 of the adenovirus, now included in an Xho l -Sph l fragment.
• the cloning of this fragment between the Sal l and Sph l sites of the plasmid pPY4 generates the plasmid pPY 15.
• the insertion of the Xhol fragment of the plasmid pKIXX, which carries a gene conferring resistance to geneticin in 293 cells, in the plasmid pPY 15 generates the plasmid pORF6Gen.
• This plasmid therefore carries a selectable gene and the open reading phases ORF6 and ORF7 of the E4 region of the adenovirus expressed from the promoter of MMTV. In this particular plasmid these two genes follow each other and the respective coding sequences are carried by the same strand of DNA.
• the first codon for initiating translation is that of the open phase ORF6 (position 34077 in the genome of Ad5). , and it is separated from the CAP site of the MMTV promoter by 235 nucleotides. Insofar as the alternative splicing is sequential and involves first of all the recognition of the 5 ′ donor site, the main 5 ′ donor site located upstream of the reading phase ORF1 (towards position 35548) has therefore not been included in the construction of the plasmid pORF6Gen to subsequently allow efficient expression of the products of the reading phases ORF6 and ORF6 / 7 (FIG. 4A).
• the plasmid pPY14 corresponds to the cloning of the Bgl2-Xba l fragment of 1.9 kb (obtained after partial digestion with the enzyme Bgl2), of the plasmid pPY6 between the corresponding sites of the plasmid pIC20H.
• This 19 kb fragment therefore includes the adenovirus type 5 sequence from position 34387 (Bgl2) to position 32490 (Bgl2, followed by the site
• the plasmid pPY14 is therefore isogenic with the plasmid pPY13 with the exception that it additionally includes a Bgl2 fragment corresponding to almost all of the open reading phase ORF4.
• This plasmid therefore contains all of the open reading phases ORF4, ORF6 and ORF7 of the adenovirus, now included in an Xho1-Sph1 fragment. The cloning of this fragment between the SalI and Sphl sites of the plasmid pPY4 generates the plasmid pPY16.
• the insertion of the Xhol fragment of the plasmid pKIXX, which carries a gene conferring resistance to geneticin in 293 cells, into the plasmid pPY16 generates the plasmid pORF4Gen.
• This plasmid therefore carries a selectable gene and the open reading phases ORF4, ORF6 and ORF7 of the E4 region of the adenovirus expressed from the promoter of MMTV.
• these two genes follow each other and the respective coding sequences are carried by the same strand of DNA.
• This example describes the construction of a plasmid comprising a functional unit of E4 (Cf example 1.2.) Under the control of a promoter derived from MMTV More particularly, this promoter is a derivative of MMTV comprising 5 elements of response to glucocorticoids, c ' ie a derivative highly inducible by glucocorticoids This plasmid was constructed in the following manner.
• the Ad5 BglII fragment (position 341 15 to 32490) includes the sequences (ORF6 + ORF7) from the E4 region. This fragment was first cloned between the BglII and BamHI sites of the plasmid pIC20H (Marsh et al., Gene 32 (1984) 481), which generates the plasmid pPY13 in which the Bglll site located upstream of ORF6 is conserved. The BglII-SalI fragment of the plasmid pPY13 therefore includes all of the sequences (ORF6 + ORF7) of the E4 region of Ad5. This fragment was cloned between the BamHI and SalI sites of the plasmid pIC20H, which generates the plasmid pPY45.
• the Xbal fragment (approximately 1 kb) of the plasmid pGRE5-1 corresponds to a promoter derived from MMTV, highly inducible by glucocorticoids.
• This fragment was isolated and cloned between the Xbal sites of the plasmid pIC20H isolated from a dam context.
• the plasmid obtained was designated pPY21.
• the BglII site originating from the cloning multisite of the plasmid pIC20H is located immediately upstream of the 5 elements of the promoter capable of binding the receptor nuclear to glucocorticoids.
• a derivative of pJY1 was also constructed containing a gene for resistance to geneticin.
• the XhoI-SalI fragment of the plasmid pMSCV contains a bacterial gene conferring resistance to eukaryotic cells to geneticin (APH), expressed from a strong and ubiquitous promoter (PGK) in cells This fragment was cloned in the plasmid pJY 1. at the soiled site The plasmid obtained was designated pJY2
• This plasmid contains the expression cassettes pGRE5 / (ORF6 + ORF7) and PGK / APH transcribed in the same direction. 1.5. Construction of the plasmid pGG0
• the assembly of the GCR (HBD) and ORF6 + ORF7 parts of the E4 region of Ad5 is carried out after PCR amplification using the plasmid pSG5HGR as matrix and the deoxyoligonucleotides SEQ ID N ° 5
• the oligonucleotide SEQ ID No. 5 therefore makes it possible to position an ATG specifying the initiation of translation upstream of the HBD domain of the GCR ranging from amino acids 539 to 777 of the GCR.
• the oligonucleotide SEQ ID No. 6 makes it possible to position a restriction site Tth1 11 1I downstream of the HBD.
• the PCR amplification fragment was first cloned into the commercial plasmid pCRII and the white-blue screening made it possible to select a clone whose identity was verified by sequencing (plasmid pCRII / GCR).
• This plasmid is therefore the source of a Smal-Xhol fragment which carries the HBD domain of the GCR which has undergone PCR amplification with the oligos SEQ ID No. 5 and SEQ ID No. 6.
• the plasmid pMEL3 is an intermediate construction which contains the "polylinker 'EcoRl-PstI of the plasmid pSL1 180 inserted between the EcoRl and PstI sites of a derivative of the plasmid pIC20H in which the Hind3 site locates in the immediate vicinity of the Nru1 site on the" polylinker "was treated with the Klenow fragment of E coli Polymerase I and then rehgated on itself, which transforms the Hind3 site into an Nhel site
• the plasmid pMEL3 also contains a BamHI fragment corresponding to the polyadenylation signal of SV40, previously inserted into the Bgl2 site of the "polylinker" of pIC20H
• the Smal -Xhol fragment of the plasmid pCRII / GCR which contains the HBD domain of the GCR was then inserted between the corresponding sites of the plasmid pMEL3, which generates
• the plasmid pPY13 ( ⁇ Hinc2-Sspl) is generated which contains a Hind3 fragment of approximately 14 kb containing the entire sequence ORF6 + ORF7 of the E4 region of Ad5, including its polyadenylation site
• the plasmid pPY13 ( ⁇ Hinc2-Ssp1) was digested with Hind3 and its ends were filled using Klenow Polymerase This plasmid is then digested with Xhol, and the ORF6 + ORF7 sequences (approximately 1.4 kb) are then cloned between the Xhol and Nrul sites of the plasmid pMEL3 / GCR, which generates the plasmid pMEL3 / GCR-Tth1 11 1- (ORF6 + ORF7).
• GRE5 the cloning of which between the Apal sites treated with the Klenow fragment and BamHI of the plasmid pMEL3 / GCR- (ORF6 + ORF7) generates the plasmid pMEL3 / GRE- (GCR- ORF6 + ORF7)
• This plasmid is the source of a Bgl2-Nhe 1 fragment corresponding to the expression cassette coding for the GCR- fusion (ORF6 + ORF7) expressed from the GRE5 promoter and the cloning of which between the Bgl2 and Xbal of the plasmid pCI-Neo (Promega) generates the plasmid pGG0.
• This example describes the construction of complementary cell lines for the E1 and E4 regions of the adenoviruses according to the invention. These lines allow the construction and propagation of recombinant adenoviruses deleted for these regions, without using a helper virus.
• the lines of the invention were constructed by co-transfection of the selected cells in the presence of calcium phosphate, by the plasmids described in Example 1 and a construct coding for the glucocorticoid receptor (Hollenberg et al., Nature, 3 18, (1985) 635-641).
• the cells of line 293 in dishes 5 cm in diameter were transfected with 1 to 5 ⁇ g of plasmid E4 (pE4Gen, pORF6Gen, pORF4Gen, pGG0 or pJY2) and optionally 5 ⁇ g of a receptor expression plasmid human with glucocorticoids expressed from the SV40 virus early promoter (plasmid pSG5HGR), in the presence of calcium phosphate, according to the protocol described by Graham and Van der Eb (Virology 52 (1973) 456).
• plasmid E4 pE4Gen, pORF6Gen, pORF4Gen, pGG0 or pJY2
• plasmid pSG5HGR a receptor expression plasmid human with glucocorticoids expressed from the SV40 virus early promoter
• Ad2d1808 (Weinberg and Ketner, J. Virol. 57 (1986) 833).
• Ad5d11004, d11007 or d11014 (Bridge and Ketner, J. Virol 63 (1989) 631), d1101 1 (Bridge et al., Virology 193 (1993) 794) are deletion mutants carrying significant deletions in the E4 region . These mutants are unable to replicate in 293 cells, but can be produced in W162 cells (Weinberg and Ketner, PNAS 80 (1983) 5383).
• the 50 clones resistant to geneticin were tested for their capacity to produce these E4 viruses and therefore to transcomplement the E4 region.
• 60 clones transfected with the plasmid pJY2, resistant to geneticin were also screened for this. ability to transcomplement the E4 region
• each clone is infected in a liquid medium with the d1808 virus at a multiplicity of infection of approximately 0.1 PFU / cell (the viral titer is obtained on the W162 line)
• the infection was carried out at an ego of 0.5 pfu / cell for the pJY2 clones, in a medium supplemented with dexamethazone (1 ⁇ M)
• dexamethazone (1 ⁇ M)
• the appearance of an amplifiable cytopathic effect by successive reinfection of the cells with the cell lysate obtained is indicative of a certain viral spread of dl808 by the cells of the clone considered
• This transcomplementation is then objectified both by analysis of the level of viral replication and the faculty of the cells to form viral plaques (a possible protocol is described by Hitt, M; Bett, AJ; Prevec, L.
• This step made it possible to highlight several particular clones exhibiting effective properties of transcomplementation of the E4 region.
• the first designates Clone # 2, results from the transfection of the plasmid pORF6Gen; the second, designated Clone # 4, was isolated after transfection of the plasmid pE4Gen.
• Stable clones capable of transcomplementing for the E4 region, and highly inducible by glucocorticoids were also obtained after transfection of the 293 cells with the plasmid pJY2.
• other stable clones capable of efficiently propagating viruses defective for the E4 region, and highly inducible by a non-steroid compound have also been prepared.
• clones were obtained by co-transfection of 293 cells with the plasmid pJY2 and by a plasmid expressing an artificial transactivator composed of a transactivating region (VP 16), of the region which binds DNA from the glucocorticoid receptor, and of a C-terminal truncated part of the progesterone receptor, such so this receiver hybrid is capable of transactivating the GRE's promoter in the presence of RU486 and not of steroids.
• the clones obtained are effectively capable of effectively propagating E4-defective viruses in the presence of RU486.
• the plasmid pGG0 alone, or an equimolecular combination of the plasmids pGG0 and pSG5HGR are transfected into permissive cells for adenovirus type 2 or 5, and the stable clones are selected in the presence of 400 mg / l of geneticin.
• transfection of the plasmid pGG0 in 293 cells generates the clone IGRP18 which allows the propagation of the doubly defective viruses for E1 and E4 in the presence of dexamethasone (1 ⁇ M), which is not the case in the absence of addition.
• 2.3. Ability to propagate adenoviruses deficient for E1.
• the capacity of the prepared lines to complement the E1 region was verified after infection with the adenovirus Ad-RSVBGal
• Ad-RSVBGal This first generation adenovirus (deficient in E1 only), comprises the LacZ gene of E.coli under the control of the virus LTR promoter RSV (Stratford-Perricaudet et al., J. Clin. Invest, 90 (1992) 626).
• the cells of clones # 2, # 4 and cells obtained with pJY2 were infected with the Ad-RSVßGal virus and viral spread was observed for each clone.
• the cells of clones # 2 and # 4 were analyzed in Southern blot to verify the expression of the viral proteins. More particularly, the Southern Blot analysis (analysis of genomic DNA hybridizing with a radioactive probe originating from the adenoviral E4 region) was carried out according to the protocol described by Maniatis et al. To this end, the genomic DNA of cells 293 and # 2 has been prepared. 2 ⁇ g of this DNA were used as a template in a PCR reaction carried out in the presence of Taq Polymerase, oligo 1 of sequence SEQ ID No. 1 (corresponding to nucleotides 52 to 71 of the promoter MMTV) and of the oligo 2 of sequence SEQ ID No. 2 (corresponding to positions 32921-32940 of the Ad5 genome).
• oligo amplify a 2617 bp fragment of the plasmid pORF6Gen
• the amplification was carried out under the following conditions: denaturation at 94 ° C. for 5 min, 30 amplification cycles by denaturation at 94 ° C for 1 min, hybridization at 60 ° C for 2 min, and extension at 70 ° C for 3 min, the extension during the last cycle being extended for 10 min
• the products amplification were then analyzed by SDS electrophoresis 1% agarose and identified by Southern Blot and hybridization with a labeled probe covering the region (ORF6 + ORF7) of the adenovirus or the MMTV region.
• oligo 2 SEQ ID No. 2
• oligo 3 SED ID No. 3
• oligo 3 SED ID No. 3
• the amplification products were analyzed in SDS 1% agarose gel and identified in Southern Blot and Hybridization with the labeled probe (ORF6 + ORF7).
• Southern blot analyzes more particularly indicate that clone # 2 contains a copy of the MMTV- (ORF6 + ORF7) cassette integrated into its genome.
• the integrity of this cassette has also been demonstrated by amplification using oligos 1 and 2 which generates an expected 2.6 Kb fragment, which can be specifically detected by a radiolabelled probe corresponding to the unit (ORF6 + ORF7) or to the MMTV promoter (FIG.
• ORF6 has also been demonstrated by immunodetection of the fiber protein
• the results obtained show that the 293 cells infected with the adenovirus Ad5 dl 1007 do not produce fibers
• a fiber-specific signal is detected in the cells of the invention (clone # 2 in particular) after infection with this mutant, and not in the other uninfected cells
• the presence of the fiber has also been demonstrated in the cells of the invention infected with the mutants d1808, d11004 (ORF1 + ), d1101 1 or d1 1014 (ORF4 + ) in the presence of dexamethazone.
• clone # 2 Another advantageous property of clone # 2 according to the invention resides in the regulated and inducible character of the expression of the E4 activity.
• the results obtained show that the formation of virus plaques is observed only in the presence of dexamethazone.
• the expression of the adenovirus fiber protein after infection with the mutant Ad5dl1007 is significantly increased under the induction conditions (FIG. 6).
• the same results were obtained with the mutants Ad5d1808, Ad5d11004, Ad5d1101 1 and Ad5d11014.
• the expression of the E4 activity in clone # 4 is constitutive.
• This example describes the use of the cell lines according to the invention for the production of deficient recombinant viruses in the E1 and E4 regions.
• These adenoviruses were produced by homologous recombination, after co-transfection, in the cells of the invention, of two DNA fragments, one bringing the left part of the genome of the recombinant virus (having a deletion in the E1 region), the other bringing the right part of the genome of the recombinant virus (having a deletion in the E4 region)
• the 293E4 cells (clone # 2 and # 4) were cotransfected with 10 mg of DNA from the Ad-RSVBGal virus (Stratford-Perricaudet et al.) Digested with Srf1 (or 5 mg of DNA from the plasmid having used for the construction of this virus and digested with Xmn 1), and 10 mg of the DNA of the virus providing the functional deletion of the E4 region (for example Ad2d1808, Ad5d11004, Ad5d11007 or Ad5d11014) digested with the enzyme Clal. After the cytopathic effect has appeared, the viruses are purified by at least two consecutive solid spreading cycles to form plaques on clone 2.
• Ad-RSVBGal virus Stratford-Perricaudet et al.
• Srf1 or 5 mg of DNA from the plasmid having used for the construction of this virus and digested with Xmn 1
• 10 mg of the DNA of the virus providing the functional deletion of the E4 region for example
• plaques corresponding to the infection of the virus sought are then amplified by consecutive infection cycles.
• High titer stocks are prepared by purification on a cesium chloride gradient.
• Viruses are stored at -80 ° C according to conventional techniques of those skilled in the art.
• it allows propagation in a liquid medium which is particularly effective for the AD5d11014 virus (E 1 + E4- but ORF4 + ) or for the E1-E4- virus constructed from the d11014 virus.
• clone 4 obtained after transfection of the plasmid pE4Gen is not very efficient in making plaques because it is difficult to maintain the cell confluence for a time long enough for the viral lysis plaques to be easily identifiable (and especially if the recombinant virus sought does not code for ⁇ -galactosidase)
• An alternative production method is based on the clonal construction of the viruses according to the invention. More particularly, according to this method, the adenoviruses ⁇ E1 ⁇ E4 are produced by homologous double-recombination in vivo after co-transfection of 3 overlapping DNA fragments, each providing part of the virus genome.
• Ad5 d11014 II overlaps fragment II on 1652 nucleotides ( Figure 7)
• This fragment provides the right part of the genome of the recombinant virus, containing the modified E4 region ( ⁇ E4, ORF4 + ). Its contamination is once again more than improbable since it was purified after complete digestion with Nsil generating 7 fragments of size between 178 nucleotides and 4 kb.
• the purified fragments were co-transfected into clone # 2 cells according to the protocol described for 293 cells, in the presence of 1 ⁇ M of dexamethazone in the medium (added every 4 days for 3 weeks).
• the cells were harvested, frozen and thawed 3 times in an ice / ethanol bath, then centrifuged at 3000 g for 20 min. The cell lysate was then added to a fresh culture of clone # 2 cells (also designated IGRP2) in the presence of dexamethazone (1 ⁇ M) After 5 days, a cytopathic effect was observed, demonstrating the production of viruses.
• a stock of this recombinant virus Ad ⁇ E1, ⁇ E4, ORF4 + was obtained after gradual amplification of the mixture inducing the cytopathic effect on 100 10 cm dishes containing clone # 2 cells under confluence, in the presence of 1 ⁇ M dexamethazone After purification on a cesium chloride gradient and dialysis at 4 ° C, the viral stock was titrated on monolayers of clone # 2 cells supplemented with dexamethazone (1 ⁇ M), before staining in vitro with X-Gal As all the plates are positive, the titer may be expressed either in pfu / ml or in viral plaque expressing ⁇ -gal.
• a stock of 10 10 pfu was prepared The absence of RCA in this stock was then checked by restriction analyzes and in southern
• the viral DNA of a recombinant of the stock was prepared according to the technique de Challberg (Virology 1 14 (1981) 196). 2 ⁇ g of this DNA were subjected to a restriction analysis and 1% agarose gel. The fragments were transferred to a Hybond-N membrane (Amersham) and hybridized with a radioactive probe corresponding to the ITRs of the adenovirus to specifically detect the fragments. located at the ends of the viral genome.
• StuI generates only 2 fragments hybridizing with the ITR probe
• One of these fragments has a mobility corresponding to that of the 1,125 bp fragment of AdRSV ⁇ gal encoding ⁇ gal, the other migrates like the StuI fragment of 2,673 bp of Ad5d11014 carrying the E4 deletion
• Prolonged exposure of the autoradiogram does not reveal any additional band having a size corresponding to the E1 + (3158 bp) or E4 + (3980 bp) fragment.
• This example describes the use of cell lines containing all or part of the E4 region of an adenoviral genome for the production of AAV. These AAVs were produced by co-transfection, in said cells, of an ITR-AAV plasmid and a Rep / Cap plasmid, and by co-infection with an adenovirus helper.
• This plasmid contains a nucleic acid of interest (E. coli ⁇ -gal expression cassette composed of the LTR promoter-
• RVS of the LacZ gene preceded by a nuclear localization signal, and of the polyadenylation site of the SV40 virus borders on 2 AAV ITRs.
• the plasmid pMA10 was obtained by digesting the plasmid pXL2582 by Spel and by closing by treatment with the DNA ligase of the bacteriophage T4. This treatment makes it possible to remove the palindromic sequences downstream of the left ITR of the AAV.
• the plasmid pXL2582 was obtained by ligating the following fragments into the EcoRI-KpnI sites of pXL2359 (described in application FR94 02445): a) EcoRI-Xbal (containing the left ITR of AAV up to nucleotide 155, site HinfI on the published AAV sequence) of pXL2580, and, b) XbaI-KpnI of pXL2359 (containing the expression cassette for the LacZ gene)
• the plasmid pXL2580 was obtained from pXL2359 as follows: pXL2359 a was digested with EcoRI-Xbal and deposited on 1% agarose gel, from which a 650 bp fragment was purified This fragment was redigested by Hinfl and treated with DNA Polymerase from bacteriophage T4, cut with PstI and then deposited on 2% agarose gel, from which a 200 bp fragment was purified This fragment,
• the plasmid used denotes p ⁇ Bal, has been described by Lebkowski et al (Mol. Cel. Biol. 8 (1988) 3988). This plasmid contains the rep and cap regions of the AAV under the control of the endogenous promoter P5. Other promoters can be substituted for the P5 promoter, such as in particular the promoter constituting the LTR-RVS.
• the adenovirus helper used is a wild Adenovirus Ad5. It is understood that other helper viruses can be used, and in particular an Ad5 defective for the E1 and / or E4 region, or a canine adenovirus.
• the advantage of using a canine adenovirus lies in their capacity to support the replication of AAVs while being defective viruses in humans. Therefore, the AAVr preparations obtained are totally devoid of RCA and of contaminating human adenovirus. .
• the production was carried out by transfection of 20 dishes of 5 cm in diameter of Clone # 2 cells (IGRP2), at a density of approximately 3.10 cells per dish, previously inoculated 24 to 48 hours in 10% FCS MEM medium.
• IGRP2 Clone # 2 cells
• In each dish were co-transfected 1 ⁇ g of plasmid pMA10 and 5 ⁇ g of plasmid p ⁇ Bal in the presence of 16 ⁇ g of peptide H1, 16.5 ⁇ l of lipofectamine (Gibco-BRL, Life Technologies) and OPTIMEM lipofectamine (Gibco- BRL, Life Technologies) according to the supplier's recommendations
• the transfection mixture was removed and the cells were infected for one hour with the adenovirus helper, with an infectivity of 10 virus pfu per cell in a final volume of 500 ⁇ l MEM10% FCS medium + 10 M dexamethasone was then added.
• the cells were harvested 5 days later, taken up in a 10 mM Tris HCl buffer, pH 8, lysed by 3 freezing and thawing, then treated with sodium deoxycholate and trypsin at the respective concentrations of 0.25% and 1% for 30 min at 37C.
• the recombinant AAVs produced were then purified on a gradient of cesium chloride at a density of 1.4, in a rotor SW55.1 at 35000rpm for 20 hours.

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