WO2004033696A2 - Vecteurs adenoviraux recombinants et leurs applications - Google Patents
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- WO2004033696A2 WO2004033696A2 PCT/FR2003/002964 FR0302964W WO2004033696A2 WO 2004033696 A2 WO2004033696 A2 WO 2004033696A2 FR 0302964 W FR0302964 W FR 0302964W WO 2004033696 A2 WO2004033696 A2 WO 2004033696A2
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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
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
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- 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
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- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10351—Methods of production or purification of viral material
- C12N2710/10352—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
Definitions
- the present invention relates to new recombinant adenoviruses, and to their preparation process, as well as to their uses as vectors for expression and gene transfer, for vaccine purposes or for therapeutic purposes such as the treatment of cancer.
- Adenoviruses are naked viruses with a linear double-stranded DNA genome of approximately 30-40 bp, flanked by short inverted repeat sequences (ITRs).
- the adenovirus genome is organized into early transcription units (E1 to E4) and into a late unit (MLTU) composed of five families of transcripts (L1 to L5) whose expression is separated by the initiation of replication viral DNA.
- E1 to E4 early transcription units
- MLTU late unit
- the early phase begins two hours after infection by transcription and sequential expression of the ElA regions then E4, almost simultaneously E3 and E1B, then E2A and finally E2B.
- the immediately early ElA region codes for transactivators of other early adenovirus genes (E1B, E2, E3 and E4) as well as cellular genes. Eight hours after infection, replication of viral DNA begins.
- the late phase which begins twelve hours after infection is characterized by the extinction of the synthesis of cellular proteins in favor of late viral proteins which enter into the structural composition of the adenoviral particle, and participate in the assembly of the virion and in its release by affecting the structural integrity of the infected cell.
- Adenoviruses are particularly attractive for the development of viral vectors, because of their characteristics and the amount of knowledge available on their genetic organization and biology.
- a replicative virus capable of multiplying in the host human or animal
- a non-replicative virus incapable of multiplying in the host The construction of a non-replicating vector involves deleting a region essential for viral replication. The resulting viruses, which are unable to replicate and therefore produce infectious particles in cells permissible to infection with the corresponding wild virus, are produced in modified cell lines capable of providing the products of the deleted genes in trans.
- a commonly used strategy consists in inserting a heterologous gene in the left part of the genome between the left ITR and the El region, in place of the promoter and the coding region of the ElA gene (partial deletion of El) and possibly of the gene. E1B (total deletion of the El region). Viruses deleted for ElA are unable to replicate in cells that do not complement the functions of ElA. However, they are able to express significant amounts of exogenous protein in infected cells.
- adenoviral vectors Ad2 and Ad5 deleted from the El region and possibly from the E3 region have been constructed, essentially for the purpose of human gene therapy. In order to improve these vectors, mutations (E2 region) or additional deletions (E2 or E4 region) have been introduced. Canine non-replicative adenoviral vectors deleted from the entire E1 region have also been developed for human gene therapy applications [KLONJKOWSKI et al. , Human Gene Therapy, 8, 2103-2115, 1997, deletion of positions 411 to 2898 of Cav2; WO 95/14101 and US Patent 5837531 in the name of RHONE POULENC RORER; KREMER et al. , J. Virol., 74, 505-512, 2000, deletion of positions 412 to 2497 of Cav2).
- non-replicating vectors have shown good gene transfer efficiency in numerous tissues. However, they have a certain number of drawbacks, in particular for the transfer of genes into cells in active division such as tumor cells. In these cells, a rapid extinction of the expression of the transferred gene linked to the loss of the extrachromosomal vector during successive divisions.
- replicative vector requires that no sequence of the viral genome essential for its replication and for the production of infectious viral particles in the host be deleted (productive viral cycle). At the present time, only a small number of heterologous sequence insertion sites are known in adenoviruses which make it possible to satisfy these requirements.
- Replicative vectors were obtained by inserting heterologous genes in nonessential regions such as the E3 region and the right part of the genome between the right ITR and the transcriptional regulatory sequences of the E4 promoter. Replicative vectors have also been obtained by insertion of a heterologous gene in the left part of the genome between the left ITR and the E1 region, provided that functional E1 genes are kept.
- a heterologous sequence between positions 455 and 917 of the human adenovirus (Ad5) which inactivates the ElA gene by deletion of the promoter and part of the coding region of ElA is compensated by inserting a copy of this gene in an ectopic position in the vector (ELOIT et al., J. Gen. Virol., 76, 1583-1589, 1995).
- Replicative vectors have been constructed in this way from human adenoviruses (ELOIT et al., Cited above), bovines (MITTAL et al., J. Gen. Virol., 76, 93-102, 1995), sheep (XU et al., Virology, 230, 62-71, 1997), avians
- replicative adenoviral vectors have been developed essentially for vaccine applications. In general, they have demonstrated great efficiency in inducing imune responses, both with regard to the antibody response and the CTL response (for a review see ELOIT, Virologie, 2, 109-120, 1998 and KLONJKOWSKI et al., In "Adenoviruses: baslc biology to gene therapy", pp. 1 63-1 73, P. Seth, Ed., R. G. Landes Company, Austin Texas, USA).
- replicative vectors have some drawbacks: - they pose biosecurity problems linked to the risk of the spread of such replicative viruses,
- adenoviruses capable of replicating their genome in a permissive host, but incapable of multiplying.
- adenoviruses will hereinafter be called “pseudo-replicative adenoviruses”.
- the positions of the different regions of the adenoviral genome are defined by reference to the positions of the corresponding regions (ie containing elements of similar function) of the genome of the canine adenovirus type 2 in the GenBank sequence. J04368.
- the region located between the end of the left ITR and the start of the sequence coding for ElA corresponds to that located between position 311 and position 499 in the genomic sequence GenBank J04368 of canine adenovirus type 2.
- the subject of the present invention is a recombinant adenovirus capable of being obtained from a replicative adenovirus by deletion of all or part of the region of the genome of said replicative adenovirus corresponding to that located in the genome of the canine adenovirus of the type 2 (GenBank J04368), between positions 311 and 499, said deletion comprising all or part of the genome region of the original replicating adenovirus corresponding to that located between positions 311 and 401 in the genome of canine adenovirus type 2.
- the deleted portion consists of all or part of the region of the adenovir genome us original replicator corresponding to that located between positions 311 and 319 in the genome of canine adenovirus type 2; this deletion makes it possible to obtain a replicative recombinant adenovirus capable of multiplying in a host permissive to infection by the original wild-type adenovirus (productive viral cycle).
- the deleted portion comprises all or part of the genome region of the original replicative adenovirus corresponding to that located between positions 318 and 401 in the genome of canine adenovirus type 2; this deletion advantageously makes it possible to obtain pseudo-replicative adenoviruses, that is to say capable of replicating but incapable of producing infectious viral particles and therefore incapable of multiplying (abortive cycle) in a host permissive to infection by l original wild adenovirus.
- Obtaining the pseudo-replicative adenoviruses in accordance with the invention notably involves the suppression of all or part of the putative packaging signals of the 5'-TTTA / G-3 'type A x , A XI , and A II (located respectively in positions 341-344, 377-380, and 388-391, in the sequence of Cav2 GenBank J04368).
- the deleted portion in these pseudo-replicative adenoviruses can further comprise: - all or part of the genome region of the original replicative adenovirus corresponding to that located between positions 311 and 319 in the genome of the canine adenovirus of type 2; and or
- the recombinant adenoviruses in accordance with the invention retain the sequences of the left ITR essential for replication, for activation of transcription (4 repeating GGTCA motifs located between positions 62 and 99 in the genome of Cav2) as well as the packaging signals Ai of type 5'TTTGN 8 CG-3 ', and n to A IX of type 5'-TTTA / G-3' (located at positions 197-200, 206-209, 213-216, 226-232, 239-242 respectively , 250-253, 258-261, 272-275 and 306-309 in the Cav2 genome). They also conserve the entire coding sequence of ElA as well as regions of the El gene located downstream thereof (polyadenylation signal of ElA and ElB region).
- a recombinant adenovirus in accordance with the invention, also comprises at least one heterologous sequence of interest inserted into its genome.
- said heterologous sequence will, in the case of a replicative adenovirus, be inserted in the region of the genome corresponding to that located between positions 311 and 319 in the genome of l canine adenovirus type 2.
- said heterologous sequence can also be inserted in this region, or in any other place in the region of the genome corresponding to that located between positions 311 and 499 in the genome of the canine adenovirus. type 2. Insertion into this region can be done in place of or near the deleted portion. It is also possible to insert a heterologous sequence into any of the sites which are usually used for this purpose for the construction of replicative adenoviruses. The insertion can for example be carried out in the region E3, or in the region located between the region E4 and the right ITR, as described in US Patent 6090393, or in the 3 'portion of the right ITR, as described in U.S. Patent 5,616,326.
- heterologous sequence means any sequence other than that between positions 311 and 499 of the genome of said wild-type adenovirus.
- a vaccine antigen for example the gag or env genes of the feline immunodeficiency virus (FIV), the protein S, M or N of the feline coronavirus, a capsid protein of canine or feline parvovirus, the glycoprotein G of the rabies virus or the Hap-1 protein of Leptospira sp., Etc.
- FV feline immunodeficiency virus
- M or N of the feline coronavirus a capsid protein of canine or feline parvovirus
- the glycoprotein G of the rabies virus or the Hap-1 protein of Leptospira sp., Etc.
- Epo erythropoietin
- VEGF vascular endothelioma growth factor
- NT-3 neurotrophin 3
- AMF atrial natriuretic factor
- genes that can be used for the treatment of cancer for example that of IL-2, IFN ⁇ , etc.
- Recombinant adenoviruses in accordance with the invention may in particular be derived from mammalian adenoviruses, and in particular from canine adenoviruses, in particular from canine type 2 adenoviruses.
- recombinant adenoviruses in accordance with the present invention can be prepared by conventional techniques, well known in themselves to those skilled in the art
- Vectors Methods Mol. Biol., 7, 109-128, 1991
- techniques comprising: (i) the generation of recombinant genomes in E. coll by conventional techniques of double homologous recombination and (ii) the transfection of the recombinant genomes thus obtained in appropriate cell lines allowing the amplification of said genomes and their packaging in infectious viral particles.
- homologous recombination techniques in E. coli, such as those described by CHARTIER et al. , (J. Virol., 70, 7, 4805-4840, 1996) and in US Patent 6110,735 in the name of TRANSGENE or else those described by CROUZET et al. (Proc. Nat. Acad. Sci.
- the inventors have now developed a method for inserting a heterologous sequence into an adenovirus, which does not require linearization of the adenovirus genome by cleavage at the insertion site.
- the heterologous DNA fragment (donor molecule) to be inserted into the adenovirus genome (recipient molecule), comprises a selection marker making it possible to isolate the recombinant plasmids on their double resistance to ampicillin and kanamycin , and
- said fragment is co-transformed with a recipient molecule, either in circular form or in linearized form by cleavage at a restriction site located outside the insertion site.
- the subject of the present invention is also a process for the preparation of a recombinant adenovirus by homologous intermolecular recombination in a prokaryotic cell characterized in that it comprises the following steps: ⁇ ) introduction into said prokaryotic cell: (i) d 'a plasmid comprising the genome of an adenovirus and a first selection gene; and (ii) a previously linearized DNA fragment comprising a heterologous sequence to be inserted into said genome flanked by sequences homologous to those flanking the site of said plasmid where must be carried out insertion, and including a second selection gene, different from the first; and ⁇ ) culture of said prokaryotic cell under selective conditions, in order to allow the generation and selection of cells containing recombinant plasmids expressing the first and second selection gene, and ⁇ ) isolation of the genome of said recombinant adenovirus from the cells selected.
- selective conditions is understood to mean culture conditions in which the first and second selection agents (for example antibiotics) are present at concentrations which do not allow the multiplication of untransformed cells but allow the multiplication of co cells. -transformed.
- the plasmid used in ⁇ is in circular form.
- the plasmid used in step ⁇ ) is previously linearized by cleavage at a restriction site located outside the insertion site.
- the first and / or the second selection gene is a gene for resistance to an antibiotic, for example a gene for resistance to ampicillin or to kanamycin.
- the second selection gene is surrounded by 2 restriction sites, identical or different, absent in the genome of the adenovirus used in step ⁇ ); such a selection gene can thus be excised from the genome sequence of the recombinant adenovirus by digestion at these sites.
- the method according to the invention comprises, after the preparation of the recombinant genome according to steps ⁇ ) to ⁇ ) above, an additional step of transfection of the recombinant genome in an appropriate cell line allowing the amplification of said genome and its packaging into infectious viral particles.
- adenoviruses in accordance with the invention, it is possible to use cell lines known per se to those skilled in the art (cf. for example GRAHAM and PREVEC, cited above), expressing the E1 region of the adenovirus. , and optionally the E4 region of the adenovirus, when the latter has been altered by insertion of a heterologous sequence of interest.
- lines which can be used there may be mentioned in particular human lines such as line 293 (GRAHAM et al., J. Gen. Virol., 36, 59-74, 1977) and canine lines such as line DK / E1- 28 (KLONJKOWSKI et al., Human Gene Therapy, supra).
- said cell line is of canine origin.
- the subject of the invention is also plasmids and nucleic acid molecules which can be used for the preparation of the genome of a recombinant adenovirus in accordance with the invention, in particular a canine adenovirus, as well as said recombinant genomes, which can be obtained. by the methods as defined above.
- the subject of the invention is in particular the following nucleic acid molecules and plasmids:
- any nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule representing the genome of a recombinant adenovirus according to the invention as defined above; b) a nucleic acid molecule constituted by a fragment of the molecule a) above, comprising between 10 and 1000 bp, preferably at least 300 bp, of the sequence of the original replicating adenovirus situated upstream of the deleted portion and between 10 and 5000 bp, preferably between 10 and 1000 bp, preferably at least 300 bp, of the sequence of the original replicating adenovirus situated downstream of the deleted portion; such a molecule may further comprise all or part of a heterologous sequence inserted in place of or near the deleted portion.
- any nucleic acid vector in particular any plasmid, containing a nucleic acid molecule a) or b) as defined above.
- the subject of the invention is also the recombinant adenoviruses in accordance with the invention for use as a medicament.
- the subject of the invention is in particular the use of adenoviruses in accordance with the invention, for the preparation of immunogenic or vaccine compositions, or of medicaments intended for gene therapy or for the treatment of cancer, as well as for the production of recombinant proteins.
- said medicament or said composition is intended to be administered to a domestic or wild Carnivore, in particular the cat, the dog or the fox, or else to humans.
- the recombinant adenoviruses in accordance with the invention are particularly well suited to therapeutic uses, for example vaccines, in humans and animals.
- the recombinant adenoviruses according to the invention replicative or pseudoreplicative, multiply significantly in the nucleus of transduced cells allowing efficient transduction of both quiescent cells and actively dividing cells such as tumor cells.
- the pseudo-replicative adenoviruses according to the invention which do not produce any infectious particle have a high biosecurity and also allow to induce a strong immune response during booster injections.
- Recombinant adenoviruses in accordance with the invention have applications for the vaccination and treatment of cancer in domestic or wild carnivores, in particular cats, dogs or foxes.
- these canine adenoviruses can be used in human gene therapy, to target tissues different from those which can be transduced by human vectors, for example cells of the central nervous system.
- fragment A the left and right ends of the genome of the Manhattan strain of Cav2 corresponding at the sequences of positions 1 to 1060 (fragment A) and 29323- 31323 (fragment B) are amplified separately by PCR from the genomic DNA of the Manhattan strain of Cav2 (APPEL et al., Am. J. Vet. Res ., 34, 543-550, 1973), using the following primers: Fragment A
- the fragments A and B obtained are cloned separately in the plasmid pCR2.1 (TA Cloning System, INVITROGEN) to give the plasmids pCR2.1 / ITR left and pCR2.1 / ITR right, respectively.
- the plasmid pCR2.1 / ITR left is digested with BamHI and Xbal and the fragment of 1111 bp thus generated is cloned between the BamHI and Xbal sites of the plasmid pPolylI Amp R (GenBank M18128, LATHE et al., Gene, 57, 193- 201, 1987) to give the plasmid called pPolylI / ITR left.
- the right plasmid pCR2.1 / ITR is cleaved with BamHI, treated with Klenow polymerase, then cleaved with Xbal; the 2052 bp fragment thus generated is cloned between the Xbal and PvuII sites of the plasmid pPolylI / ITR left to give the plasmid pPolyII.ITRs.Cav2.
- This plasmid contains the left and right ends of the genome of the Manhattan strain of Cav2 cloned in the form of an Ascl-Ascl fragment of 3073 bp comprising an Xbal site at position 1066 of said fragment, allowing the plasmid to be linearized at the site of DNA insertion.
- the genomic DNA of the Manhattan strain of Cav2 and the DNA of pPolyII.ITRs.Cav2 linearized at the Xbal site are co-transformed into the strain of __. coli BJ5183 recBC s
- pCav2 A recombinant plasmid of 33425 bp, called pCav2, is isolated from colonies resistant to ampicillin.
- the plasmid pCav2 contains the complete genome of Cav2 (Manhattan strain) cloned as a fragment of 31331 bp flanked by two Ascl sites which are unique in this plasmid, these sites being absent in the genome of Cav2 (Manhattan and Toronto strains) as well as in that of the ovine adenovirus OAV strain.
- Cav2 Manhattan strain
- Ascl sites which are unique in this plasmid, these sites being absent in the genome of Cav2 (Manhattan and Toronto strains) as well as in that of the ovine adenovirus OAV strain.
- Shuttle plasmids bi) pNavette / 311-439.CMVeGFP
- This 6111 bp plasmid is derived from the pBluescript KS plasmid (STRATAGENE) by insertion of the sequences of
- This plasmid is constructed according to the following steps:
- the PCR amplification product is cloned into the plasmid pCR2.1 to give the plasmid pCR2.1 / UpRecSeq (1- 311).
- the PCR amplification product is cloned into the plasmid pCR2.1 to give the plasmid pCR2.1 / DownRecSeq (439-1060).
- the plasmid pEGFP-1 (CLONTECH) is cleaved with BamHI, treated with Klenow polymerase and then digested with
- the 741 bp fragment thus obtained is cloned between the Xhol sites (previously repaired by treatment with the Klenow polymerase) and NotI of the pCI plamide (PROMEGA), to give the plasmid pCMVeGFP.
- the plasmid pCMVeGFP is then cleaved with BglII, treated with Klenow polymerase and then digested with BamHI to generate a 2050 bp fragment (fragment E).
- fragment E is inserted between the SmaI and BamHI sites of the plasmid pBLUESCRIPT KS to give the plasmid pKS / CMVeGFP.
- the plasmid pCR2.1 / UpRecSeq (1-311) is cleaved by Kpnl and Sali and the fragment of 371 bp thus obtained (fragment C) is cloned between the Kpnl and Sali sites of the plasmid pKS / CMVeGFP, to give the plasmid pKS / CMVeGFP-C.
- the plasmid pCR2.1 / DownRecSeq (439-1060) is cleaved by Xbal and the 650 bp fragment thus obtained (fragment D) is inserted at the Xbal site of the plasmid pKS / CMVeGFP-C, to give the plasmid called pNavette 311-439 / CMVeGFP.
- the shuttle plasmid pNavette 311-401 / CMVeGFP is constructed from the plasmid pNavette 311-439 / CMVeGFP, according to the following steps:
- the sequence 401-1060 (DownRecSeq) is amplified by PCR using the primers:
- the PCR amplification product is cloned into the plasmid pCR2.1 to give the plasmid pCR2.1 / DownRecSeq (401-1060).
- This plasmid pCR2.1 / DownRecSeq (401-1060) is digested with EcoRI then treated with Klenow polymerase and the fragment 401-1060 thus obtained is substituted for the fragment 439-1060 of the plasmid pNavette 311-439 / CMVeGFP previously digested with Xbal then treated with Klenow polymerase, to give the plasmid pNavette 311-401 / CMVeGFP.
- the shuttle plasmid pNavette 311-319 / CMVeGFP is constructed from the plasmid pNavette 311-439 / CMVeGFP, according to the following steps:
- the sequence 319-1060 (DownRecSeq) is amplified by PCR using the primers:
- This plasmid pCR2.1 / DownRecSeq (319-1060) is digested with EcoRI then treated with Klenow polymerase and the fragment 319-1060 thus obtained is substituted for the fragment 439-1060 of the plasmid pNavette 311-439 / CMVeGFP previously digested with Xbal then treated with Klenow polymerase, to give the plasmid pNavette 311-319 / CMVeGFP.
- the PCR amplification product is cloned into the plasmid pCR2.1 to give the plasmid pCR2.1-Kana / PmeI.
- the plasmid pCR2.1-Kana / PmeI is cleaved by EcoRI, treated with Klenow polymerase and the fragment of approximately 959 bp containing the coding phase of the Kana gene is inserted at the EcoRV site of the plasmid pNavette 311-439 / CMVeGFP to give the plasmid pNavette 311-439 / CMVeGFP / Kana.
- the Kana gene which is then excised from the recombinant plasmid by digestion at the 2 Pmel sites, is absent from the sequence of the recombinant adenovirus generated from this plasmid.
- b 4 shuttle plasmid pPoly II 311-439 / CMVeGFP / Kana ( Figure 2)
- This 6332 bp plasmid is obtained by cloning between the Kpnl (position 42) and PvuII (position 63) sites of the plasmid pPoly II, of the Kpnl-PvuII fragment of 4292 bp from pNavette 311-439 / CMVeGFP, as illustrated in FIG. 2 c) pCav 311-439 / CMVeGFP Plasmid
- This plasmid is obtained by homologous recombination in the strain of E. coli BJ5183 according to the following 2 alternatives cl and c2, illustrated respectively in Figures 3 and 4: Ci) recombination of pNavette 311-439 / CMVeGFP. Kana with the plasmid pCav2 in circular form ( Figure 3)
- the donor molecule containing the upstream (UpRecSeq 1-311) and downstream (DownRecSeq 439-1060) and the CMVeGFP and Kana cassettes sequences is prepared from the plasmid pNavette 311-439 / CMVeGFP. Kana by digestion with the restriction enzymes Kpnl and EcoRV,
- the donor molecule is prepared from the plasmid pPolylI 311-439 / CMVeGFP. Kana by digestion with the restriction enzyme Swa I,
- the recombinant plasmids are isolated on the criterion of double resistance to ampicillin and kanamycin.
- the sequence of one of them, pCav 311-439 / CMVeGFP.Kana is confirmed by enzymatic restriction and by sequencing.
- the plasmid pNavette 311-401 / CMVeGFP is digested with KpnI and SwaI and the fragment of 3167 bp thus obtained and the plasmid pCav 311-439 CMVeGFP linearized at the Pmel site are co-transformed into the strain of E. coli BJ5185.
- the recombinant plasmid pCav 311-401 CMVeGFP, generated by homologous recombination, is selected on the criterion of double resistance to ampicillin and to kanamycin. d) pCav 311-319 / CMVeGFP Plasmid
- the plasmid pNavette 311-319 / CMVeGFP is digested with KpnI and SwaI and the fragment of 3249 bp thus obtained and the plasmid pCav 311-439 CMVeGFP linearized at the Pmel site are co-transformed into the strain of E. coli BJ5185.
- the recombinant plasmid pCav 311-319 CMVeGFP, generated by homologous recombination, is selected on the criterion of double resistance to ampicillin and to kanamycin. 2) Production of recombinant viruses
- the plasmids pCav 311-439 / CMVeGFP, pCav 311-401 / CMVeGFP or pCav 311-319 / CMVeGFP are digested with the restriction enzyme Ascl in order to excise the genome sequences of the recombinant adenovirus.
- the excised adenoviral genome is then transfected into the canine cell line DK / E1-28 which constitutively expresses the El region of Cav2 (KLONJKOWSKI et al., Human Gene Therapy, cited above), in the presence of Lipofectamine (GIBCO), according to the usual techniques. , well known in themselves to those skilled in the art (cf.
- the virus is harvested from the transfected cells, then amplified in the same cell line DK / El-28 and purified by centrifugation on a cesium chloride gradient, according to a conventional protocol, as described for example in GRAHAM and PREVEC, supra.
- the genomic sequence of the Cav 311- 439 / CMVeGFP, Cav 311-401 / CMVeGFP and Cav 311-319 / CMVeGFP viruses is confirmed by enzyme restriction and by partial sequencing of the viral DNA extracted from the infected DK / E1-28 cells. according to the HIRT technique (J. Mol. Biol., 26, 365-369, 1967).
- the recombinant Cav virus preparations are titrated by limiting dilution in a 96-well plate, according to the method of SPEARMAN and KARBER (Virology Methods Manual, Brian WJ Mahy and Hillar 0 Kangro, 1996, Académie Press, Harcourt Brace & Company).
- the titer in TCID 50 / ml obtained by this method is equivalent to the titer in ufp / ml obtained by the method of plaques on DK cells, according to the protocol described in KLONJKOWSKI et al. , supra.
- the results obtained are as follows: the Cav 311-439 / CMVeGFP, Cav 311-401 / CMVeGFP, Cav 311-319 / CMVeGFP viruses isolated have a restriction profile and a sequence in accordance with those expected, the Cav 311-439 / viruses Purified CMVeGFP, Cav 311-401 / CMVeGFP and Cav 311-319 / CMVeGFP have a titre of approximately 10 9 ' 2 pfu / ml.
- EXAMPLE 2 CHARACTERISA ION OF THE RECOMBINANT VIRUS CAV 311-439
- Canine (DK / EI-28 and DK) or feline (CRFK) cell lines are infected with the Cav 311-439 CMVeGFP virus, at a multiplicity of infection of 10 pfu / cell. Uninfected cells and cells infected with wild-type Cav virus are used as controls.
- the intracellular viral DNA is prepared according to the HIRT technique (J. Mol. Biol., 26, 365-369, 1967), digested with the enzyme EcoRI, then visualized on agarose gel after electrophoretic migration.
- the level of replication is greater in canine cells than in feline cells, - the peak of replication is reached at 24 hours in DK / El-28 cells and at 48 hours in cells DK, presumably due to cellular expression of the E1 region in DK / E1-28 cells.
- Canine DK cell lines are infected with the vector Cav 311-439 CMVeGFP, at a multiplicity of infection of 0.1, 1 and 10 pfu / cell respectively. Uninfected cells and cells infected with wild type Cav virus are used as a control.
- the infected cells are harvested 2 hours and 6 days after infection, lysed by several cycles of freezing and thawing.
- the cell lysate is titrated by the aforementioned limit dilution technique.
- the amount of virus present in the cells is presented in Table III below.
- Cav 311-439 virus does not produce infectious viral particles in canine cells which do not express the E1 region as DK cells: Cav 311-439's viral cycle is abortive in canine cells.
- FIG. 6 Titers of anti-eGFP (Ac) serum antibodies in cats, on days D7, D14, D21 and D31 after the inoculation of different doses of the virus Cav 311- 439.
- CMVeGFP -B- 9.6 10 7 pfu / ml (pfu: range forming units), - ⁇ - 9.6 10 7 pfu / ml, ••• D "- 9.6 10 6 pfu / ml, - O ••• 9.6 10 6 pfu / ml.
- EXAMPLE 3 CONSTRUCTION OF A LINE OF CANINE ORIGIN CONSTITUTIVELY EXPRESSING THE El CAV2 REGION.
- a new line expressing the E1 region is constructed from the DK line (immortalized dog kidney cell line; ATCC CRL 6247) by following the following steps:
- the PCR amplification product is cloned into the plasmid pCR2.1 to give the plasmid pCR2.1 / E1.
- the pTRE plasmid (CLONTECH) is digested with BamHI, treated with Klenow polymerase and recircularized to give the plasmid pTRE / d1 BamHI.
- the plasmid pCR2.1 / El is digested with EcoRI, the fragment of 3187 bp thus obtained is cloned at the EcoRI site of the plasmid pTRE / dl BamHI to give the plasmid pTRE El Cav2.
- This plasmid pTRE El Cav2 contains the coding sequence of the protein ElA under the control of a promoter
- Responsive Element or TRE the coding sequences of the ElB proteins (133R and 438R; SHIBATA et al., Virology, 172,
- the DK cells are cotransfected using the plasmid pTRE El Cav2 linearized at the Aatll site and the plasmid pTK-Hyg (CLONTECH) linearized at the Asel site.
- Clones are selected in the presence of hygromycin (150 ⁇ g / ml) and then analyzed by Southern blot, Northern blot, RT-PCR and Western blot. 4 clones expressing the El region (ElA and ElB), capable of efficiently producing the deleted vectors according to the invention were isolated.
- mice divided into three groups were inoculated intramuscularly with the dose of 10 8 pfu of the following viruses:
- the Cav 311-319 CONTROL virus is isogenic with the Cav 311-319 eGFP virus with the exception of the inserted heterologous gene (in place of the gene coding for GFP, a heterologous gene coding for a protein having no antigenic relationship with is inserted. GFP).
- Cav 311-319 makes it possible to induce in the mouse an immune (humoral) response specific to the heterologous gene inserted in this adenovirus.
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EP03775478A EP1549751A2 (fr) | 2002-10-08 | 2003-10-08 | Vecteurs adenoviraux recombinants et leurs applications |
AU2003283504A AU2003283504B2 (en) | 2002-10-08 | 2003-10-08 | Recombinant adenoviral vectors and applications thereof |
JP2004542569A JP4951204B2 (ja) | 2002-10-08 | 2003-10-08 | 組換えアデノウイルスベクターとその応用 |
US10/530,712 US8119396B2 (en) | 2002-10-08 | 2003-10-08 | Recombinant adenoviral vectors and applications thereof |
CA002501633A CA2501633A1 (fr) | 2002-10-08 | 2003-10-08 | Vecteurs adenoviraux recombinants et leurs applications |
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US10619169B2 (en) | 2016-09-20 | 2020-04-14 | Boehringer Ingelheim Vetmedica Gmbh | EHV insertion site ORF70 |
MX2019003116A (es) | 2016-09-20 | 2019-08-29 | Boehringer Ingelheim Vetmedica Gmbh | Nueva vacuna contra la gripe porcina. |
EP3515504B1 (fr) | 2016-09-20 | 2023-07-12 | Boehringer Ingelheim Vetmedica GmbH | Adénovirus vecteur canin |
EP3516064A1 (fr) | 2016-09-20 | 2019-07-31 | Boehringer Ingelheim Vetmedica GmbH | Nouveaux promoteurs |
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Non-Patent Citations (6)
Title |
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ALEMANY R ET AL: "COMPLEMENTATION OF HELPER-DEPENDENT ADENOVIRAL VECTORS: SIZE EFFECTS AND TITER FLUCTUATIONS" JOURNAL OF VIROLOGICAL METHODS, AMSTERDAM, NL, vol. 68, no. 2, novembre 1997 (1997-11), pages 147-159, XP001070256 ISSN: 0166-0934 * |
ELOIT M ET AL: "ISOGENIX ADENOVIRUSES TYPE 5 EXPRESSING OR NOT EXPRESSING THE E1A GENE: EFFICIENCY AS VIRUS VECTORS IN THE VACCINATION OF PERMISSIVE AND NON-PERMISSIVE SPECIES" JOURNAL OF GENERAL VIROLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 76, no. 7, 1 juillet 1995 (1995-07-01), pages 1583-1589, XP002028963 ISSN: 0022-1317 cité dans la demande * |
GRABLE M ET AL: "CIS AND TRANS REQUIREMENTS FOR THE SELECTIVE PACKAGING OF ADENOVIRUS TYPE 5 DNA" JOURNAL OF VIROLOGY, NEW YORK, US, US, vol. 66, no. 2, 1 février 1992 (1992-02-01), pages 723-731, XP002012398 ISSN: 0022-538X * |
KREMER ERIC J ET AL: "Canine adenovirus vectors: An alternative for adenovirus-mediated gene transfer" JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 74, no. 1, janvier 2000 (2000-01), pages 505-512, XP002167110 ISSN: 0022-538X cité dans la demande * |
MORRISON MARK D ET AL: "Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria" VIROLOGY, RAVEN PRESS, NEW YORK, NY, US, vol. 293, no. 1, 1 février 2002 (2002-02-01), pages 26-30, XP002206291 ISSN: 0042-6822 cité dans la demande * |
SOUDAIS C ET AL: "Characterization of cis-acting sequences involved in canine adenovirus packaging." MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY. UNITED STATES APR 2001, vol. 3, no. 4, avril 2001 (2001-04), pages 631-640, XP002242928 ISSN: 1525-0016 * |
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US20060233756A1 (en) | 2006-10-19 |
JP4951204B2 (ja) | 2012-06-13 |
FR2845395B1 (fr) | 2008-05-30 |
FR2845395A1 (fr) | 2004-04-09 |
JP2006501835A (ja) | 2006-01-19 |
US8119396B2 (en) | 2012-02-21 |
EP1549751A2 (fr) | 2005-07-06 |
WO2004033696A3 (fr) | 2004-05-27 |
CA2501633A1 (fr) | 2004-04-22 |
AU2003283504A1 (en) | 2004-05-04 |
AU2003283504B2 (en) | 2009-06-04 |
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