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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • 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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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/10—Vectors comprising a non-peptidic targeting moiety
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
• • C—CHEMISTRY; METALLURGY
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
  • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

• nucleic acid The major obstacle to the penetration of a nucleic acid into a target cell or organ, rests on the size and polyanionic nature of this nucleic acid which oppose its passage through cell membranes.
• Adenoviridae The family of Adenoviridae is widespread in mammals and birds and includes more than one hundred different serotypes of non-enveloped double-stranded DNA viruses, having a capsid of icosahedral symmetry (Horwitz, In: Fields BN, Knipe DM, Howley PM, ed Virology, Third Edition, Philadelphia Ed: Raven Publishers, 1996: 2149-2171).
• Their genome includes in particular a repeated inverted sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes.
• ITR inverted sequence
• Psi encapsidation sequence
• the main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the E1 region are necessary for viral propagation.
• the main late genes are contained in regions L1 to L5.
• the adenovirus has a very wide cellular tropism. Unlike the retrovirus whose cycle is dependent on cell division, it can advantageously infect cells in active division such as quiescent cells and its genome is maintained in episomal form. In addition it can be produced with high titers (10 ⁇ pfu / ml). These major assets have made it a vector of choice for the cloning and expression of heterologous genes.
• Group C adenoviruses, in particular type 2 and 5, as well as canine adenoviruses of CAV-2 type, the molecular biology of which is best known, are at the origin of the vectors currently used.
• adenoviral vectors have been used for the cloning and expression of genes in vitro (Gluzman et al., Cold Spring Harbor, New York 1 1724, p. 187), for the creation of transgenic animals ( WO95 / 22616) for the transfer of genes into cells ex vivo (WO95 / 14785; WO95 / 06120) or also for the transfer of genes into cells in vivo (see in particular WO93 / 19191, WO94 / 24297, WO94 / 08026) .
• AAV adeno-associated viruses
• a first objective of the present invention aims precisely to increase the transfection efficiency of these viral vectors by optimizing their internalization at the level of the target cell to be treated.
• Another objective targeted and achieved by the present invention relates to the reduction or even the suppression of the immune response conventionally manifested by the cells treated against the viral vector.
• the administration of recombinant viruses such as recombinant adenoviruses defective for replication (Yang et al., PNAS (1994) 4407) induces an important immune response.
• One of the major roles of the immune system is to destroy the non-self or self-altered elements.
• the administration of a gene therapy vector of viral origin introduces non-self motifs into the body.
• cells infected with such a vector and thereby expressing an exogenous gene become self-altering elements.
• DOTMA lipophilic group associated with an amino group via a so-called "spacer” arm
• DOTMA lipophilic group associated with an amino group via a so-called "spacer” arm
• DOTAP, DOBT or ChOTB can in particular be cited as representative of this category of cationic lipids.
• Other compounds, such as DOSC and ChOSC are characterized by the presence of a choline group in place of the quaternary ammonium group.
• the CMLV are obtained by enzymatic digestion of rabbit aorta according to a method adapted from Chamley et al (Cell Tissue Res. 197,177, 503-522) and then placed in primary culture. To do this, the rabbit aorta is removed and incubated for 45 minutes in the presence of collagenase (Collagenase II, Cooper Biomedical) at 37 ° C. A second digestion in the presence of collagenase and elastase (Biosys) is carried out for 2 hours at 37 ° C. These two digestions make it possible to obtain a cell suspension consisting essentially of CMLV.
• collagenase Collagenase II, Cooper Biomedical
• the Ad-Luc carries an expression cassette containing the luciferase gene under the control of the CMV promoter.
• This expression cassette comes from a commercial plasmid pUT650 (Cayla, Toulouse France) and contains the luciferase gene fused to the zeo resistance gene under the control of the CMV promoter.
• the expression cassette was inserted into the E1 region of the adenovirus In340 (Feldman et al., Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors Gene Ther, 1997. 4: 189-98 ; Robert, JJ et al Gene Neurochemistry., 1997, Vol 68, pp 2152-2160; Hearing et al 1983, Cell 33 p695-703.).
• the evaluation of the transfer is made either by measuring the luciferase activity (AVi.oCMVluc) or by histochemistry (AVi.oCMV ⁇ Gal) 3 days post infection. Several doses of lipofectamine were tested for a constant MOI of AVi . oCMVluc.
• one of the major obstacles to optimal efficiency of a recombinant adenovirus is its neutralization by the immune system. Indeed, following a cell lysis after a first transfer to animals or following a first contact with an adenovirus in humans, the immune system can effectively neutralize the virus in the circulation thus reducing the possibilities of reinjection of virus.
• This example reproduces in vitro the neutralization of the recombinant adenovirus by a pool of human sera, and advantageously shows that the association of a recombinant adenovirus with liposomes makes it possible to repeal this neutralization and, depending on the dose of liposomes, the transduction may be greater than that observed with the virus alone.
• FIGS. 4A and B use CMLVs transduced with Ad-Luc at MOI 100 in the presence of fetal calf serum (SVF) or adult human serum (SHA) in the DMEM culture medium.
• SVF fetal calf serum
• SHA adult human serum
• This example therefore shows that the claimed composition makes it possible, on the one hand, to completely repeal the neutralization of a recombinant adenovirus by neutralizing antibodies or serum and, on the other hand, to increase the transduction efficiency, in the presence of neutralizing serum. , as already shown in the previous examples.
• This example shows the increase in transduction of a therapeutic gene (gax) in rabbit smooth muscle cells by association of the adenovirus.
• AVi.oCMVrGax and a cationic lipid such as lipofectin (CLAAT).
• CLAAT lipofectin
• Smooth rabbit muscle cells are seeded at the rate of 5 ⁇ 10 4 cells per well of a MW 48 in DMEM 10% FCS medium.
• the infection is carried out 24 hours later in DMEM medium without serum.
• Different dilutions of adenovirus are mixed with 60 ng of lipofectamine in a final volume of 25 ⁇ l adjusted with PBS. This solution is incubated for 30 min at room temperature.
• the culture medium is replaced with 175 ⁇ l of serum-free medium per well and the mixture of adenovirus and lipofectamine is added to the cells.
• the infection lasts 1 hour at 37 ° C.
• the medium containing the virus is replaced by DMEM 0.5% SVF.
• 24 h after infection cell proliferation is induced by adding medium rich in serum (DMEM 10% FCS).
• Cell viability is estimated 72 h post-infection using the Alamar Blue test (Biosource).
• the MOIs vary from 0 to 3 10 4 PV / cell with the same viruses (Il-a and b). After infection, the cells are incubated in DMEM 0.5% FCS medium for 24 hours. The medium is then changed to medium rich in serum. Cell viability is estimated 72 hours post infection.
• This example describes the transfer of genes to fragments of arteries isolated using the association of an adenovirus encoding luciferase or ⁇ Galactosidase and lipofectamine (CLAAT).
• CLAAT adenovirus encoding luciferase or ⁇ Galactosidase and lipofectamine
• the artery fragments are ground in 500 ⁇ l of lysis buffer containing anti-proteases and then incubated for 20 min at room temperature with shaking. They are then centrifuged for 5 min at 10,000 revolutions. The reading is carried out on 10 ⁇ l of extract in the presence of 50 ⁇ l of substrate for 10 seconds using a kit (Luciférase assay Kit, Promega) and a luminometer (Berthold).
• This example demonstrates that gene transfer is possible on isolated arteries using CLAAT.
• this technique makes it possible on the one hand to improve gene transfer and on the other hand to reach the cells of the adventitia. He can be consider using this technique for the treatment of venous grafts using genes of interest, such as for example the FGF factor, carried by an adenoviral vector.

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• 1996
  • 1996-11-29 FR FR9614693A patent/FR2756491B1/fr not_active Expired - Fee Related
• 1997
  • 1997-11-28 KR KR1019990704738A patent/KR20000057307A/ko not_active Application Discontinuation
  • 1997-11-28 JP JP52437898A patent/JP2001514485A/ja active Pending
  • 1997-11-28 SK SK709-99A patent/SK70999A3/sk unknown
  • 1997-11-28 CZ CZ991882A patent/CZ188299A3/cs unknown
  • 1997-11-28 IL IL13005397A patent/IL130053A0/xx unknown
  • 1997-11-28 AU AU74010/98A patent/AU737846B2/en not_active Ceased
  • 1997-11-28 BR BR9713434-1A patent/BR9713434A/pt not_active IP Right Cessation
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  • 1997-11-28 CA CA002272637A patent/CA2272637A1/fr not_active Abandoned
  • 1997-11-28 EP EP97948959A patent/EP0948636A1/fr not_active Withdrawn
• 1999
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