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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
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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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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
  • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the subject of the present invention is a cassette comprising a DNA sequence coding for a human IFN, the expression of which is placed under the control of elements which can be regulated by the human immunodeficiency virus (HIV).
• HIV human immunodeficiency virus
• HIV the etiological agent of AIDS (acquired immunodeficiency syndrome)
• T helper T lymphocytes
• the disease progresses to a deficit in cellular immunity, which makes sick individuals particularly susceptible to opportunistic infections. So far, no treatment has proven completely satisfactory despite the multiple efforts invested in the development of new antiviral molecules.
• HIV is a retrovirus belonging to the lentivirus family. Its genetic material, consisting of an RNA molecule, is packaged in a capsid of a protein nature, itself surrounded by a viral envelope.
• structural genes gag, pol and env
• capsid proteins gag, pol and env
• the HIV genome includes regulatory genes that control viral replication. Among these The latter include the tat and rev genes coding for the TAT and REV proteins respectively.
• the TAT protein has a trans-activating role in the expression of all of the HIV genes (Arya et al., 1985, Science, 229, 69-73). It seems that TAT intervenes on both a transcriptional and post-transcriptional level. It interacts specifically with a short nucleotide sequence, designated TAR sequence (for Trans-activation Responsive Region), and located at the 5 'end of the genome and viral transcripts (position +1 to +80; +1 representing the site of initiation of transcription) (Rosen et al., 1985, Cell, 41, 813-823).
• TAR sequence for Trans-activation Responsive Region
• the REV protein promotes the transport of messenger RNA (mRNA) coding for structural proteins to the cytoplasm, for translation.
• REV specifically recognizes an RRE sequence (for REV Responsive Element) on mRNAs. This is localized in the env gene near another sequence called CRS (Cis-acting Repression Sequence), involved in the nuclear retention of mRNA carrying it. It is assumed that the binding of the REV protein at its RRE target sequence has the effect of counterbalancing the inhibitory effect of CRS on the passage of large viral mRNAs to the cytoplasm.
• the sequences essential for the transcription of viral genes are located at the two ends of the genome at the level of LTRs (Long Terminal Repeat). While the 5 'LTR contains the promoter sequences as well as the TAR sequences, the 3' LTR includes the sequences involved in the termination of transcription.
• LTRs Long Terminal Repeat
• IFNs interferons
• the synthesis of cellular (endogenous) IFNs is suppressed in cells infected with HIV. This repression prevents cells infected with HIV and surrounding cells from defending themselves effectively against viral infection (Mark, 1992, AIDS Res. Hum. Retrovirus, 8, 199-207).
• the aim of the present invention is to propose different vectors for expression of human IFN which can be regulated by the TAT and / or REV proteins of HIV. This approach takes advantage of the replication characteristics of the HIV virus.
• the subject of the present invention is an expression cassette comprising at least one DNA sequence coding for a human IFN, placed under the control of clements capable of ensuring its expression in a mammalian cell, characterized in that that said elements are regulable by the human immunodeficiency virus (HIV).
• HAV human immunodeficiency virus
• a DNA sequence in use in the present invention can encode a human IFN of the u, p or y type. These may be the sequences disclosed in the prior art and in particular in Shaw et al. (1983, Nucleic Acid Res., 11, 555-573) for IFN ⁇ , Higashi et al. (1983, J. Biol. Chem., 258, 9522-9529) for the TFN ⁇ and Gray et al. (1982, Nature, 295, 503-508) for IFN ⁇ . These sequences can be isolated from the gene or cDNA. By sequence coding for a human IFN is also understood to denote a sequence coding for an analog of an IFN.
• analog is meant a molecule which would have an amino acid sequence slightly different from a native IFN but which would retain most of the antiviral function.
• an analog can be obtained by deletion, substitution and / or addition of one or more amino acids of the native protein or of a fragment thereof. Those skilled in the art know the techniques which allow these modifications to be made without abolishing the biological function of the protein.
• the elements ensuring the expression of a DNA sequence in use in the context of the present invention are the conventional elements allowing the transcription of the DNA sequence into mRNA and the translation of the latter into protein, such as for example a promoter and the translation initiation and termination codons.
• a cassette according to the invention comprises sequences which can be regulated by the HIV virus.
• a promoter is chosen which is functional in a mammalian cell and, preferably, in a human cell. It can come from any eukaryotic gene or from a viral genome and be of ubiquitous or regulable nature, in particular in response to certain tissue-specific or event-specific cellular signals. The choice of promoter is very wide and within the reach of those skilled in the art. However, the promoter of the murine PGK (PhosphoGlycerate kinase) and ⁇ -actin genes or the SV40 promoter (Simian Virus 40) will advantageously be used. According to a particularly preferred mode, the sequences regulable by HIV consist of a TAR and / or RRE / CRS type sequence.
• a TAR-like sequence interacts with the TAT protein to activate the expression of genes under its control.
• an RRE / CRS type sequence interacts with the REV protein to promote the transport of mRNAs in the cytoplasm. It is indicated that it naturally contains a splice acceptor site.
• these sequences may be slightly different from the native TAR and RRE / CRS sequences (as published by Wain-Hobson et al., 1985, Cell, 40, 9-17), provided however d '' be able to interact with viral TAT and REV proteins.
• TAR type sequence Being a TAR type sequence, it is inserted 3 'to the promoter and, most preferably, in position +1 (+1 being the site of initiation of transcription).
• the 5 'HIV LTR which naturally includes a functional promoter in cells mammal followed by the TAR sequence (in position +1 to +80). It is also possible to use a portion of it, which comprises the minimal promoter sequences (Spl sequences, CAAT box and TATA box) and the TAR sequence.
• a 200 bp fragment corresponding to the 3 'end of the LTR and extending from positions -120 to +80 of the viral genome is designated by the name of mini LTR.
• a cassette according to the invention comprises a conventional promoter and an RRE / CRS type sequence capable of interacting with the viral protein REV of HIV.
• the mRNAs will be blocked in the nucleus under the action of CRS.
• the RRE / CRS sequence is introduced 3 'to the DNA sequence coding for an IFN.
• the expression of a DNA sequence used in the present invention can be subjected to double regulation by TAT and REV, which makes it possible to circumvent the problems of escape from expression observed commonly. with certain promoters.
• an expression cassette according to the invention can comprise a TAR type sequence and an RRE / CRS type sequence; so that in the event of escape of the expression, the few mRNAs produced will remain blocked in the nucleus. Only the penetration of HIV and the synthesis of the viral proteins TAT and REV can trigger the synthesis of IFN.
• an expression cassette according to the invention may, in addition, contain other elements contributing to the expression of the DNA sequence coding for IFN, in particular, an intron, suitable splicing signals and elements transcription termination (polyadenylation signal). The choice and location of these elements is within the reach of the skilled person.
• cassette comprising from 5 'to 3':
• the 5 'HIV LTR a DNA sequence encoding an IFN and the polyadenylation signal of SV40
• the HIV mini LTR a DNA sequence encoding an IFN and the polyadenylation signal of SV40
• the PGK promoter the TAR sequence, a DNA sequence coding for an IFN and the polyadenylation signal of SV40;
• SV40 promoter a DNA sequence coding for an IFN, the RRE / CRS sequence and the polyadenylation signal of SV40;
• the HIV mini LTR a DNA sequence coding for an IFN, the RRE / CRS sequence and the polyadenylation signal of SV40; and.
• the invention also extends to vectors comprising an expression cassette according to the invention.
• vectors comprising an expression cassette according to the invention.
• vectors are well known to those skilled in the art, as are the methods for constructing and propagating them.
• viral vectors derived from retroviruses, herpes virus, adenovirus or viruses associated with adenovirus will be used.
• synthetic vectors can also be used.
• vectors defective for replication, and in particular retroviral vectors are particularly preferred.
• a cassette according to the invention is positioned in reverse orientation relative to the LTRs of the retroviral vector, in order to position the promoters in the opposite direction to avoid interference.
• a vector according to the invention can, in addition, comprise a gene encoding for a selection marker, such as, for example, the neomycin and puromycin acetyl transferase genes conferring resistance to the antibiotics G418 and puromycin.
• the invention also covers the viruses and viral vector particles obtained by transfection of the vector according to the invention, in an encapsidation cell line, in particular amphotropic, producing the structural proteins of the virus in question.
• the invention also relates to a eukaryotic cell comprising, an expression cassette or a vector according to the invention. These can be introduced either in vitro into a cell taken from the patient or directly in vivo.
• the cell is advantageously a human cell and, preferably, a cell of the hematopoietic line (stem cells or primary lymphocytes).
• the invention also relates to the therapeutic use of an expression cassette, a vector or a cell according to the invention, for the manufacture of a medicament intended for the prevention or treatment of viral infections and, in particular, HIV infections.
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising, as a therapeutic or prophylactic agent, an expression cassette, a vector or a cell according to the invention, in combination with a vehicle which is acceptable from a pharmaceutical point of view.
• a pharmaceutical composition according to the invention can be manufactured in a conventional manner.
• a therapeutically effective amount of such a therapeutic or prophylactic agent is combined with an acceptable carrier, diluent or adjuvant. It can be administered by any conventional route used in the field of art. Administration can take place in a single or repeated dose after a certain interval of interval.
• the amount to be administered will be chosen according to various criteria, in particular the route of administration, the patient, the state of the disease and the duration of treatment.
• a pharmaceutical composition according to the invention contains 10 3 to 10 14 pfu (range forming unit), advantageously from 10 5 to 10 13 pfu, preferably from 10 6 to 10 12 pfu and, most preferably, from 10 7 to 10 10 pfu of viral vector particles.
• the invention relates to a method of treatment of viral infections, and in particular to HIV, according to which a therapeutically effective amount of an expression cassette, a vector or a cell according to the invention is administered. to a patient in need of such treatment.
• a therapeutically effective amount of an expression cassette, a vector or a cell according to the invention is administered.
• they can be administered directly in vivo, for example by the intravenous route.
• a somatic gene therapy protocol which consists in taking bone marrow stem cells or lymphocytes from the peripheral blood of a patient, transfecting them with a vector according to the invention, cultivating them in vitro according to the techniques of art and administer them to the patient.
• FIG. 1 shows schematically the vector pTG4313 allowing the expression of a DNA sequence coding for IFN ⁇ under the control of the HIV LTR.
• FIG. 2 represents the vector pTG4344 co-expressing the IFN ⁇ gene and the puromycin selection gene.
• FIG. 3 schematizes the retroviral vector pTG4379 for the transfer of an IFN gene and its expression inducible by TAT.
• FIG. 4 shows the evolution of the infection in U937 cells infected with HIV III B and transfected with the expression vectors of IFN ⁇ ( ⁇ ), ⁇ ( ⁇ ) and ⁇ ( ⁇ ), compared to the control untransfected cells ( ⁇ ).
• FIG. 5 illustrates the retroviral vector pTG6324 comprising the expression cassettes of IFN ⁇ (under the control of the HIV mini LTR) and of the neo selection gene (under the control of the retroviral LTR 5 ′).
• FIG. 6 illustrates the evolution of the infection in human CD8-PBLs transduced by the vector pTG6327 ( ⁇ ) then infected with the HIV III B virus, compared with the non-transduced cells ( ⁇ ).
• the human genes coding for the ⁇ and ⁇ IFNs were cloned by PCR (Polymerase Chain Reaction) from the genomic DNA of lymphoid cells, for example CEM-A3 cells (derived from a CEM line available at ATCC under number CCLl 19). Such banks are commercially available. Amplification of DNA fragments by PCR can be carried out using commercial kits according to the manufacturer's specifications or according to the technology described in PCR Protocols-A (edited by Innis, Gelfand, Sninsky and White, Académie Press Inc) using primers complementary to the published sequences.
• the human gene coding for IFN ⁇ is obtained from pTG1 1 described in the French patent application published under the number 2.583.429.
• protruding 5 ′ restriction sites can be converted into blunt sites by treatment with the large Klenow fragment of the DNA polymerase of E. coli while the protruding 3 'sites are treated with T4 polymerase.
• EXAMPLE 1 Construction of Inducible Interferon Expression Vectors by the TAT protein of HIV and in which the selection gene consists of the puromycin gene.
• vectors in which the expression of the IFN ⁇ , ⁇ , and ⁇ genes is placed under the control of the HIV 5 'LTR.
• These vectors were constructed from the vector pTG1322 described in Mehtali (1992, AIDS and Human Retroviruses, 8, 1959-1965) comprising a heterologous gene placed under the control of the HIV 5 'LTR (corresponding XhoI-HindIII fragment of 725 bp at positions -645 to +80 of the HIV genome).
• This vector also carries the intron and the polyadenylation signal of the SV40 virus. It is digested with HindIII treated with Klenow and then digested with SmaI.
• pTG4314 is obtained by inserting the SmaI-EcoRV fragment carrying the IFN ⁇ gene into pTG1322 obtained as above.
• PTG4315 is generated by insertion of the ⁇ FN ⁇ gene in the form of an HpaI-PvuII fragment between the same pTG 1322 sites.
• a selection gene conferring resistance to puromycin (puromycin R) in the form of a 1.2 kb BamHI-BglII treated Klenow fragment is introduced into the EcoRI site of these various IFN expression vectors. .
• This fragment contains the SV40 virus early promoter, the puromycin R gene and the SV40 polyadenylation signal.
• the vectors pTG4344 (represented in FIG. 2), pTG4345 and pTG4346, are obtained, which allow the co-expression of the puromycin R selection gene and of the IFN ⁇ , ⁇ or ⁇ gene respectively.
• the vector at the base of the constructs is pLXSP which is derived from pLXSN (Miller and Rossman, 1989, Biotechniques, 7, 980-988) after replacement of the gene for neomycin selection by the puromycin R gene and the 3 'LTR of MSV (Myelo Sarcoma Virus) by the 3' LTR of MPSV (Myelo Proliferative Sarcoma Virus).
• a 200 bp ScaI-HindIII fragment corresponding to the mini LTR (position -120 to +80) is isolated from the HIV genome, which is then introduced upstream of each of the IFNs genes. Then, at 3 ′, genes are introduced, the polyadenylation signal of SV40.
• the vector thus obtained is digested with BglII, treated with Klenow.
• the fragment comprising the expression cassette "mini LTR - IFN - polyadenylation signal" is introduced between the HpaI-BglII sites of the retroviral vector pTG4056.
• the clones in which this fragment is positioned in reverse orientation with respect to the 5 'and 3' LTRs are selected.
• pTG4056 originates from pLXSP following the deletion of the SV40-puromycin R promoter expression block and the reinsertion of the puromycin R gene in the correct orientation to be placed under the control of the 5 'LTR.
• the retroviral vectors pTG4392, pTG4391 and pTG4379 ( Figure 3) allowing the transfer and the expression of the sequences coding respectively for the IFNs ⁇ , ⁇ and ⁇ . They can be transfected into an amphotropic packaging cell line, for example the line PA317 (Miller et al., 1986, Mol. Cell., Biol. 6, 2895-2902), Gp Env. Am (Markowitz et al., 1988, Virology, 167, 400-406) or PG13 (Miller et al., 1991, J. Virol, 65, 2220-2224).
• the transfected cells are placed in a selective medium (puromycin 6 ⁇ g / ml). Resistant cells are isolated, which will form lines producing amphotropic particles capable of infecting human cells, from which a viral stock can be built up which can be used in anti-AIDS gene therapy.
• the TAR sequence (corresponding to nucleotides +1 to +80 of the HIV genome) is generated by chemical synthesis. Then, the double stranded oligonucleotide thus obtained is introduced by insertional mutagenesis in position +1 of the murine PGK promoter, the sequence of which is published in Adra et al. (1987, Gene, 60, 65-74).
• the fragment comprising the sequence coding for human IFN ⁇ , ⁇ or ⁇ is inserted downstream of the TAR sequence, followed by the polyadenylation signal of SV40.
• each IFN ⁇ , ⁇ or ⁇ sequence corresponds to a retroviral vector.
• an amphotropic packaging line producing each type of viral particles is established.
• the preceding example is reproduced with the following variant:
• the synthetic TAR sequence is introduced in position +1 of the rat ⁇ -actin promoter, the sequence of which is described in Nudel et al., (1983, Nucleic Acids Res., 11, 1759 -1771)
• the HIV mini LTR is isolated from the viral genome after ScaI-HindIII digestion, inserted into any plasmid to be treated as a SmaI-HindIII fragment and then cloned between the same sites of the vector pTG2359.
• the latter corresponds to the cloning of the polyadenylation site of the SV40 virus in the plasmid p Bluescript KS (Stratagene).
• PTG4347 is obtained in which a SmaI-HpaI fragment carrying the sequences coding for IFN ⁇ is introduced, after digestion with BalI
• the "mini LTR-IFN ⁇ -polyA SV40" expression cassette is purified from vector pTG4355 obtained in the previous step in the form of a BamHI-BglII fragment, which is cloned in the BamHI site. of pTG6320 to give pTG6324 ( Figure 5).
• the latter therefore includes the MSV LTR directing the expression of the neo selection gene, followed by the cassette "mini LTR - IFN ⁇ - polyA SV40" positioned in reverse orientation in order to avoid interference phenomena between the promoters and finally the MPSV retroviral 3 'LTR.
• pTG6320 comes from pLXSP following the detection of the expression cassette of the puromycin gene and replacement by the neomycin gene (Colbère - Garapin et al., 1981, J. Mol. Biol., 150, 1 - 14).
• the cloning strategy is quite similar to what is described above.
• the vector pTG631 1 comes from the insertion of a SmaI-HpaI fragment carrying the sequence coding for IFN ⁇ in the BalI site of pTG4347.
• the retroviral vector pTG6328 is obtained by cloning the purified BamHI.-BglII fragment from pTG631 1 into the vector pTG6320 previously digested with BamHI. This is the equivalent of pTG6324 with the difference that it carries the IFN ⁇ gene in place of the IFN ⁇ gene.
• the SmaI-HindIII fragment carrying the HIV mini LTR is inserted between the same sites of pTG4356, to generate pTG6312.
• the vector pTG4356 corresponds to the vector pTG2359 into which the IFN ⁇ gene (SmaI-EcoRV fragment) has been cloned (site BalI).
• site BalI the expression cassette for lTFN ⁇ is purified from pTG6312 after digestion with BamHI and BglII and then subcloned into the BamHI site of pTG6320 to give pTG6327.
• Stocks of infectious viral particles can be prepared from each of the above vectors pTG6324, pTG6327 and pTG6328. To do this, these the latter are transfected in a conventional manner into an ecotropic packaging line, for example the GP + E 86 line (Markowitz et al., 1988, J. Virol., 62, 1120-1124). 48 h after transfection, the cells are cultured in a selective medium (G418 1 mg / ml).
• the mixture of clones resistant to G418 is maintained for 3 additional days in a selective medium.
• the culture supernatant is harvested to conventionally infect an amphotropic packaging line such as line PA3 17. After selection, the lines thus obtained will constitute the cells producing infectious viral particles allowing the transfer and expression of the various IFNs in as part of human gene therapy.
• pHMG-TAT is a vector for expression of the native TAT protein and results from the cloning of the sequence coding for this in the vector pHMG (Mehtali et al., 1990, Gene, 91, 179-184).
• a good clone producing virions titrating 5 ⁇ 10 to 10 pfu / ml is selected after infection of murine NIH-3T3 cells (ATCC CRL 1658) with an aliquot of culture supernatant of the clone. We check its ability to produce IFN as indicated above. A clonal dilution is again carried out from the previous clones in order to generate subclones. We select for each of them a good subclone producer of virions and IFN after titration and assay of IFN.
• EXAMPLE 3 Construction of expression vectors of a human IFN inducible by the HIV REV protein.
• a retroviral vector is described by insertion into the vector pLXSP and in the reverse orientation with respect to the LTRs, of an expression cassette comprising from 5 'to 3': - the promoter of the SV40 virus;
• the latter is isolated from the genome of HIV-1 isolate Lai in the form of a BglII-BamHI fragment (extending from nucleotides 7178 to 8032 of the sequence as published by Wain-Hobson et al., Supra). This fragment can be treated with Klenow before being introduced into an adequate restriction site; and
• EXAMPLE 4 Construction of expression vectors of a human IFN inducible by the TAT and REV proteins of HIV.
• the BamHI-BglII fragment treated with Klenow and carrying the HIV RRE / CRS sequence is inserted into the vectors pTG4392, pTG4391 and pTG4379 between the sequence coding for one of the IFNs and the polyadenylation signal of SV40.
• the vectors thus obtained can be modified by the insertion of a splicing donor site between the HIV LTR and the IFN gene. As before, it is possible to generate an amphotropic line producing infectious particles corresponding to each of the vectors thus obtained.
• EXAMPLE 5 Resistance to viral infection of cell lines transfected with TAT-inducible IFN expression vectors.
• the experiments described below are carried out in cell lines naturally infected with HIV, such as: - the U937 line (ATCC CRL 1593) derived from human monocytes; and - the CEM-A3 line derived from CD4 + human lymphocyte cells (ATCC CCL1 19).
• the cells are cultured according to the supplier's recommendations and transfected by electroporation of at least 20 ⁇ g of DNA using appropriate equipment (Puiser Biorad gene, voltage 210V, capacitance 960 ⁇ F).
• pTG4314 is co-electropore into U937 cells (2 ⁇ 10 7 cells) with the vector pLXSP in a DNA concentration ratio of 20: 1.
• the cells are returned to culture at 37 ° C. in the presence of 5% CO 2 and the following day, placed in a selective medium (puromycin 0.5 ⁇ g / ml).
• Antibiotic resistant cells are selected. Their resistance to infection is tested by infecting them with an HIV preparation at a high TCID 50 (at least 1000).
• the TCID 50 is determined according to the method described in Reed et al., (1938, Am. J. Hyg., 27, 493-497), on cell lines that can be infected with HIV, such as CEM cells. It corresponds to the dose at which 50% of the cells are infected and 50% are not. It is checked for each batch of HIV used.
• a fraction of the electroporated U937 cell culture corresponding to approximately 10 6 cells, is removed and centrifuged at low speed. After washing, the cell pellet is taken up in 200 ⁇ l of culture medium in the absence of serum. Then the cells are treated with different dilutions of the viral preparation. The infection is allowed to continue for 1 hour at 37 ° C.
• the cells are then washed twice in culture medium and cultured in 5 ml of the same medium, supplemented with 10% of FCS (fetal calf serum).
• FCS fetal calf serum
• the medium is renewed 2 or 3 times a week.
• the spread of the virus is evaluated by measuring the reverse transcriptase activity at regular intervals. in the culture supernatant according to the method described in Spire et al. (1985, Lancet, i, 188-189).
• the presence and localization of the viral protein gp120 is evaluated by immunofluorescence using an anti-gp120 antibody (obtained from serum from seropositive patients). It is found that in the cells expressing IFN, the protein gp120 is in intracellular form, whereas it should be sub-membrane, in order to allow the budding of the viral particles. Taken together, these results indicate that IFN acts at the late stages of the viral cycle and probably at the level of virion assembly.
• EXAMPLE 7 Method of treatment of AIDS by gene therapy.
• - CD4 + lymphocytes from peripheral blood which can be collected in particular by leukopheresis
• - hematopoietic stem cells CD34 + which can be obtained from '' a bone marrow or peripheral blood sample (mobilization by chemotherapy or by action of hematopoietic growth factors).
• the patient is subjected to a blood sample or leukopheresis. After eliminating the red blood cells, the mononuclear cells are isolated by centrifugation on Ficoll and cultured according to the techniques of the art. First, we get rid of the cells adhering to the plastic of the culture flasks. The cell suspension is recovered and cultured in flasks coated with anti-CD8 antibodies (Immunotech) in order to eliminate the CD8 + cells. The cell suspension thus obtained is mainly composed of CD4 + lymphocytes. Of course, the other enrichment techniques can also be used.
• the growth factors that can be used for this purpose are varied. As an indication, one can cite a mixture of anti-human CD3 antibodies (Immunotech, about 10 ng / ml) and IL-2 (Cetus; 100 U / ml) or PHA (phytohemagglutidine, Sigma, at a concentration about 5 ⁇ g / ml) and IL-2.
• the cells are transduced by the retroviral vector.
• Different methodologies are applicable: - co-culture of CD4 + cells and amphotropic producing cells of examples 1 to 4.
• - co-culture of the 2 cell types, the CD4 + cells being deposited in the culture wells and the producer cells in a Transwell (Costar) - infection of the CD4 + cells with an aliquot of culture supernatant of the producer cells according to an ego (multiplicity of infection) preferably between 1 and 5.
• the transduction is carried out in the presence of protamine sulphate (at a concentration of approximately 5 ⁇ g / ml) to promote the attachment of the virus to the cell surface .
• the cell culture is passed through a selective medium (puromycin in the context of the vectors of Example 1 or G418 in the context of the vectors of Example 2) and continued for 7 to 8 days until the number is obtained. of appropriate cells. It is indicated that it may be advantageous to use the AIM V medium (Gibco BRL) supplemented with L-glutamine (2 mM) and IL-2 (100 U / ml) and devoid of serum.
• a selective medium puromycin in the context of the vectors of Example 1 or G418 in the context of the vectors of Example 2
• the stages of purification of the CD34 cells are as follows: - isolation of the mononuclear cells from the peripheral blood or bone marrow sample by Ficoll centrifugation, - depletion of adherent cells, and - capture of CD34 + cells by culture in flasks coated with anti-CD34 antibody (Immunotech) or in the presence of beads coated with this antibody.
• the suspended cells are eliminated and the adherent cells are recovered after action, for example, of papain.
• the CD34 + cells thus obtained are stimulated by various growth factors to place them in the division cycle and thus promote retroviral infection.
• a mixture of IL-3 (approximately 10 ng / ml), IL-6 (approximately 50 U / ml) and SCF (Stem cell factor, approximately 50 U / ml) can be used for this purpose.
• the culture medium for 48 hours before carrying out the transduction according to the protocol indicated above. These factors are commercially available.
• the cells are then re-administered to the patient.

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• 1993
  • 1993-12-13 FR FR9314915A patent/FR2713657B1/fr not_active Expired - Fee Related
• 1994
  • 1994-12-13 WO PCT/FR1994/001458 patent/WO1995016784A1/fr not_active Application Discontinuation
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