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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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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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
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

• the present invention relates to a new polypeptide designated ⁇ P62, its variants, the corresponding nucleic sequences, and their therapeutic uses, in particular in anti-cancer gene therapy.
• oncogenes and suppressor genes are involved in the control of cell division.
• the ras genes and their products generally designated p21 proteins, play a key role in controlling cell proliferation in all the eukaryotic organisms where they have been sought.
• certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic.
• a large number of human tumors have been associated with the presence of modified ras genes.
• overexpression of these p21 proteins can lead to disruption of cell proliferation. Understanding the exact role of these p21 proteins in cells, their mode of operation and their characteristics therefore constitutes a major challenge for the understanding and therapeutic approach of carcinogenesis.
• the GAP protein was the first whose involvement in transduction signal has been documented. It is a cytosolic protein present in all eukaryotic organisms which has the ability to greatly accelerate the hydrolysis of GTP, linked to the normal protein. It has two areas ensuring distinct functions. Its carboxy-terminal end carries the catalytic activity which interacts with the p21 proteins and which increases their GTPase activity.
• SH2 and SH3 domains which participate in the transduction of the message and interact with other proteins.
• proteins are two proteins, p62 and p190, of 62kDa and 190 kDa respectively, which are strongly phosphorylated to tyrosine.
• p62 and p190 proteins which are strongly phosphorylated to tyrosine.
• These two proteins form a specific complex with GAP and are immunoprecipitated by antibodies directed against different epitopes of GAP.
• Amino acids 271 to 443 of p62 contain phosphorylated tyrosines and appear to be involved in these interactions.
• the p62 protein (or even Sam68) has been identified by Wong et al. (Cell 69 (1992) 551). It contains 443 amino acids, the sequence of which has been described in the literature (see SEQ ID No. 1). In addition to the characteristics mentioned above, the p62 protein has several characteristics specific to hnRNP ("heterogeneous nuclear RiboNucleoProtein”):
• a first object of the invention therefore relates to any p62 derivative capable of at least partially inhibiting the interaction between a GAP protein and p62.
• the derivatives according to the invention are capable of at least partially inhibiting the oncogenic power of the ras and / or src proteins.
• the derivatives according to the invention are capable of inducing cell death by apoptosis.
• the derivatives according to the invention are also characterized by the loss of the ability to interact with the RNA of p62.
• the present invention describes in particular the detection, cloning and characterization of a natural isoform of the protein p62.
• This isoform designated ⁇ p62 (or ⁇ Sam ⁇ )
• ⁇ p62 has a deletion in the zone of homology to the GRP33 protein, which covers the KH domain. Due to this deletion, ⁇ p62 does not have all of the properties of p62. Thus, ⁇ p62 has an interaction domain with GAP and GRB2 intact as well as the various sequences rich in prolines partners of SH3 ( Figure 1). However, ⁇ p62 is no longer able to interact with nucleic acids due to the deletion of the domain of homology to the GRP33 protein.
• ⁇ p62 therefore interferes with the proliferation and differentiation processes and leads, in the various cell models, to cell death by apoptosis.
• the invention relates more particularly to any p62 derivative carrying at least one deletion in the region of homology to the GRP33 protein. More particularly, the derivatives according to the invention comprise at least one deletion in the region between residues 145 to 247 of the protein p62 as shown in the sequence SEQ ID No. 1, and which covers the KH domain.
• the deletion advantageously relates to more than 10 amino acids and, more preferably, to more than 30 acids amines. It can relate to one or more sites inside this region, as soon as the resulting derivative exhibits the properties described above.
• the derivative according to the invention is a polypeptide comprising all or part of the sequence SEQ ID No. 2 or a variant thereof.
• the term variant within the meaning of the invention denotes any polypeptide whose structure is distinguished from the sequence SEQ ID No. 2 by one or more modifications of a genetic, biochemical and / or chemical nature. It may in particular be any mutation, substitution, deletion, addition and / or modification of one or more residues.
• Such derivatives can be generated for different purposes, such as in particular that of increasing the affinity of the peptide for its interaction site, that of improving its production levels, that of increasing its resistance to proteases or of '' improve its passage through cell membranes, that of increasing its therapeutic efficacy or reducing its side effects, or that of giving it new pharmacokinetic and / or biological properties.
• the variants include deletions or mutations relating to amino acids the presence of which is not decisive for the activity of the derivative.
• Such amino acids can be identified, for example, by cell activity tests as described in the examples.
• the derivatives of the invention retain at least part of the p62 protein allowing interaction with the SH2 domain of GAP.
• This part of p62 is more particularly composed of phosphorylated tyrosines, localized between residues 200 to 450 of the protein p62 (Cf SEQ ID No. 1).
• a preferred derivative according to the invention therefore comprises at least (i) a deletion in the region between residues 145 to 247 of p62 and (ii) a part of p62 allowing interaction with the SH2 domain of GAP. More preferably, the deletion relates to residues 1 to 202.
• derivatives according to the invention having particularly advantageous properties may consist of polypeptides essentially comprising the region carrying the phosphorylated tyrosines of p62.
• a particularly preferred example of a polypeptide according to the invention is represented by the ⁇ p62 polypeptide of sequence SEQ ID No. 2, having a deletion of residues 170-208 of the sequence of p62.
• Another example is represented by the polypeptide p62-C comprising the residues 203 to 443 of p62 (sequence SEQ ID No. 3).
• ⁇ p62 can enter into competition with p62 with respect to GAP.
• GAP being one of the effectors of the Ras proteins
• ⁇ p62 blocks the mitogenic pathways which depend on it.
• Overexpressed by gene transfer (transfection, infection, microinjection, etc.) ⁇ p62 induces cell death by apoptosis in normal (fibroblasts NIH3T3 and Swiss 3T3) or tumor (H460; HCT116) cells, and is capable of inhibiting the formation of foci induced by ras.
• the p62-C derivative (essentially comprising the C-terminal part of ⁇ p62, which covers the region between amino acids 203 and 443 and which corresponds to the domain of interaction with the SH2 domains of GAP and of GRB2).
• This C-terminal part also contains three sites for interaction with the SH3 domains, those having the most affinity for Fyn.
• the significant therapeutic activity of the derivatives according to the invention is linked to their multiple properties, and in particular their capacity for titrating the SH3 domains of proteins of the src family (example: fyn), their capacity for inhibiting the recruitment of GRB2 by titrating its SH2 domain, and their ability to inhibit the effector function of the GAP protein for signaling pathways which depend on Ras.
• the nucleic acid according to the invention can be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• gDNA genomic DNA
• cDNA complementary DNA
• It can be of human, animal, viral, synthetic or semi-synthetic origin. It can be obtained in different ways and in particular by chemical synthesis using the sequences presented in the application and for example a nucleic acid synthesizer. It can also be obtained by screening libraries using specific probes, in particular as described in the application (see sequences SEQ ID No. 6 and 7 for example).
• nucleic acids of the invention can be prepared according to any technique known to those skilled in the art.
• the nucleic acid according to the invention is a cDNA or an RNA.
• the nucleic acid according to the invention is advantageously chosen from: (a) all or part of the sequence SEQ ID No. 2 or SEQ ID No. 3 or their complementary strand, (b) any sequence hybridizing with the sequences (a ) and coding for a derivative according to the invention,
• the present invention also relates to any expression cassette comprising an acid nucleic acid as defined above, a promoter allowing its expression and a transcription termination signal.
• the promoter is advantageously chosen from functional promoters in mammalian cells, preferably human. More preferably, it is a promoter allowing the expression of a nucleoic acid in a hyperproliferative cell (cancerous, restenosis, etc.).
• promoters can be used. It may for example be the own promoter of the p62 gene. They can also be regions of different origin (responsible for the expression of other proteins, or even synthetic). It can thus be any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific way or not, inducible or not, strong or weak. Mention may in particular be made of the promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the target cell.
• ubiquitous promoters can be used in particular (promoter of the HPRT, PGK genes, ⁇ -actin, tubulin, etc.), promoters of intermediate filaments (promoter of the GFAP genes, desmin, vimentin, neurofilaments, keratin, etc. ), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII, ApoAl genes, etc.), tissue-specific promoters (promoter of the pyruvate kinase gene, villin, intestinal fatty acid binding protein, ⁇ - smooth muscle actin, etc.) or promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, etc.).
• they may be promoter sequences originating from the genome of a virus, such as for example the promoters of the E1A and MLP genes of adenovirus, the early promoter of CMV, or also the promoter of the RSV LTR, etc. .
• these promoter regions can be modified by adding activation or regulatory sequences, or allowing tissue-specific or majority expression.
• the present invention now provides new therapeutic agents which, by their iterative antiprol properties and / or apoptotics to interfere with many cellular dysfunctions.
• the nucleic acids or cassettes according to the invention can be injected as such at the site to be treated, or incubated directly with the cells to be destroyed or treated.
• nucleic acid or the cassette is incorporated into a vector.
• the vector used can be of chemical origin (liposome, nanoparticle, peptide complex, cationic lipids or polymers, etc.) viral (retrovirus, Adenovirus, herpes virus, AAV, vaccinia virus, etc.) or plasmid.
• the use of viral vectors is based on the natural properties of transfection of viruses.
• adenoviruses for example, adenoviruses, herpes viruses, retroviruses and associated adeno viruses. These vectors are particularly effective in terms of transfection.
• a preferred object according to the invention resides in a defective recombinant retrovirus whose genome comprises a nucleic acid as defined above.
• Another particular object of the invention resides in a defective recombinant adenovirus whose genome comprises a nucleic acid as defined above.
• the vector according to the invention can also be a non-viral agent capable of promoting the transfer and expression of nucleic acids in eukaryotic cells.
• Chemical or biochemical vectors, synthetic or natural represent an interesting alternative to natural viruses in particular for reasons of convenience, security and also by the absence of theoretical limit as regards the size of the DNA to be transfected.
• These synthetic vectors have two main functions, to compact the nucleic acid to be transfected and to promote its cell fixation as well as its passage through the plasma membrane and, where appropriate, the two nuclear membranes.
• the non-viral vectors all have polycationic charges.
• the nucleic acid or vector used in the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
• the nucleic acid or the vector is used in an injectable form. It can therefore be mixed with any pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection at the site to be treated. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes.
• a direct injection of the nucleic acid into the patient's tumor is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues.
• the doses of nucleic acid used can be adapted according to different parameters, and in particular according to the gene, the vector, the mode of administration used, the pathology concerned or even the duration of the treatment sought.
• the invention also relates to any pharmaceutical composition comprising at least one nucleic acid.
• composition comprising at least one vector as defined above. It also relates to any pharmaceutical composition comprising at least one p62 derivative as defined above.
• the pharmaceutical compositions according to the invention are very particularly suitable for the treatment of hyperproliferative disorders, such as in particular cancers and restenosis.
• the present invention thus provides a particularly effective method for the destruction of cells, in particular of hyperproliferative cells. It can be used in vitro or ex vivo. Ex vivo, it essentially consists in incubating the cells in the presence of one or more nucleic acids (or of a vector, or cassette or directly of the derivative). In vivo, it consists in administering to the organism an active quantity of a vector (or of a cassette) according to the invention, preferably directly at the level of the site to be treated (tumor in particular).
• the subject of the invention is also a method of destroying hyperproliferative cells comprising bringing said cells or a part of them into contact with a nucleic acid as defined above.
• the present invention is advantageously used in vivo for the destruction of cells in hyperprol iteration (ie in abnormal proliferation). It is thus applicable to the destruction of tumor cells or smooth muscle cells of the vascular wall (restenosis). It is particularly suitable for the treatment of cancers in which an activated oncogene is involved.
• colon adenocarcinomas thyroid cancers, lung carcinomas, myeloid leukemias, colorectal cancers, breast cancers, lung cancers, gastric cancers, cancer of the lungs esophagus, B lymphomas, ovarian cancer, bladder cancer, glioblastoma, hepatocarcinoma, bone, skin, pancreatic cancer, kidney and prostate cancer, etc.
• the products of the invention are also useful for the identification of other partners in oncogenic signaling pathways, by searching for inhibitors, agonists, competitors or molecules interacting in vivo with these products.
• the invention also relates to the antisense sequences, the expression of which in a target cell makes it possible to control the transcription and / or the translation of cellular mRNA coding for p62 or ⁇ p62.
• sequences can for example be transcribed in the target cell into RNA complementary to the cellular mRNA ⁇ p62 or p62 and thus block their translation into protein according to the technique described in patent EP 140 308.
• sequences may consist of all or part of the nucleic sequences SEQ ID No. 1, 2 or 3, transcribed in the reverse orientation .
• the present invention also relates to the use of any compound capable of inducing the expression or the overexpression of ⁇ p62 in a cell for the preparation of a pharmaceutical composition intended for the treatment of hyperproliferative disorders.
• Figure 1 Schematic representation of the structural domains of p62 and ⁇ p62.
• Figure 2 Effect of p62 and ⁇ p62 on transactivation by ras proteins of an RRE derived from the enhancer of the polyome virus.
• Figure 3 Demonstration of cell death induced by ⁇ p62 in NIH3T3 fibroblasts.
• Figure 4 Demonstration of the expression of ⁇ p62 in embryonic fibroblasts treated with different cytotoxic agents and by deprivation of serum.
• Figure 5 Inhibition of the formation of foci induced by oncogenes.
• 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, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.
• the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. 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 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.
• PCR Polymerase-catalyzed Chain Reaction, Saiki RK et al., Science 22 ⁇ (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Enzym. 155 (1987) 335-350]
• DNA thermal cycler Perkin Elmer Cetus
• Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
• Example 1 Isolation of the DNA complementary to ⁇ o62.
• the complementary DNA of ⁇ p62 was isolated by PCR on a population of complementary DNA synthesized from poly A + RNA extracted from human placenta. 1 ⁇ g of DNA was used in conjunction with the primers derived from the sequence of p62 and which cover amino acids 123 to 131 on the one hand (oligo 5 ') and 437 to 443 on the other hand (oligo 3').
• the sequences of these primers are as follows: 5 'oligo: CAGCTGCTGACGGCAGAAATTGAG (SEQ ID No. 4) 3' oligo: TTAATAACGTCCATATGGGTGCTC (SEQ ID No. 5)
• this p62 isoform was confirmed by screening for a DNA library complementary to human placenta established in the vector ⁇ gt 1 1.
• the oligonucleotide used for this screening is a 24-mer corresponding to the specific junction of the deletion present in
• Example 2 Construction of expression vectors of ⁇ p62 and p62-C.
• This example describes the construction of vectors usable for the transfer of the nucleic acids of the invention in vitro or in vivo.
• Plasmid vector
• This vector is a eukaryotic expression vector.
• the nucleic acids coding for the variants p62-C and ⁇ p62 were inserted into this vector in the form of EcoRI fragments. They are thus placed under the control of the promoter of the enhancer of the SV40 virus.
• the vector pcDNA3 (Invitrogen). It is also a eukaryotic expression vector. The nucleic acids coding for the variants p62-C and ⁇ p62, were inserted into this vector in the form of fragments
• the invention resides in the construction and the use of viral vectors allowing the transfer and the expression in vivo of nucleic acids as defined above.
• adenoviruses various serotypes, whose structure and properties vary somewhat, have been characterized.
• serotypes it is preferred to use, in the context of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application WO 94/26914).
• adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service).
• the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example].
• adenoviruses of human or canine or mixed origin are used.
• the defective adenoviruses of the invention comprise ITRs, a sequence allowing the encapsidation and a nucleic acid according to the invention.
• the E1 region at least is non-functional.
• the viral gene considered can be made non-functional by any technique known to a person skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the gene (s) considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents.
• the adenovirus according to the invention comprises a deletion in the E1 and E4 regions.
• it comprises a deletion in region E1 at the level of which the region E4 and the nucleic acid of the invention are inserted (Cf FR94 13355).
• the deletion in the E1 region preferably extends from nucleotides 455 to 3329 on the sequence of the adenovirus Ad5.
• the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the DNA sequence of interest. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
• the cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
• a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%) or lines capable of complementing the E1 and E4 functions as described in particular in applications No. WO 94/26914 and WO95 / 02697. Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
• AAV adeno-associated viruses
• the defective recombinant AAVs according to the invention can be prepared by cotransfection, in a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing a nucleic sequence of the invention of interest bordered by two inverted repeat regions (ITR) of AAV, and of a plasmid carrying the packaging genes (rep and cap genes) of AAV.
• a cell line which can be used is, for example, line 293.
• the recombinant AAVs produced are then purified by conventional techniques.
• retroviruses are integrative viruses, selectively infecting dividing cells. They therefore constitute vectors of interest for cancer applications.
• the genome of retroviruses essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
• gag, pol and env genes are generally deleted, in whole or in part, and replaced by a heterologous nucleic acid sequence of interest.
• retroviruses such as in particular MoMuLV
• murine moloney leukemia virus also designated MoMLV
• MSV murine moloney sarcoma virus
• HaSV harvey sarcoma virus
• SNV spleen necrosis virus
• RSV rous sarcoma virus
• a plasmid comprising in particular the LTRs
• the packaging sequence and said nucleic acid is constructed, then used to transfect a cell line called packaging, capable to provide trans deficient retroviral functions in the plasmid.
• packaging lines are therefore capable of expressing the gag, pol and env genes.
• packaging lines have been described in the prior art, and in particular the line PA317 (US 4,861, 719); the PsiCRIP line (WO 90/02806) and the GP + envAm-12 line (WO 89/07150).
• the recombinant retroviruses may include modifications at the level of the LTRs to suppress transcriptional activity, as well as extended packaging sequences comprising a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639).
• the recombinant retroviruses produced are then purified by conventional techniques.
• a defective recombinant adenovirus or retrovirus indeed have properties which are particularly advantageous for the transfer of genes into tumor cells.
• cationic polymers of polylysine type (LKLK) n, (LKKL) n, immine polyethylene and DEAE dextran or alternatively cationic or lipofectant lipids. They have the property of condensing DNA and promoting its association with the cell membrane.
• lipopolyamines lipofectamine, transfectam, etc.
• DOTMA cationic or neutral lipids
• DOGS DOGS
• DOPE DOPE
• NIH 3T3 fibroblasts were transfected with a reporter gene, that of chloramphenicol acetyl transferase, placed under the control of Ras response elements derived from the enhancer of the polyome virus. These elements are stimulated 15 to 20 times when the cells are cotransfected with an expression vector carrying the cDNA of the SV40 Middle T (MT) oncogene. This stimulation is only slightly affected when a cotransfection provides the expression vectors of p62-C and of ⁇ p62 (see example 2). When cotransfection is performed, no longer with MT but with the activated form of the Ha-ras oncogene (Val 12), the CAT activity is stimulated 30 to 40 times above the basic level.
• MT Middle T
• p62-C and ⁇ p62 almost completely inhibit any activity due to oncogenic Ras.
• the stimulation obtained by cotransfection with the v-src oncogene is strongly inhibited by the proteins p62-C and ⁇ p62, but not by p62.
• Example 4 Demonstration of cell death induced by ⁇ p62 in NIH3T3 fibroblasts ( Figure 3).
• NIH3T3 fibroblasts were transfected with an efficiency of 60% with 5 ⁇ g of ⁇ p62 expression vector (example 2). 24 hours after transfection, the cells show a significant alteration in their viability compared to the control. Analysis of their DNA reveals after migration on agarose gel degradation scales characteristic of apoptosis phenomena. The same phenomena are observed when p62-C is transfected under the same conditions as ⁇ p62.
• Example 5 Demonstration of the expression of ⁇ p62 in embryonic fibroblasts treated with different cytotoxic agents and by deprivation of serum (Fi ⁇ ure 4).
• This example describes another study showing that ⁇ p62 interferes with the transformation process induced by oncogenes. More particularly, this example demonstrates that ⁇ p62 is capable of inhibiting the formation of foci induced by different oncogenes (oncogenic ras, v-src) in NIH-3T3 cells, while p62 does not affect this phenomenon.
• NIH3T3 fibroblasts were co-transfected with 0.1 ⁇ g of v-Src or Ha-Ras Val12 expression vector and with 4 mg of p62, ⁇ p62 or empty expression vector (Example 2).
• the cells were maintained in a medium containing 10% newborn calf serum and the number of outbreaks was determined after fixing and staining of the cells in the presence of fuchsin phenol. The experiments were carried out in triplicate.
• Example 7 Demonstration of an interaction with src in vivo
• NIH3T3 fibroblasts were transfected with a p62 or ⁇ p62 expression vector comprising a myc marker ("myc teg") (Example 2).
• the transfected cells were kept in asynchronous growth or blocked in the mitotic phase by treatment by nocodazole.
• the cells were then co-transfected with a v-Src or empty expression vector. 48 hours later, the cells were lysed and the complexes formed were immunodetected using anti-myc antibodies (antibody 9E10) and anti-Src antibodies (antibody N16).
• NAME RHONE POULENC RORER S.A.
• GCA CCA AGA GCA CGG ACA GCG GGC ATC CAG AGG ATA CCT TTG CCT CCA 960
• GCA CGG ACA GCG
• GGC ATC CAG AGG ATA CCT TTG CCT CCA CCT CCT GCA 480

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• 1995
  • 1995-06-01 FR FR9506533A patent/FR2734826B1/fr not_active Expired - Fee Related
• 1996
  • 1996-05-29 BR BR9608632A patent/BR9608632A/pt not_active Application Discontinuation
  • 1996-05-29 CZ CZ19973759A patent/CZ291533B6/cs not_active IP Right Cessation
  • 1996-05-29 JP JP8536252A patent/JPH11506325A/ja not_active Ceased
  • 1996-05-29 SK SK1613-97A patent/SK283071B6/sk unknown
  • 1996-05-29 US US08/952,899 patent/US6544948B1/en not_active Expired - Fee Related
  • 1996-05-29 ZA ZA964392A patent/ZA964392B/xx unknown
  • 1996-05-29 HU HU9901346A patent/HUP9901346A3/hu unknown
  • 1996-05-29 AU AU61291/96A patent/AU718889B2/en not_active Ceased
  • 1996-05-29 WO PCT/FR1996/000802 patent/WO1996038556A2/fr not_active Application Discontinuation
  • 1996-05-29 CA CA002219861A patent/CA2219861A1/fr not_active Abandoned
  • 1996-05-29 EP EP96918728A patent/EP0828832A2/fr not_active Withdrawn
  • 1996-05-29 KR KR1019970708672A patent/KR19990022193A/ko not_active Application Discontinuation
  • 1996-05-30 IL IL11849396A patent/IL118493A0/xx unknown
• 1997
  • 1997-11-25 NO NO975409A patent/NO975409L/no not_active Application Discontinuation

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