WO2001062937A1 - Skin infection-related retroviral sequences - Google Patents

Skin infection-related retroviral sequences Download PDF

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Publication number
WO2001062937A1
WO2001062937A1 PCT/FR2001/000543 FR0100543W WO0162937A1 WO 2001062937 A1 WO2001062937 A1 WO 2001062937A1 FR 0100543 W FR0100543 W FR 0100543W WO 0162937 A1 WO0162937 A1 WO 0162937A1
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Prior art keywords
seq
sequence
nucleotide
nucleic acid
polypeptide
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PCT/FR2001/000543
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French (fr)
Inventor
Jean-Jacques Guilhou
Jean-Pierre Moles
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Guilhou Jean Jacques
Moles Jean Pierre
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Application filed by Guilhou Jean Jacques, Moles Jean Pierre filed Critical Guilhou Jean Jacques
Priority to AU37490/01A priority Critical patent/AU3749001A/en
Publication of WO2001062937A1 publication Critical patent/WO2001062937A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the demonstration of retroviral nucleotide sequences associated with skin conditions, such as psoriasis.
  • Psoriasis is a frequent pathology (2 to 3% of the Caucasian population), cutaneous, chronic, of unknown origin and characterized by a genetic predisposition, an epidermal hyperproliferation associated with abnormalities in the differentiation of keratinocytes, and abnormalities of the local or systemic immunity close to that observed in autoimmune pathologies.
  • keratinocyte hyperproliferation is due to the activation of psoriatic lymphocytes.
  • the origin of this activation remains unknown.
  • retroviruses Guilhou et al, 1978 and Guilhou, 1981.
  • sequences correspond to a partial sequence of two human retroviruses (namely a virus being hosted by a human) coding, on the one hand, for the protease and the terminal NH 2 part of the RNA-dependent DNA polymerase called PsoRVl and, on the other hand, for the envelope called PsoRV2.
  • These retroviruses are endogenous retroviruses since their presence in genomic DNA has been detected not only in all patients with psoriasis but also in healthy subjects.
  • the authors of the present invention have further shown that homologous retroviral nucleotide sequences, in particular those relating to the PsoRVl polymerase gene, are also present in other skin diseases.
  • PsoRV designates any pathogenic agent, associated with the pathologies mentioned above, in particular a viral species, the attenuated strains of said viral species, or the detecting particles which are interfering or containing co-encapsidated genomes or else genomes recombined with part of the PsoRV genome, derived from this species. It is known that viruses and more particularly viruses containing RNA have a variability, in particular following high rates of spontaneous mutation, which will be taken into account in the context of the present invention.
  • the invention therefore relates to the nucleotide sequences of the PsoRV1 and PsoRV2 retroviruses, and the polypeptides encoded by these sequences, it being understood that the equivalents of these sequences also form part of the invention.
  • Two nucleotide or peptide sequences are said to be equivalent or derived from one another, or from a reference sequence, if functionally the corresponding biopolymers can play substantially the same role, without being identical, with respect to 'application or use considered, or in the technique in which they occur.
  • Two sequences obtained in particular due to natural variability, in particular spontaneous mutation of the species from which they have been identified or induced, are equivalent, as are two homologous sequences, the homology being defined below.
  • both DNA and viral RNA will be used to characterize the sequences relating to a virus having such reverse transcriptase activity, called PsoRV according to the present description.
  • sequence listing is appended to this description.
  • the sequence SEQ 1D n ° 1 represents the nucleotide sequence of PsoRVl.
  • sequence SEQ ID No. 2 represents the nucleotide sequence of PsoRV2.
  • sequences SEQ ID n ° 1 and n ° 2 being retroviral sequences, they can be read in the six possible reading phases and can moreover be overlapping with respect to each other
  • sequence SEQ ID No. 3 represents the peptide sequence of the protease of PsoRV1 corresponding to reading frame 1 of the nucleotide sequence SEQ ID No. 1 (base pairs No. 1 to 291).
  • sequence SEQ ID No. 4 represents the peptide sequence of the PsoRV1 polymerase corresponding to the reading frame 3 of the nucleotide sequence SEQ ID No. 1 (base pairs No. 207 to 950).
  • sequence SEQ ID No. 5 represents the peptide sequence of the envelope of PsoRV2a corresponding to the reading frame 3 of the sequence SEQ ID No. 2. (base pairs # 3 to 260).
  • sequences SEQ ID No. 6 to 14 are oligonucleotide primers, as described in the examples below.
  • the sequence SEQ ID No. 15 represents the nucleotide sequence of PsoRV2b, which can be considered as a variant of PsoRV2a.
  • sequence SEQ ID No. 16 is the corresponding amino acid sequence.
  • the subject of the invention is therefore an isolated nucleic acid, the sequence of which is chosen from SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 15, or the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID # 1, or going from nucleotide 207 to nucleotide 950 on SEQ ID # 1, or going from nucleotide 3 to nucleotide 260 on SEQ ID # 2, or a homologous sequence, defined as i) a sequence identical to at least 80% of the SEQ sequence
  • SEQ ID no 15 or the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID no 1, or going from nucleotide 207 to nucleotide 950 on SEQ ID no 1, or going from nucleotide 3 to nucleotide 260 on SEQ ID No. 2, or their complementary sequences, under stringent hybridization conditions.
  • a homologous nucleotide sequence according to the invention is identical to at least 85% of the sequences SEQ ID No. 1 or No. 2 or No. 15 or of their fragments indicated above, more preferably at least 90%, or at least 95%.
  • such a homologous nucleotide sequence specifically hybridizes to the sequences complementary to the sequences SEQ ID No. 1 or No. 2 or No. 15 or their fragments indicated above, under stringent conditions.
  • the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
  • Tm 81.5 + 0.41 (% G + C) +16.6Log (cation concentration) - 0.63 (% formamide) - ( 600 / number of bases) (Sambrook et al., 1989).
  • Tm 4 (G + C) + 2 (A + T).
  • the hybridization temperature is approximately 5 to 30 ° C, preferably 5 to 10 ° C below Tm, and the hybridization buffers used are preferably solutions of high ionic strength such as a 6xSSC solution for example.
  • the homologous nucleotide sequences of interest include any nucleotide sequence which differs from the sequences SEQ ID No. 1 or No. 2 or No. 15 or from their fragments indicated above by mutation, insertion, deletion or substitution of one or more bases , or by the degeneration of the genetic code.
  • the present invention also relates to an isolated polypeptide, comprising the amino acid sequence SEQ ID No. 3 to SEQ ID No. 5 or No. 16, or a homologous sequence, defined as i) a sequence identical to at least 80 % of the sequence SEQ ID No. 3 to No. 5 or No. 16; or ii) a sequence encoded by a nucleic acid sequence hybridizing with the sequence SEQ ID No. 1 or 2 or No. 15 or their fragments indicated above, or its complementary sequence, under stringent hybridization conditions. More generally, by “homologous amino acid sequence” is meant any amino acid sequence which differs from the sequence SEQ ID No. 3 to 5 or No.
  • substitutions are preferably conservative substitutions, that is to say substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid); amino acids with apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan, and cysteine).
  • such a homologous amino acid sequence is identical to at least 85% of the sequence SEQ ID No. 3 to No. 5 or No. 16, preferably at least 90%, more preferably at least 95%.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705. Similar amino acid sequences are aligned to obtain the maximum degree of homology (ie identity). To this end, it may be necessary to introduce artificially spaces ("gaps") in the sequence. Once the optimal alignment has been achieved, the degree of homology (ie identity) is established by recording all the positions for which the amino acids of the two sequences compared are identical, relative to the total number of positions.
  • polypeptide of the present invention can be synthesized by any method well known to those skilled in the art.
  • the polypeptide of the invention can for example be synthesized by the techniques of synthetic chemistry, such as Merrifield-type synthesis which is advantageous for reasons of purity, antigenic specificity, absence of unwanted side products and for its ease of production.
  • Recombinant polypeptides can also be produced by a method, in which a vector containing a nucleic acid comprising the sequence SEQ ID No. 1 or 2 or No. 15 or their fragments indicated above, or a homologous sequence is transferred into a host cell which is cultured under conditions allowing expression of the corresponding polypeptide.
  • the polypeptide produced can then be recovered and purified.
  • the purification methods used are known to those skilled in the art.
  • the recombinant polypeptide obtained can be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific mono- or polyclonal antibodies, etc.
  • the nucleic acid sequence of interest can be inserted into a cloning vector or an expression vector, in which it is operably linked to elements allowing the regulation of its expression, such as in particular promoters, activators and / or transcription terminators.
  • the signals controlling the expression of the nucleotide sequences are chosen as a function of the cell host used.
  • the nucleotide sequences according to the invention can be inserted into autonomous replication vectors within the chosen host, or integrative vectors of the chosen host.
  • Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example electroporation or precipitation with calcium phosphate.
  • the cloning and / or expression vectors as described above, containing one of the nucleotide sequences defined according to the invention also form part of the present invention.
  • the invention further relates to host cells transfected, transiently or stable, with these expression vectors.
  • These cells can be obtained by introducing into host cells, prokaryotic or eukaryotic, a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.
  • host cells examples include mammalian cells, insect cells, bacteria or yeast strains.
  • the different nucleotide sequences of the invention can be of artificial origin or not. They may be DNA or RNA sequences, obtained by screening of sequence banks using probes prepared on the basis of sequences SEQ ID No. 1 or 2 or No. 15. Such libraries can be prepared by conventional molecular biology techniques known to those skilled in the art.
  • nucleotide sequences according to the invention can also be prepared by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening libraries.
  • nucleotide sequences allow the production of probes or primers, hybridizing specifically with a sequence SEQ ID No. 1 or 2 or No. 15 or their fragments, according to the invention, or its strand complementary.
  • the appropriate hybridization conditions correspond to the temperature and ionic strength conditions usually used by those skilled in the art, preferably under stringent conditions as defined above.
  • the subject of the present invention is therefore the use of a nucleic acid as defined above for the manufacture of nucleic probes or oligonucleotide primers, capable of specifically hybridizing, under stringent conditions, with all or part of said nucleic acid such as defined above.
  • the present invention also relates to a nucleic acid having at least 10 nucleotides, which specifically hybridizes with one of the nucleic acid sequences SEQ ID No. 1 or 2 or No. 15 or their fragments, or its complement, in stringent hybridization conditions.
  • nucleic acids can be used as a probe for the detection of a retroviral sequence associated in particular with psoriasis, lichen and atopy.
  • These nucleic acids of the invention useful as probes comprise at least 10 nucleotides, preferably at least 20 nucleotides, more preferably at least 100 nucleotides.
  • the nucleic acids of the invention can be useful as primers for the amplification of all or part of a retroviral sequence associated in particular with psoriasis, lichen and atopy.
  • These primers preferably comprise in this case at least 10 nucleotides, preferably at least 14 nucleotides, and preferably less than 40 nucleotides. It is possible, for example, to use as primers, for amplification (for example by PCR), the nucleic acids consisting of the sequences SEQ ID No. 6 to 14.
  • the probes or primers of the invention are marked, prior to their use.
  • several techniques are within the reach of those skilled in the art such as, for example, fluorescent, radioactive, chemiluminescent or enzymatic labeling.
  • the probes can also be used as capture probes, immobilized on a support solid by any suitable means, directly or indirectly, for example by covalence or passive adsorption.
  • the subject of the invention is also a method of in vitro detection of the presence of a retroviral nucleic sequence associated with a skin condition in a patient, comprising the steps consisting in: ai) isolating the DNA or RNA from a biological sample; b1) optionally producing cDNA from said RNA; d) bringing said DNA or cDNA into contact with nucleic acids as defined above, as primers, under conditions allowing the specific amplification of a sequence SEQ ID No. 1 or
  • the invention further relates to a method of in vitro diagnosis of a skin condition, such as in particular psoriasis, lichen or atopic eczema, in a patient, in which the presence of 'a retroviral nucleic acid sequence associated with one of these pathologies, by means of the detection method described above.
  • biological sample is meant in particular a sample of the biological fluid, living tissue, tissue fragment, mucus, organ or organ fragment type or any culture supernatant obtained using a sample.
  • biological fluid is meant in particular serum, plasma, urine or cerebrospinal fluid.
  • the probes used for diagnostic purposes of the invention can be used in all known techniques, and in particular the techniques known as “DOT-BLOT”, “SOUTHERN-BLOT”, “NORTHERN-BLOT” and the “SANDWICH technique ".
  • the "SANDWICH” technique is used in the present invention, comprising a specific capture probe and / or a specific detectably labeled probe, it being understood that the capture probe and the detection probe must have at least one nucleotide sequence partially different.
  • the subject of the invention is also antibodies directed against the polypeptides as defined above.
  • Polyclonal antibodies may be poly- or monoclonal antibodies or their fragments, chimeric antibodies, in particular humanized or immunoconjugated.
  • Polyclonal antibodies can be obtained from the serum of an animal immunized against a polypeptide according to the usual procedures.
  • an appropriate peptide fragment can be used as the antigen, which can be coupled via a reactive residue to a protein or another peptide.
  • Rabbits are immunized with the equivalent of 1 mg of the peptide antigen according to the procedure described by Benoit et al. (1982). At four-week intervals, the animals are treated with injections of 200 ⁇ g of antigen and bled 10-14 days later. After the third injection, the antiserum is examined to determine its capacity to bind to the antigen peptide radiolabelled with iodine, prepared by the chloramine-T method and is then purified by chromatography on an ion exchange column carboxymethyl cellulose (CMC). Antibody molecules are then collected from mammals and isolated to the desired concentration by methods well known to those skilled in the art.
  • CMC carboxymethyl cellulose
  • the antibodies can be purified by immunoaffinity chromatography using solid phase immunizing polypeptides.
  • the antibody is brought into contact with the immunizing polypeptide in solid phase for a sufficient time so as to make the polypeptide immunoreact with the antibody molecule in order to form an immunological complex in solid phase.
  • the monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by Kôhler and Milstein (1975).
  • the antibodies or antibody fragments of the invention can be, for example, chimeric antibodies, humanized antibodies, Fab and F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies.
  • the antibodies of the invention in particular the monoclonal antibodies, can in particular be used for the analysis by immunohistochemistry of the polypeptides on sections of specific tissues, for example by immunofluorescence, gold labeling, immunoperoxidase ...
  • the antibodies thus
  • the products can advantageously be used in any situation where the expression of the polypeptides must be observed.
  • the invention also relates to the use of at least one antibody thus produced for the detection or purification of a polypeptide as defined above in a biological sample.
  • the invention relates to an in vitro method for detecting or measuring the level of expression of the PsoRV1 or PsoRV2 polypeptides as defined above in a biological sample comprising the contacting of at least one antibody as defined previously with said biological sample under conditions allowing the possible formation of specific immunological complexes between the PsoRV1 or PsoRV2a or PsoRV2b polypeptides and the said antibody or antibodies and the detection of any specific immunological complexes possibly formed.
  • the invention also relates to a kit for the implementation of this method comprising:
  • the subject of the invention is also a pharmaceutical composition
  • a nucleic acid as defined above in association with a pharmaceutically acceptable vehicle, said nucleic acid being capable of hybridizing in vivo or in vitro on RNA and / or on PsoRV1 or PsoRV2 DNA (a or b) to block the replication phenomena, in particular translation and / or transcription, and / or to degrade said DNA and / or RNA.
  • the composition is intended for use in gene therapy.
  • the nucleic acid preferably inserted into a generally viral vector (such as adenoviruses and retroviruses) can be administered in naked form, free of any vehicle promoting transfer to the target cell, such as anionic liposomes, cationic lipids, microparticles, for example gold microparticles, precipitating agents, for example calcium phosphate, or any other agent which facilitates transfection.
  • the polynucleotide can be simply diluted in a physiologically acceptable solution, such as a sterile solution or a sterile buffer solution, in the presence or in the absence of a vehicle.
  • a nucleic acid of the invention can be combined with agents which facilitate transfection. It can be, inter alia, (i) associated with a chemical agent which modifies cell permeability such as bupivacaine; (ii) encapsulated in liposomes, possibly in the presence additional substances that facilitate transfection; or (iii) associated with cationic lipids or microparticles of silica, gold or tungsten.
  • agents which facilitate transfection can be, inter alia, (i) associated with a chemical agent which modifies cell permeability such as bupivacaine; (ii) encapsulated in liposomes, possibly in the presence additional substances that facilitate transfection; or (iii) associated with cationic lipids or microparticles of silica, gold or tungsten.
  • nucleic acid constructs of the invention cover microparticles, these can be injected intradermally or intraepidermally by the gene gun technique, "gene gun” (WO 94/24263).
  • the amount to be used as a drug depends in particular on the construction of nucleic acid itself, the individual to whom this nucleic acid is administered, the mode of administration and the type of formulation, and the pathology.
  • a therapeutically or prophylactically effective amount varying from approximately 0.1 ⁇ g to approximately 1 mg, preferably from approximately 1 ⁇ g to approximately 800 ⁇ g and, preferably from approximately 25 ⁇ g to approximately 250 ⁇ g, can be administered to human adults.
  • the nucleic acid constructs of the invention can be administered by any conventional route of administration.
  • the choice of route of administration depends in particular on the formulation chosen. Skin application and intradermal injection are preferred, however.
  • a subject of the invention is also a method of therapeutic treatment of a skin condition in which an effective quantity of a nucleic acid blocking the expression of the sequences PsoRV1 or PsoRV2a or PsoRV2b is administered to a patient in need of such treatment.
  • the target patient is generally a human being, but the application can also be extended to any mammal if necessary.
  • Ex vivo therapy can also be implemented.
  • the invention more specifically provides an in vitro method for preparing a cell assembly intended to be implanted in a recipient patient, comprising the incorporation of a nucleotide sequence of PsoRV1 or PsoRV2a or PsoRV2b as defined above in cells in culture .
  • the targeted cells can be autologous cells of the recipient patient, but also heterologous cells (from another donor of the same species) or xenologists (from a donor of another animal species). It may preferably be skin cells, more particularly keratinocytes.
  • nucleotide sequences can be incorporated by any transfer technique known to a person skilled in the art, by “gun gene”, in naked form, or in association with anionic liposomes, cationic lipids, microparticles, for example microparticles of or, or any other agent facilitating transfection.
  • the nucleotide sequences are preferably inserted beforehand in a vector, of the retrovirus or adenovirus type for example.
  • the methods of implantation of the modified cells depend in particular on the type of cells and the disease targeted.
  • Another object of the invention is a method of surgical treatment in which the cells thus modified are implanted in a recipient patient requiring such treatment.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide as defined above, in association with a pharmaceutically acceptable vehicle.
  • a pharmaceutical composition of this type can be particularly useful for stimulating or abrogating the immune responses of the patient vis-à-vis the endogenous PsoRV1 and PsoRV2a or PsoRV2b polypeptides, in the context of vaccination or desensitization, respectively.
  • the invention also relates to a pharmaceutical composition comprising an antibody as defined above, preferably a monoclonal antibody, where appropriate humanized, in association with a pharmaceutically acceptable vehicle.
  • An antibody-based composition according to the invention may be particularly useful for inactivating the PsoRV1 or PsoRV2a or PsoRV2b polypeptides in the context of a pathology involving these polypeptides, such as skin diseases.
  • the modes of administration, the dosages and the galenical forms of the pharmaceutical compositions according to the invention, containing at least one antibody or a polypeptide can be determined in the usual way by a person skilled in the art, in particular according to the criteria generally taken into account. for the establishment of a therapeutic treatment adapted to a patient, such as for example the age or the body weight of the patient, the seriousness of his general state, the tolerance to the treatment, and the observed side effects, etc.
  • a therapeutically or prophylactically effective amount of said antibody or polypeptide varying from about 0.1 ⁇ g to about 1 mg can be administered to human adults.
  • the pathologies targeted are all those in which the expression of PsoRV1 or PsoRV2a or PsoRV2b is observed.
  • Skin diseases are more particularly targeted, and among these in particular dermatoses with an immune component.
  • psoriasis As examples of the pathologies concerned, mention may be made of psoriasis, lichen, atopic eczema, lupus erythematosus, and skin cancers (carcinomas), including for example basal cell carcinoma and squamous cell carcinoma.
  • the subject of the invention is finally the use of a PsoRV1 or PsoRV2a or PsoRV2b polypeptide as defined above or of a nucleic acid coding for the polypeptide, for the screening of compounds (isolated or in mixture) capable of inhibiting the activity of said polypeptide.
  • a method for screening for compounds, isolated or as a mixture, capable of inhibiting the PsoRVl polymerase can thus be implemented, method comprising:
  • inhibitors of other known polymerases such as nucleotide analogs.
  • Clone 58-1 3 was obtained by reverse transcription and degenerate PCR reaction according to the method published by Tuke et al, (1997) from total RNA extracted from biopsy sections of lesional skin of patients with psoriasis and cloning. in the vector pCR® 2.1 -Topo according to the conditions recommended by the manufacturer (TOPO TM TA-cloning® system; Invitrogen, The Netherlands).
  • the clone sequence using the "DyeTerminator sequencing kit" (Perkin-Elmer, France) and an AB1373 sequencer (Applied Biosystems, USA) made it possible to define a specific primer for PsoRV, called 58-1 3 (SEQ ID No. 6) .
  • CONS SEQ ID No. 7 The clone CONS-58-13 was obtained by reverse transcription and PCR using the primers defined below and identified as the sequences SEQ ID No. 6 and SEQ ID No. 7, from total RNAs extracted from sections of lesional skin biopsies of patients with psoriasis and cloning into the vector pCR® 2.1 -Topo according to the conditions recommended by the manufacturer (TOPO TM TA-cloning® system; Invitrogen, The Netherlands).
  • primer 58-13 SEQ ID n ° 6:
  • the search for the expression of the PsoRVl sequence was obtained by reverse transcription techniques from total RNAs extracted from biopsy sections of lesional skin of patients suffering from psoriasis.
  • the cDNA synthesis was carried out with an oligo dT 16 primer and reverse transcriptase (GIBCO-BRL) according to the conditions recommended by the supplier.
  • a PCR was then carried out using the primers defined from the sequence of the clone CONS-58-13. Two pairs of primers were used: - the first pair called WALE-PIVA, SEQ ID n ° 8 and SEQ
  • Table 1 represents the frequency of detection of the expression of PsoRVl in biopsies of lesions of patients with psoriasis, lichen or atopy, or basal or squamous cell carcinoma, and in controls on healthy subjects.
  • Env-PsoRV2a clone coding for the envelope of the retrovirus The clones Env-PsoRV2-5 'and Env-PsoRV2-3' were obtained by reverse transcription and degenerate PCR reaction using primers conserved within the HERV-K family, identified by the sequences Env5 'SEQ ID n ° 11, LTR3 'SEQ ID n ° 12, Env3' SEQ ID n ° 13 and LTR5 'SEQ ID n ° 14, from total RNA extracted from biopsy sections of lesional skin of patients with psoriasis. The PCR products were then cloned and sequenced according to the methods described in Example 1.

Abstract

The invention concerns the isolation of retroviral nucleotide sequences called PsoRV1 and PsoRV2, associated with skin infections, in particular with psoriasis, and their diagnostic and therapeutic uses.

Description

SEQUENCES RETROVIRALES ASSOCIEES A DES AFFECTIONS CUTANEES RETROVIRAL SEQUENCES ASSOCIATED WITH SKIN CONDITIONS
L'invention a trait à la mise en évidence de séquences nucleotidiques rétrovirales associées à des affections cutanées, telles que le psoriasis.The invention relates to the demonstration of retroviral nucleotide sequences associated with skin conditions, such as psoriasis.
Le psoriasis est une pathologie fréquente (2 à 3 % de la population caucasienne), cutanée, chronique, d'origine inconnue et caractérisée par une prédisposition génétique, une hyperprolifération épidermique associée à des anomalies de la différenciation des kératinocytes, et des anomalies de l'immunité locale ou systémique proches de celles observées dans les pathologies auto-immunes. Récemment, il a été démontré que l'hyperprolifération kératinocytaire était due à l'activation des lymphocytes psoriasiques. Mais, l'origine de cette activation reste inconnue. Une théorie antérieure suggérait que cette activation soit générée par la présence de rétrovirus (Guilhou et al, 1978 et Guilhou, 1981 ). La présence de particules ressemblant à des rétrovirus a par ailleurs été observée dans les fluides biologiques de patients atteints de psoriasis (Guilhou et al, 1982 ; Iversen, 1983 et Dalen et al, 1983), sans qu'une activité transcriptase inverse ne puisse être démontrée. Les auteurs de la présente invention sont maintenant parvenus à caractériser des séquences nucleotidiques spécifiquement exprimées dans les lésions de patients atteints de psoriasis. Ces séquences correspondent à une séquence partielle de deux rétrovirus humains (à savoir un virus étant hébergé par un humain) codant, d'une part, pour la protéase et la partie NH2 terminale de l'ADN polymérase ARN-dépendante dénommée PsoRVl et, d'autre part, pour l'enveloppe dénommée PsoRV2. Ces rétrovirus sont des rétrovirus endogènes puisque leur présence dans l'ADN genomique a pu être détectée non seulement chez tous les patients atteints de psoriasis mais aussi chez les sujets sains. Les auteurs de la présente invention ont en outre montré que des séquences nucleotidiques rétrovirales homologues, en particulier celles concernant le gène de la polymérase de PsoRVl , étaient également présentes dans d'autres maladies de la peau. Le terme "PsoRV" utilisé dans la présente description désigne tout agent pathogène, associé aux pathologies citées ci-dessus, notamment une espèce virale, les souches atténuées de ladite espèce virale, ou les particules détectives interférentes ou contenant des génomes co-encapsidés ou encore des génomes recombinés avec une partie du génome PsoRV, dérivées de cette espèce. Il est connu que les virus et plus particulièrement les virus contenant de l'ARN ont une variabilité, consécutive notamment à des taux élevés de mutation spontanée, dont on tiendra compte dans le cadre de la présente invention. L'invention porte donc sur les séquences nucleotidiques des rétrovirus PsoRVl et PsoRV2, et les polypeptides codés par ces séquences, étant entendu que font également partie de l'invention les équivalents de ces séquences.Psoriasis is a frequent pathology (2 to 3% of the Caucasian population), cutaneous, chronic, of unknown origin and characterized by a genetic predisposition, an epidermal hyperproliferation associated with abnormalities in the differentiation of keratinocytes, and abnormalities of the local or systemic immunity close to that observed in autoimmune pathologies. Recently, it has been shown that keratinocyte hyperproliferation is due to the activation of psoriatic lymphocytes. However, the origin of this activation remains unknown. A previous theory suggested that this activation is generated by the presence of retroviruses (Guilhou et al, 1978 and Guilhou, 1981). The presence of particles resembling retroviruses has also been observed in the biological fluids of patients suffering from psoriasis (Guilhou et al, 1982; Iversen, 1983 and Dalen et al, 1983), without any reverse transcriptase activity being able to be demonstrated. The authors of the present invention have now succeeded in characterizing nucleotide sequences specifically expressed in the lesions of patients suffering from psoriasis. These sequences correspond to a partial sequence of two human retroviruses (namely a virus being hosted by a human) coding, on the one hand, for the protease and the terminal NH 2 part of the RNA-dependent DNA polymerase called PsoRVl and, on the other hand, for the envelope called PsoRV2. These retroviruses are endogenous retroviruses since their presence in genomic DNA has been detected not only in all patients with psoriasis but also in healthy subjects. The authors of the present invention have further shown that homologous retroviral nucleotide sequences, in particular those relating to the PsoRVl polymerase gene, are also present in other skin diseases. The term "PsoRV" used in the present description designates any pathogenic agent, associated with the pathologies mentioned above, in particular a viral species, the attenuated strains of said viral species, or the detecting particles which are interfering or containing co-encapsidated genomes or else genomes recombined with part of the PsoRV genome, derived from this species. It is known that viruses and more particularly viruses containing RNA have a variability, in particular following high rates of spontaneous mutation, which will be taken into account in the context of the present invention. The invention therefore relates to the nucleotide sequences of the PsoRV1 and PsoRV2 retroviruses, and the polypeptides encoded by these sequences, it being understood that the equivalents of these sequences also form part of the invention.
Deux séquences nucleotidiques ou peptidiques sont dites équivalentes ou dérivées l'une par rapport à l'autre, ou par rapport à une séquence de référence, si fonctionnellement les biopolymères correspondants peuvent jouer sensiblement le même rôle, sans être identiques, vis à vis de l'application ou utilisation considérée, ou dans la technique dans laquelle elles interviennent. Sont notamment équivalentes deux séquences obtenues du fait de la variabilité naturelle, notamment mutation spontanée de l'espèce à partir de laquelle elles ont été identifiées ou induites, ainsi que deux séquences homologues, l'homologie étant définie ci-après.Two nucleotide or peptide sequences are said to be equivalent or derived from one another, or from a reference sequence, if functionally the corresponding biopolymers can play substantially the same role, without being identical, with respect to 'application or use considered, or in the technique in which they occur. Two sequences obtained in particular due to natural variability, in particular spontaneous mutation of the species from which they have been identified or induced, are equivalent, as are two homologous sequences, the homology being defined below.
Etant donné qu'un virus possédant une activité enzymatique transcriptase inverse peut être génétiquement caractérisé aussi bien sous forme d'ARN que d'ADN, il sera fait mention aussi bien de l'ADN que de l'ARN viral pour caractériser les séquences relatives à un virus possédant une telle activité transcriptase inverse, dit PsoRV selon la présente description.Since a virus possessing reverse transcriptase enzymatic activity can be genetically characterized in the form of both RNA and DNA, both DNA and viral RNA will be used to characterize the sequences relating to a virus having such reverse transcriptase activity, called PsoRV according to the present description.
Un listage de séquences est annexé à la présente description. La séquence SEQ 1D n° 1 représente la séquence nucléotidique de PsoRVl .A sequence listing is appended to this description. The sequence SEQ 1D n ° 1 represents the nucleotide sequence of PsoRVl.
La séquence SEQ ID n° 2 représente la séquence nucléotidique de PsoRV2. Les séquences SEQ ID n°1 et n°2 étant des séquences rétrovirales, elles peuvent être lues dans les six phases de lecture possibles et peuvent en outre être chevauchantes les unes par rapport aux autresThe sequence SEQ ID No. 2 represents the nucleotide sequence of PsoRV2. The sequences SEQ ID n ° 1 and n ° 2 being retroviral sequences, they can be read in the six possible reading phases and can moreover be overlapping with respect to each other
La séquence SEQ ID n° 3 représente la séquence peptidique de la protéase de PsoRVl correspondant au cadre de lecture 1 de la séquence nucléotidique SEQ ID n°1 (paires de bases n°1 à 291 ).The sequence SEQ ID No. 3 represents the peptide sequence of the protease of PsoRV1 corresponding to reading frame 1 of the nucleotide sequence SEQ ID No. 1 (base pairs No. 1 to 291).
La séquence SEQ ID n° 4 représente la séquence peptidique de la polymérase de PsoRVl correspondant au cadre de lecture 3 de la séquence nucléotidique SEQ ID n°1 (paires de bases n°207 à 950). La séquence SEQ ID n° 5 représente la séquence peptidique de l'enveloppe de PsoRV2a correspondant au cadre de lecture 3 de la séquence SEQ ID n°2. (paires de bases n°3 à 260).The sequence SEQ ID No. 4 represents the peptide sequence of the PsoRV1 polymerase corresponding to the reading frame 3 of the nucleotide sequence SEQ ID No. 1 (base pairs No. 207 to 950). The sequence SEQ ID No. 5 represents the peptide sequence of the envelope of PsoRV2a corresponding to the reading frame 3 of the sequence SEQ ID No. 2. (base pairs # 3 to 260).
Les séquences SEQ ID n° 6 à 14 sont des amorces oligonucléotidiques, telles que décrites dans les exemples ci-après. La séquence SEQ ID n° 15 représente la séquence nucléotidique de PsoRV2b, qui peut être considéré comme un variant de PsoRV2a.The sequences SEQ ID No. 6 to 14 are oligonucleotide primers, as described in the examples below. The sequence SEQ ID No. 15 represents the nucleotide sequence of PsoRV2b, which can be considered as a variant of PsoRV2a.
La séquence SEQ ID n° 16 est la séquence d'acides aminés correspondante.The sequence SEQ ID No. 16 is the corresponding amino acid sequence.
L'invention a donc pour objet un acide nucléique isolé, dont la séquence est choisie parmi SEQ ID n° 1 , SEQ ID n° 2 ou SEQ ID n° 15, ou la séquence des fragments allant du nucleotide 1 au nucleotide 291 sur SEQ ID n°1 , ou allant du nucleotide 207 au nucleotide 950 sur SEQ ID n°1 , ou allant du nucleotide 3 au nucleotide 260 sur SEQ ID n°2, ou une séquence homologue, définie comme i) une séquence identique à au moins 80 % de la séquence SEQThe subject of the invention is therefore an isolated nucleic acid, the sequence of which is chosen from SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 15, or the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID # 1, or going from nucleotide 207 to nucleotide 950 on SEQ ID # 1, or going from nucleotide 3 to nucleotide 260 on SEQ ID # 2, or a homologous sequence, defined as i) a sequence identical to at least 80% of the SEQ sequence
ID n° 1 , SEQ ID n° 2 ou SEQ ID n° 15 ou de la séquence des fragments allant du nucleotide 1 au nucleotide 291 sur SEQ ID n°1 , ou allant du nucleotide 207 au nucleotide 950 sur SEQ ID n°1 , ou allant du nucleotide 3 au nucleotide 260 sur SEQ !D n°2, ii) une séquence hybridant avec la séquence SEQ ID n° 1 , SEQID no.1, SEQ ID no.2 or SEQ ID no.15 or of the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID no.1, or going from nucleotide 207 to nucleotide 950 on SEQ ID no.1 , or going from nucleotide 3 to nucleotide 260 on SEQ! D n ° 2, ii) a sequence hybridizing with the sequence SEQ ID n ° 1, SEQ
ID n° 2, SEQ ID n° 15 ou la séquence des fragments allant du nucleotide 1 au nucleotide 291 sur SEQ ID n°1 , ou allant du nucleotide 207 au nucleotide 950 sur SEQ ID n°1 , ou allant du nucleotide 3 au nucleotide 260 sur SEQ ID n°2, ou leurs séquences complémentaires, dans des conditions stringentes d'hybridation.ID no 2, SEQ ID no 15 or the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID no 1, or going from nucleotide 207 to nucleotide 950 on SEQ ID no 1, or going from nucleotide 3 to nucleotide 260 on SEQ ID No. 2, or their complementary sequences, under stringent hybridization conditions.
De préférence, une séquence nucléotidique homologue selon l'invention est identique à au moins 85 % des séquences SEQ ID n° 1 ou n°2 ou n° 15 ou de leurs fragments indiqués ci-dessus, de préférence encore au moins 90 %, ou au moins 95 %.Preferably, a homologous nucleotide sequence according to the invention is identical to at least 85% of the sequences SEQ ID No. 1 or No. 2 or No. 15 or of their fragments indicated above, more preferably at least 90%, or at least 95%.
De manière préférentielle, une telle séquence nucléotidique homologue hybride spécifiquement aux séquences complémentaires des séquences SEQ ID n° 1 ou n°2 ou n° 15 ou de leurs fragments indiqués ci- dessus, dans des conditions stringentes. Les paramètres définissant les conditions de stringence dépendent de la température à laquelle 50% des brins appariés se séparent (Tm).Preferably, such a homologous nucleotide sequence specifically hybridizes to the sequences complementary to the sequences SEQ ID No. 1 or No. 2 or No. 15 or their fragments indicated above, under stringent conditions. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
Pour les séquences comprenant plus de 30 bases, Tm est définie par la relation : Tm=81 , 5+0,41 (%G+C)+16, 6Log(concentration en cations) - 0,63(%formamide) -(600/nombre de bases) (Sambrook et al., 1989).For sequences comprising more than 30 bases, Tm is defined by the relationship: Tm = 81.5 + 0.41 (% G + C) +16.6Log (cation concentration) - 0.63 (% formamide) - ( 600 / number of bases) (Sambrook et al., 1989).
Pour les séquences de longueur inférieure à 30 bases, Tm est définie par la relation : Tm= 4(G+C) + 2 (A+T).For sequences of length less than 30 bases, Tm is defined by the relation: Tm = 4 (G + C) + 2 (A + T).
Dans des conditions de stringence appropriées, auxquelles les séquences aspécifiques n'hybrident pas, la température d'hybridation est approximativement de 5 à 30°C, de préférence de 5 à 10°C en dessous de Tm, et les tampons d'hybridation utilisés sont de préférence des solutions de force ionique élevée telle qu'une solution 6xSSC par exemple.Under appropriate stringency conditions, to which the aspecific sequences do not hybridize, the hybridization temperature is approximately 5 to 30 ° C, preferably 5 to 10 ° C below Tm, and the hybridization buffers used are preferably solutions of high ionic strength such as a 6xSSC solution for example.
Les séquences nucleotidiques homologues d'intérêt incluent toute séquence nucléotidique qui diffère des séquences SEQ ID n° 1 ou n°2 ou n° 15 ou de leurs fragments indiqués ci-dessus par mutation, insertion, délétion ou substitution d'une ou plusieurs bases, ou par la dégénérescence du code génétique.The homologous nucleotide sequences of interest include any nucleotide sequence which differs from the sequences SEQ ID No. 1 or No. 2 or No. 15 or from their fragments indicated above by mutation, insertion, deletion or substitution of one or more bases , or by the degeneration of the genetic code.
La présente invention a également pour objet un polypeptide isolé, comprenant la séquence d'acides aminés SEQ ID n° 3 à SEQ ID n°5 ou n° 16, ou une séquence homologue, définie comme i) une séquence identique à au moins 80% de la séquence SEQ ID n° 3 à n°5 ou n° 16; ou ii) une séquence codée par une séquence d'acide nucléique hybridant avec la séquence SEQ ID n° 1 ou 2 ou n° 15 ou de leurs fragments indiqués ci-dessus, ou sa séquence complémentaire, dans des conditions stringentes d'hybridation. Plus généralement, par « séquence d'acides aminés homologue », on entend toute séquence d'acides aminés qui diffère de la séquence SEQ ID n° 3 à 5 ou n° 16 par substitution, délétion et/ou insertion d'un acide aminé ou d'un nombre réduit d'acides aminés, notamment par substitution d'acides aminés naturels par des acides aminés non naturels ou pseudoacides aminés à des positions telles que ces modifications ne portent pas significativement atteinte aux propriétés antigéniques ou biologiques du polypeptide. Ces propriétés sont notamment des propriétés protéasiques pour les homologues de SEQ ID n°3, des propriétés de polymérase pour les homologues de SEQ ID n°4 et des propriétés de protéine de structure pour les homologues de SEQ ID n°5.The present invention also relates to an isolated polypeptide, comprising the amino acid sequence SEQ ID No. 3 to SEQ ID No. 5 or No. 16, or a homologous sequence, defined as i) a sequence identical to at least 80 % of the sequence SEQ ID No. 3 to No. 5 or No. 16; or ii) a sequence encoded by a nucleic acid sequence hybridizing with the sequence SEQ ID No. 1 or 2 or No. 15 or their fragments indicated above, or its complementary sequence, under stringent hybridization conditions. More generally, by “homologous amino acid sequence” is meant any amino acid sequence which differs from the sequence SEQ ID No. 3 to 5 or No. 16 by substitution, deletion and / or insertion of an amino acid or of a reduced number of amino acids, in particular by substitution of natural amino acids with non-natural amino acids or pseudo-amino acids at positions such that these modifications do not significantly affect the antigenic or biological properties of the polypeptide. These properties are in particular protease properties for the counterparts of SEQ ID No. 3, polymerase properties for the counterparts of SEQ ID No. 4 and structural protein properties for the counterparts of SEQ ID No. 5.
Lesdites substitutions sont de préférence des substitutions conservatives, c'est-à-dire des substitutions d'acides aminés de même classe, telles que des substitutions d'acides aminés aux chaînes latérales non chargées (tels que l'asparagine, la glutamine, la serine, la thréonine, et la tyrosine), d'acides aminés aux chaînes latérales basiques (tels que la lysine, l'arginine, et l'histidine), d'acides aminés aux chaînes latérales acides (tels que l'acide aspartique et l'acide glutamique) ; d'acides aminés aux chaînes latérales apolaires (tels que la glycine, l'alanine, la valine, la leucine, l'isoleucine, la praline, la phénylalanine, la méthionine, le tryptophane, et la cystéine). De préférence, une telle séquence d'acides aminés homologue est identique à au moins 85 % de la séquence SEQ ID n°3 à n°5 ou n° 16, de préférence au moins 90 %, de préférence encore au moins 95%.Said substitutions are preferably conservative substitutions, that is to say substitutions of amino acids of the same class, such as substitutions of amino acids with uncharged side chains (such as asparagine, glutamine, serine, threonine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid); amino acids with apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan, and cysteine). Preferably, such a homologous amino acid sequence is identical to at least 85% of the sequence SEQ ID No. 3 to No. 5 or No. 16, preferably at least 90%, more preferably at least 95%.
L'homologie est généralement déterminée en utilisant un logiciel d'analyse de séquence (par exemple, Séquence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Des séquences d'acides aminés similaires sont alignées pour obtenir le maximum de degré d'homologie (i.e. identité). A cette fin, il peut être nécessaire d'introduire de manière artificielle des espaces (« gaps ») dans la séquence. Une fois l'alignement optimal réalisé, le degré d'homologie (i.e. identité) est établi par enregistrement de toutes les positions pour lesquelles les acides aminés des deux séquences comparées sont identiques, par rapport au nombre total de positions.Homology is usually determined using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (ie identity). To this end, it may be necessary to introduce artificially spaces ("gaps") in the sequence. Once the optimal alignment has been achieved, the degree of homology (ie identity) is established by recording all the positions for which the amino acids of the two sequences compared are identical, relative to the total number of positions.
Le polypeptide de la présente invention peut être synthétisé par toutes les méthodes bien connues de l'homme du métier. Le polypeptide de l'invention peut par exemple être synthétisé par les techniques de la chimie de synthèse, telles que la synthèse de type Merrifield qui est avantageuse pour des raisons de pureté, de spécificité antigénique, d'absence de produits secondaires non désirés et pour sa facilité de production.The polypeptide of the present invention can be synthesized by any method well known to those skilled in the art. The polypeptide of the invention can for example be synthesized by the techniques of synthetic chemistry, such as Merrifield-type synthesis which is advantageous for reasons of purity, antigenic specificity, absence of unwanted side products and for its ease of production.
Des polypeptides recombinants peuvent également être produits par un procédé, dans lequel un vecteur contenant un acide nucléique comprenant la séquence SEQ ID n° 1 ou 2 ou n° 15 ou de leurs fragments indiqués ci-dessus, ou une séquence homologue est transféré dans une cellule hôte qui est mise en culture dans des conditions permettant l'expression du polypeptide correspondant.Recombinant polypeptides can also be produced by a method, in which a vector containing a nucleic acid comprising the sequence SEQ ID No. 1 or 2 or No. 15 or their fragments indicated above, or a homologous sequence is transferred into a host cell which is cultured under conditions allowing expression of the corresponding polypeptide.
Le polypeptide produit peut ensuite être récupéré et purifié. Les procédés de purification utilisés sont connus de l'homme du métier. Le polypeptide recombinant obtenu peut être purifié à partir de lysats et extraits cellulaires, du surnageant du milieu de culture, par des méthodes utilisées individuellement ou en combinaison, telles que le fractionnement, les méthodes de chromatographie, les techniques d'immunoaffinité à l'aide d'anticorps mono- ou polyclonaux spécifiques, etc.The polypeptide produced can then be recovered and purified. The purification methods used are known to those skilled in the art. The recombinant polypeptide obtained can be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific mono- or polyclonal antibodies, etc.
La séquence d'acide nucléique d'intérêt peut être insérée dans un vecteur de clonage ou un vecteur d'expression, dans lequel elle est liée de manière opérante à des éléments permettant la régulation de son expression, tels que notamment des promoteurs, activateurs et/ou terminateurs de transcription.The nucleic acid sequence of interest can be inserted into a cloning vector or an expression vector, in which it is operably linked to elements allowing the regulation of its expression, such as in particular promoters, activators and / or transcription terminators.
Les signaux contrôlant l'expression des séquences nucleotidiques (promoteurs, activateurs, séquences de terminaison...) sont choisis en fonction de l'hôte cellulaire utilisé. A cet effet, les séquences nucleotidiques selon l'invention peuvent être insérées dans des vecteurs à réplication autonome au sein de l'hôte choisi, ou des vecteurs intégratifs de l'hôte choisi. De tels vecteurs seront préparés selon les méthodes couramment utilisées par l'homme du métier, et les clones en résultant peuvent être introduits dans un hôte approprié par des méthodes standard, telles que par exemple l'électroporation ou la précipitation au phosphate de calcium.The signals controlling the expression of the nucleotide sequences (promoters, activators, termination sequences, etc.) are chosen as a function of the cell host used. To this end, the nucleotide sequences according to the invention can be inserted into autonomous replication vectors within the chosen host, or integrative vectors of the chosen host. Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as for example electroporation or precipitation with calcium phosphate.
Les vecteurs de clonage et/ou d'expression tels que décrits ci- dessus, contenant une des séquences nucleotidiques définies selon l'invention font également partie de la présente invention.The cloning and / or expression vectors as described above, containing one of the nucleotide sequences defined according to the invention also form part of the present invention.
L'invention vise en outre les cellules hôtes transfectées, de manière transitoire ou stable, par ces vecteurs d'expression. Ces cellules peuvent être obtenues par l'introduction dans des cellules hôtes, procaryotes ou eucaryotes, d'une séquence nucléotidique insérée dans un vecteur tel que défini ci-dessus, puis la mise en culture desdites cellules dans des conditions permettant la réplication et/ou l'expression de la séquence nucléotidique transfectée.The invention further relates to host cells transfected, transiently or stable, with these expression vectors. These cells can be obtained by introducing into host cells, prokaryotic or eukaryotic, a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.
Des exemples de cellules hôtes incluent notamment des cellules de mammifères, des cellules d'insectes, des bactéries ou des souches de levures.Examples of host cells include mammalian cells, insect cells, bacteria or yeast strains.
Les différentes séquences nucleotidiques de l'invention peuvent être d'origine artificielle ou non. Il peut s'agir de séquences d'ADN ou d'ARN, obtenues par criblage de banques de séquences au moyen de sondes élaborées sur la base des séquences SEQ ID n° 1 ou 2 ou n° 15. De telles banques peuvent être préparées par des techniques classiques de biologie moléculaire, connues de l'homme de l'art.The different nucleotide sequences of the invention can be of artificial origin or not. They may be DNA or RNA sequences, obtained by screening of sequence banks using probes prepared on the basis of sequences SEQ ID No. 1 or 2 or No. 15. Such libraries can be prepared by conventional molecular biology techniques known to those skilled in the art.
Les séquences nucleotidiques selon l'invention peuvent également être préparées par synthèse chimique, ou encore par des méthodes mixtes incluant la modification chimique ou enzymatique de séquences obtenues par criblage des banques.The nucleotide sequences according to the invention can also be prepared by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening libraries.
Ces séquences nucleotidiques permettent la réalisation de sondes ou amorces, hybridant spécifiquement avec une séquence SEQ ID n° 1 ou 2 ou n° 15 ou leurs fragments, selon l'invention, ou son brin complémentaire. Les conditions d'hybridation appropriées correspondent aux conditions de température et de force ionique usuellement utilisées par l'homme du métier, de préférence dans des conditions stringentes telles que définies précédemment. La présente invention a ainsi pour objet l'utilisation d'un acide nucléique tel que défini précédemment pour la fabrication de sondes nucléiques ou d'amorces oligonucléotidiques, capables d'hybrider spécifiquement, dans des conditions stringentes, avec tout ou partie dudit acide nucléique tel que défini précédemment. La présente invention a également pour objet un acide nucléique ayant au moins 10 nucléotides, qui hybride spécifiquement avec l'une des séquences d'acide nucléique SEQ ID n° 1 ou 2 ou n° 15 ou de leurs fragments, ou son complémentaire, dans des conditions stringentes d'hybridation.These nucleotide sequences allow the production of probes or primers, hybridizing specifically with a sequence SEQ ID No. 1 or 2 or No. 15 or their fragments, according to the invention, or its strand complementary. The appropriate hybridization conditions correspond to the temperature and ionic strength conditions usually used by those skilled in the art, preferably under stringent conditions as defined above. The subject of the present invention is therefore the use of a nucleic acid as defined above for the manufacture of nucleic probes or oligonucleotide primers, capable of specifically hybridizing, under stringent conditions, with all or part of said nucleic acid such as defined above. The present invention also relates to a nucleic acid having at least 10 nucleotides, which specifically hybridizes with one of the nucleic acid sequences SEQ ID No. 1 or 2 or No. 15 or their fragments, or its complement, in stringent hybridization conditions.
De tels acides nucléiques peuvent servir comme sonde pour la détection d'une séquence rétrovirale associée notamment au psoriasis, au lichen et à l'atopie. Ces acides nucléiques de l'invention utiles comme sondes comportent au minimum 10 nucléotides, préférentiellement au moins 20 nucléotides, préférentiellement encore au moins 100 nucléotides.Such nucleic acids can be used as a probe for the detection of a retroviral sequence associated in particular with psoriasis, lichen and atopy. These nucleic acids of the invention useful as probes comprise at least 10 nucleotides, preferably at least 20 nucleotides, more preferably at least 100 nucleotides.
Les acides nucléiques de l'invention peuvent être utiles comme amorces pour l'amplification de tout ou partie d'une séquence rétrovirale associée notamment au psoriasis, au lichen et à l'atopie. Ces amorces comportent de préférence dans ce cas au minimum 10 nucléotides, de préférence au moins 14 nucléotides, et préférentiellement moins de 40 nucléotides. On peut par exemple utiliser comme amorces, pour une amplification (par exemple par PCR), les acides nucléiques constitués des séquences SEQ ID n° 6 à 14.The nucleic acids of the invention can be useful as primers for the amplification of all or part of a retroviral sequence associated in particular with psoriasis, lichen and atopy. These primers preferably comprise in this case at least 10 nucleotides, preferably at least 14 nucleotides, and preferably less than 40 nucleotides. It is possible, for example, to use as primers, for amplification (for example by PCR), the nucleic acids consisting of the sequences SEQ ID No. 6 to 14.
Préférentiellement, les sondes ou amorces de l'invention sont marquées, préalablement à leur utilisation. Pour cela, plusieurs techniques sont à la portée de l'homme du métier comme par exemple le marquage fluorescent, radioactif, chimioluminescent ou enzymatique. On peut également utiliser les sondes comme sondes de capture, immobilisées sur un support solide par tout moyen approprié, directement ou indirectement, par exemple par covalence ou adsorption passive.Preferably, the probes or primers of the invention are marked, prior to their use. For this, several techniques are within the reach of those skilled in the art such as, for example, fluorescent, radioactive, chemiluminescent or enzymatic labeling. The probes can also be used as capture probes, immobilized on a support solid by any suitable means, directly or indirectly, for example by covalence or passive adsorption.
L'invention a également pour objet un procédé de détection in vitro de la présence d'une séquence nucléique rétrovirale associée à une affection cutanée chez un patient, comprenant les étapes consistant à : ai ) isoler l'ADN ou l'ARN d'un échantillon biologique ; b1 ) éventuellement produire de l'ADNc à partir dudit ARN ; d ) mettre en présence ledit ADN ou ADNc avec des acides nucléiques tels que définis précédemment, comme amorces, dans des conditions permettant l'amplification spécifique d'une séquence SEQ ID n° 1 ouThe subject of the invention is also a method of in vitro detection of the presence of a retroviral nucleic sequence associated with a skin condition in a patient, comprising the steps consisting in: ai) isolating the DNA or RNA from a biological sample; b1) optionally producing cDNA from said RNA; d) bringing said DNA or cDNA into contact with nucleic acids as defined above, as primers, under conditions allowing the specific amplification of a sequence SEQ ID No. 1 or
2, ou de leurs fragments, ou d'une séquence homologue telle que définie précédemment, d1 ) soumettre ledit ADN ou ADNc à une réaction d'amplification, e1 ) détecter les produits d'amplification, indicateurs de la présence d'une séquence rétrovirale associée à une affection cutanée, et/ou a2) la mise en contact d'un ADN ou ARN d'un échantillon biologique, avec un acide nucléique tel que défini précédemment, comme sonde, marquée de manière détectable, dans des conditions permettant l'hybridation spécifique dudit ADN ou ARN avec ladite sonde ; b2) la détection des complexes d'hybridation formés, indicateurs de la présence d'une séquence rétrovirale associée à une affection cutanée.2, or their fragments, or of a homologous sequence as defined above, d1) subjecting said DNA or cDNA to an amplification reaction, e1) detecting the amplification products, indicators of the presence of a retroviral sequence associated with a skin condition, and / or a2) bringing a DNA or RNA of a biological sample into contact with a nucleic acid as defined above, as a probe, detectably labeled, under conditions allowing the specific hybridization of said DNA or RNA with said probe; b2) the detection of the hybridization complexes formed, indicators of the presence of a retroviral sequence associated with a skin condition.
L'invention a en outre pour objet un procédé de diagnostic in vitro d'une affection cutanée, telle que notamment un psoriasis, un lichen ou un eczéma atopique, chez un patient, dans lequel on détecte, dans un échantillon biologique, la présence d'une séquence nucléique rétrovirale associée à l'une de ces pathologies, au moyen du procédé de détection décrit ci-dessus. Par "échantillon biologique", on entend notamment un prélèvement du type fluide biologique, tissu vivant, fragment de tissu, mucosité, organe ou fragment d'organe ou tout surnageant de culture obtenu à l'aide d'un prélèvement. Par "fluide biologique", on entend notamment le sérum, le plasma, l'urine ou le liquide céphalo-rachidien.The invention further relates to a method of in vitro diagnosis of a skin condition, such as in particular psoriasis, lichen or atopic eczema, in a patient, in which the presence of 'a retroviral nucleic acid sequence associated with one of these pathologies, by means of the detection method described above. By "biological sample" is meant in particular a sample of the biological fluid, living tissue, tissue fragment, mucus, organ or organ fragment type or any culture supernatant obtained using a sample. By "biological fluid" is meant in particular serum, plasma, urine or cerebrospinal fluid.
Les sondes utilisées à des fins de diagnostic de l'invention peuvent être mises en œuvre dans toutes les techniques connues, et notamment les techniques dites "DOT-BLOT", "SOUTHERN-BLOT", "NORTHERN-BLOT" et la technique "SANDWICH". Avantageusement, on utilise la technique "SANDWICH" dans la présente invention, comprenant une sonde de capture spécifique et/ou une sonde marquée de manière détectable spécifique, étant entendu que la sonde de capture et la sonde de détection doivent présenter une séquence nucléotidique au moins partiellement différente.The probes used for diagnostic purposes of the invention can be used in all known techniques, and in particular the techniques known as "DOT-BLOT", "SOUTHERN-BLOT", "NORTHERN-BLOT" and the "SANDWICH technique ". Advantageously, the "SANDWICH" technique is used in the present invention, comprising a specific capture probe and / or a specific detectably labeled probe, it being understood that the capture probe and the detection probe must have at least one nucleotide sequence partially different.
On peut également mettre en oeuvre une hybridation in situ, par exemple au moyen de la technique "FISH", sur des tissus ou cellules.It is also possible to carry out an in situ hybridization, for example by means of the "FISH" technique, on tissues or cells.
L'invention a également pour objet des anticorps dirigés contre le polypeptides tels que définis précédemment.The subject of the invention is also antibodies directed against the polypeptides as defined above.
Il peut s'agir d'anticorps poly- ou monoclonaux ou de leurs fragments, d'anticorps chimériques, notamment humanisés ou immunoconjugués. Les anticorps polyclonaux peuvent être obtenus à partir du sérum d'un animal immunisé contre un polypeptide selon les modes opératoires usuels.They may be poly- or monoclonal antibodies or their fragments, chimeric antibodies, in particular humanized or immunoconjugated. Polyclonal antibodies can be obtained from the serum of an animal immunized against a polypeptide according to the usual procedures.
Selon un mode de réalisation de l'invention, on peut utiliser comme antigène un fragment peptidique approprié, pouvant être couplé par l'intermédiaire d'un résidu réactif à une protéine ou un autre peptide. Des lapins sont immunisés avec l'équivalent de 1 mg de l'antigène peptidique selon la procédure décrite par Benoit et al. (1982). A des intervalles de quatre semaines, les animaux sont traités par des injections de 200 μg d'antigène et saignés 10 à 14 jours plus tard. Après la troisième injection, l'anti-sérum est examiné pour déterminer sa capacité à se lier au peptide antigène radiomarqué à l'iode, préparé par la méthode chloramine-T et est ensuite purifié par une chromatographie sur colonne échangeuse d'ion carboxyméthyl cellulose (CMC). Les molécules d'anticorps sont ensuite recueillies dans les mammifères et isolées jusqu'à la concentration souhaitée par les méthodes bien connues de l'homme de l'art.According to one embodiment of the invention, an appropriate peptide fragment can be used as the antigen, which can be coupled via a reactive residue to a protein or another peptide. Rabbits are immunized with the equivalent of 1 mg of the peptide antigen according to the procedure described by Benoit et al. (1982). At four-week intervals, the animals are treated with injections of 200 μg of antigen and bled 10-14 days later. After the third injection, the antiserum is examined to determine its capacity to bind to the antigen peptide radiolabelled with iodine, prepared by the chloramine-T method and is then purified by chromatography on an ion exchange column carboxymethyl cellulose (CMC). Antibody molecules are then collected from mammals and isolated to the desired concentration by methods well known to those skilled in the art.
Afin d'augmenter la spécificité du sérum polyclonal, les anticorps peuvent être purifiés par une chromatographie d'immuno-affinité en utilisant des polypeptides immunisant en phase solide. L'anticorps est mis en contact avec le polypeptide immunisant en phase solide pendant une durée suffisante de façon à faire immuno-réagir le polypeptide avec la molécule d'anticorps afin de former un complexe immunologique en phase solide.In order to increase the specificity of the polyclonal serum, the antibodies can be purified by immunoaffinity chromatography using solid phase immunizing polypeptides. The antibody is brought into contact with the immunizing polypeptide in solid phase for a sufficient time so as to make the polypeptide immunoreact with the antibody molecule in order to form an immunological complex in solid phase.
Les anticorps monoclonaux peuvent être obtenus selon la méthode classique de culture d'hybridomes décrite par Kôhler et Milstein (1975).The monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by Kôhler and Milstein (1975).
Les anticorps ou fragments d'anticorps de l'invention peuvent être par exemple des anticorps chimériques, des anticorps humanisés, des fragments Fab et F(ab')2. Ils peuvent également se présenter sous forme d'immunoconjugués ou d'anticorps marqués.The antibodies or antibody fragments of the invention can be, for example, chimeric antibodies, humanized antibodies, Fab and F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies.
Les anticorps de l'invention, en particulier les anticorps monoclonaux, peuvent notamment être utilisés pour l'analyse par immunohistochimie des polypeptides sur des coupes de tissus spécifiques, par exemple par immunofluorescence, marquage à l'or, immunoperoxydase... Les anticorps ainsi produits peuvent être avantageusement mis en oeuvre dans toute situation où l'expression des polypeptides doit être observée.The antibodies of the invention, in particular the monoclonal antibodies, can in particular be used for the analysis by immunohistochemistry of the polypeptides on sections of specific tissues, for example by immunofluorescence, gold labeling, immunoperoxidase ... The antibodies thus The products can advantageously be used in any situation where the expression of the polypeptides must be observed.
L'invention a également pour objet l'utilisation d'au moins un anticorps ainsi produit pour la détection ou la purification d'un polypeptide tel que défini précédemment dans un échantillon biologique.The invention also relates to the use of at least one antibody thus produced for the detection or purification of a polypeptide as defined above in a biological sample.
Plus précisément, l'invention concerne une méthode in vitro pour la détection ou la mesure du taux d'expression des polypeptides de PsoRVl ou PsoRV2 tels que définis précédemment dans un prélèvement biologique comprenant la mise en contact d'au moins un anticorps tel que défini précédemment avec ledit prélèvement biologique dans des conditions permettant la formation éventuelle de complexes immunologiques spécifiques entre les polypeptides de PsoRVl ou PsoRV2a ou PsoRV2b et le ou lesdits anticorps et la détection des complexes immunologiques spécifiques éventuellement formés.More specifically, the invention relates to an in vitro method for detecting or measuring the level of expression of the PsoRV1 or PsoRV2 polypeptides as defined above in a biological sample comprising the contacting of at least one antibody as defined previously with said biological sample under conditions allowing the possible formation of specific immunological complexes between the PsoRV1 or PsoRV2a or PsoRV2b polypeptides and the said antibody or antibodies and the detection of any specific immunological complexes possibly formed.
L'invention a également pour objet un kit pour la mise en œuvre de cette méthode comprenant :The invention also relates to a kit for the implementation of this method comprising:
- au moins un anticorps spécifique des polypeptides de PsoRVl et PsoRV2a ou PsoRV2b, éventuellement fixé sur un support ;- at least one antibody specific for the polypeptides of PsoRV1 and PsoRV2a or PsoRV2b, optionally fixed on a support;
- des moyens de révélation de la formation de complexes antigènes/anticorps spécifiques entre les polypeptides et ledit anticorps et/ou des moyens de quantification de ces complexes.- Means for revealing the formation of specific antigen / antibody complexes between the polypeptides and said antibody and / or means for quantifying these complexes.
L'invention a également pour objet une composition pharmaceutique comprenant un acide nucléique tel que défini précédemment en association avec un véhicule pharmaceutiquement acceptable, ledit acide nucléique étant capable de s'hybrider in vivo ou in vitro sur l'ARN et/ou sur l'ADN de PsoRVl ou PsoRV2 (a ou b) pour bloquer les phénomènes de réplication, notamment traduction et/ou transcription, et/ou pour dégrader ledit ADN et/ou ARN.The subject of the invention is also a pharmaceutical composition comprising a nucleic acid as defined above in association with a pharmaceutically acceptable vehicle, said nucleic acid being capable of hybridizing in vivo or in vitro on RNA and / or on PsoRV1 or PsoRV2 DNA (a or b) to block the replication phenomena, in particular translation and / or transcription, and / or to degrade said DNA and / or RNA.
Ladite composition est destinée à être utilisée en thérapie génique. L'acide nucléique de préférence inséré dans un vecteur généralement viral (tels que les adenovirus et les rétrovirus) peut être administré sous forme nue, exempt de tout véhicule favorisant le transfert à la cellule cible, tels que des liposomes anioniques, des lipides cationiques, des microparticules, par exemple des microparticules d'or, des agents de précipitation, par exemple du phosphate de calcium, ou tout autre agent facilitant la transfection. Dans ce cas, le polynucléotide peut être simplement dilué dans une solution physiologiquement acceptable, telle qu'une solution stérile ou une solution stérile tampon, en présence ou en l'absence d'un véhicule.The composition is intended for use in gene therapy. The nucleic acid preferably inserted into a generally viral vector (such as adenoviruses and retroviruses) can be administered in naked form, free of any vehicle promoting transfer to the target cell, such as anionic liposomes, cationic lipids, microparticles, for example gold microparticles, precipitating agents, for example calcium phosphate, or any other agent which facilitates transfection. In this case, the polynucleotide can be simply diluted in a physiologically acceptable solution, such as a sterile solution or a sterile buffer solution, in the presence or in the absence of a vehicle.
De manière alternative, un acide nucléique de l'invention peut être associé à des agents qui facilitent la transfection. Il peut être, entre autres , (i) associé à un agent chimique qui modifie la perméabilité cellulaire tel que la bupivacaïne ; (ii) encapsulé dans des liposomes, éventuellement en présence de substances supplémentaires facilitant la transfection ; ou (iii) associé à des lipides cationiques ou des microparticules de silice, d'or ou de tungstène.Alternatively, a nucleic acid of the invention can be combined with agents which facilitate transfection. It can be, inter alia, (i) associated with a chemical agent which modifies cell permeability such as bupivacaine; (ii) encapsulated in liposomes, possibly in the presence additional substances that facilitate transfection; or (iii) associated with cationic lipids or microparticles of silica, gold or tungsten.
Lorsque les constructions d'acide nucléique de l'invention recouvrent des microparticules, celles-ci peuvent être injectées par voie intradermique ou intraépidermique par la technique du canon à gènes, "gène gun" (WO 94/24263).When the nucleic acid constructs of the invention cover microparticles, these can be injected intradermally or intraepidermally by the gene gun technique, "gene gun" (WO 94/24263).
La quantité à utiliser comme médicament dépend notamment de la construction d'acide nucléique elle-même, de l'individu auquel cet acide nucléique est administré, du mode d'administration et du type de formulation, et de la pathologie. De manière générale, une quantité thérapeutiquement ou prophylactiquement efficace variant d'environ 0, 1 μg à environ 1 mg, de préférence d'environ 1 μg à environ 800 μg et, de manière préférentielle d'environ 25 μg à environ 250 μg, peut être administrée à des adultes humains.The amount to be used as a drug depends in particular on the construction of nucleic acid itself, the individual to whom this nucleic acid is administered, the mode of administration and the type of formulation, and the pathology. In general, a therapeutically or prophylactically effective amount varying from approximately 0.1 μg to approximately 1 mg, preferably from approximately 1 μg to approximately 800 μg and, preferably from approximately 25 μg to approximately 250 μg, can be administered to human adults.
Les constructions d'acides nucléiques de l'invention peuvent être administrées par toute voie d'administration conventionnelle. Le choix de la voie d'administration dépend en particulier de la formulation choisie. L'application cutanée et l'injection intradermique sont toutefois préférées.The nucleic acid constructs of the invention can be administered by any conventional route of administration. The choice of route of administration depends in particular on the formulation chosen. Skin application and intradermal injection are preferred, however.
L'invention a également pour objet une méthode de traitement thérapeutique d'une affection cutanée dans laquelle on administre à un patient nécessitant un tel traitement, une quantité efficace d'un acide nucléique bloquant l'expression des séquences PsoRVl ou PsoRV2a ou PsoRV2b.A subject of the invention is also a method of therapeutic treatment of a skin condition in which an effective quantity of a nucleic acid blocking the expression of the sequences PsoRV1 or PsoRV2a or PsoRV2b is administered to a patient in need of such treatment.
Le patient visé est généralement un être humain, mais l'application peut également être étendue à tout mammifère le cas échéant.The target patient is generally a human being, but the application can also be extended to any mammal if necessary.
Une thérapie ex vivo peut être également mise en place.Ex vivo therapy can also be implemented.
L'invention fournit plus précisément une méthode in vitro de préparation d'un ensemble cellulaire destiné à être implanté à un patient receveur, comprenant l'incorporation d'une séquence nucléotidique de PsoRVl ou PsoRV2a ou PsoRV2b telle que définie précédemment dans des cellules en culture.The invention more specifically provides an in vitro method for preparing a cell assembly intended to be implanted in a recipient patient, comprising the incorporation of a nucleotide sequence of PsoRV1 or PsoRV2a or PsoRV2b as defined above in cells in culture .
Les cellules visées peuvent être des cellules autologues du patient receveur, mais aussi des cellules hétérologues (d'un autre donneur de la même espèce) ou xénologues (d'un donneur d'une autre espèce animale). Il peut s'agir de préférence de cellules cutanées, plus particulièrement de kératinocytes.The targeted cells can be autologous cells of the recipient patient, but also heterologous cells (from another donor of the same species) or xenologists (from a donor of another animal species). It may preferably be skin cells, more particularly keratinocytes.
Les séquences nucleotidiques peuvent être incorporées par toute technique de transfert connue de l'homme du métier, par « gène gun », sous forme nue, ou en association avec des liposomes anioniques, des lipides cationiques, des microparticules, par exemple des microparticules d'or, ou tout autre agent facilitant la transfection.The nucleotide sequences can be incorporated by any transfer technique known to a person skilled in the art, by “gun gene”, in naked form, or in association with anionic liposomes, cationic lipids, microparticles, for example microparticles of or, or any other agent facilitating transfection.
Les séquences nucleotidiques sont pour cela de préférence préalablement insérées dans un vecteur, de type rétrovirus ou adenovirus par exemple.The nucleotide sequences are preferably inserted beforehand in a vector, of the retrovirus or adenovirus type for example.
Les modes d'implantation des cellules modifiées (greffe, injection, etc.) dépendent notamment du type de cellules et de l'affection visée.The methods of implantation of the modified cells (graft, injection, etc.) depend in particular on the type of cells and the disease targeted.
Un autre objet de l'invention est une méthode de traitement chirurgical dans laquelle on implante chez un patient receveur nécessitant un tel traitement les cellules ainsi modifiées.Another object of the invention is a method of surgical treatment in which the cells thus modified are implanted in a recipient patient requiring such treatment.
L'invention a également pour objet une composition pharmaceutique comprenant un polypeptide tel que défini précédemment, en association avec un véhicule pharmaceutiquement acceptable. Une composition pharmaceutique de ce type peut être particulièrement utile pour stimuler ou abroger les réponses immunitaires du patient vis-à-vis des polypeptides PsoRVl et PsoRV2a ou PsoRV2b, endogènes, dans le cadre d'une vaccination ou d'une désensibilisation, respectivement. L'invention a également pour objet une composition pharmaceutique comprenant un anticorps tel que défini précédemment, de préférence un anticorps monoclonal, le cas échéant humanisé, en association avec un véhicule pharmaceutiquement acceptable.The invention also relates to a pharmaceutical composition comprising a polypeptide as defined above, in association with a pharmaceutically acceptable vehicle. A pharmaceutical composition of this type can be particularly useful for stimulating or abrogating the immune responses of the patient vis-à-vis the endogenous PsoRV1 and PsoRV2a or PsoRV2b polypeptides, in the context of vaccination or desensitization, respectively. The invention also relates to a pharmaceutical composition comprising an antibody as defined above, preferably a monoclonal antibody, where appropriate humanized, in association with a pharmaceutically acceptable vehicle.
Une composition à base d'un anticorps selon l'invention peut être particulièrement utile pour inactiver les polypeptides PsoRVl ou PsoRV2a ou PsoRV2b dans le cadre d'une pathologie impliquant ces polypeptides, telle que les maladies de la peau. Les modes d'administration, les posologies et les formes galéniques des compositions pharmaceutiques selon l'invention, contenant au moins un anticorps ou un polypeptide, peuvent être déterminées de manière usuelle par l'homme du métier, notamment selon les critères généralement pris en compte pour l'établissement d'un traitement thérapeutique adapté à un patient, comme par exemple l'âge ou le poids corporel du patient, la gravité de son état général, le tolérance au traitement, et les effets secondaires constatés, etc.An antibody-based composition according to the invention may be particularly useful for inactivating the PsoRV1 or PsoRV2a or PsoRV2b polypeptides in the context of a pathology involving these polypeptides, such as skin diseases. The modes of administration, the dosages and the galenical forms of the pharmaceutical compositions according to the invention, containing at least one antibody or a polypeptide, can be determined in the usual way by a person skilled in the art, in particular according to the criteria generally taken into account. for the establishment of a therapeutic treatment adapted to a patient, such as for example the age or the body weight of the patient, the seriousness of his general state, the tolerance to the treatment, and the observed side effects, etc.
De manière générale, une quantité thérapeutiquement ou prophylactiquement efficace dudit anticorps ou polypeptide variant d'environ 0,1 μg à environ 1 mg peut être administrée à des adultes humains.Generally, a therapeutically or prophylactically effective amount of said antibody or polypeptide varying from about 0.1 μg to about 1 mg can be administered to human adults.
Les pathologies visées sont toutes celles dans lesquelles l'expression de PsoRVl ou PsoRV2a ou PsoRV2b est observée. Les maladies de la peau sont plus particulièrement ciblées, et parmi celles-ci notamment les dermatoses à composante immunitaire.The pathologies targeted are all those in which the expression of PsoRV1 or PsoRV2a or PsoRV2b is observed. Skin diseases are more particularly targeted, and among these in particular dermatoses with an immune component.
On peut citer, à titre d'exemples de pathologies concernées, le psoriasis, le lichen, l'eczéma atopique, le lupus érythémateux, et les cancers cutanés (carcinomes), dont par exemple le carcinome basocellulaire et le carcinome spinocellulaire.As examples of the pathologies concerned, mention may be made of psoriasis, lichen, atopic eczema, lupus erythematosus, and skin cancers (carcinomas), including for example basal cell carcinoma and squamous cell carcinoma.
L'invention a enfin pour objet l'utilisation d'un polypeptide PsoRVl ou PsoRV2a ou PsoRV2b tel que défini précédemment ou d'un acide nucléique codant pour le polypeptide, pour le criblage de composés (isolés ou en mélange) susceptibles d'inhiber l'activité dudit polypeptide.The subject of the invention is finally the use of a PsoRV1 or PsoRV2a or PsoRV2b polypeptide as defined above or of a nucleic acid coding for the polypeptide, for the screening of compounds (isolated or in mixture) capable of inhibiting the activity of said polypeptide.
Une méthode de criblage de composés, isolés ou en mélange, susceptibles d'inhiber la polymérase de PsoRVl , peut ainsi être mise en œuvre, méthode comprenant :A method for screening for compounds, isolated or as a mixture, capable of inhibiting the PsoRVl polymerase, can thus be implemented, method comprising:
- la purification de la polymérase (par exemple au moyen de la technique des "Tags", ou étiquettes (Kevin J. Petty Chapitre X, "Short Protocols in Molecular Biology", 3eme Edition, Editions Aussubel et al, imprimerie John Willey and Son Corp.) ; - suivie des tests in vitro de transcription inverse selon le protocole de PERT (Pyra et al, 1994).- the purification of the polymerase (for example by means of the technique of "Tags", or labels (Kevin J. Petty Chapter X, "Short Protocols in Molecular Biology", 3 rd Edition, Editions Aussubel et al, printing John Willey and His body.) ; - followed by in vitro reverse transcription tests according to the PERT protocol (Pyra et al, 1994).
Parmi les composés à tester, on peut notamment citer des inhibiteurs d'autres polymérases connues, tels que des analogues nucleotidiques.Among the compounds to be tested, mention may in particular be made of inhibitors of other known polymerases, such as nucleotide analogs.
Les exemples suivants illustrent l'invention sans en limiter la portée.The following examples illustrate the invention without limiting its scope.
EXEMPLE 1 :EXAMPLE 1:
Obtention du clone CONS-58-13 codant pour la protéase et la partie 5' de l'ADN polymérase ARN dépendante (PsoRVl).Obtaining of the clone CONS-58-13 coding for the protease and the 5 ′ part of the DNA RNA dependent polymerase (PsoRVl).
Le clone 58-1 3 a été obtenu par transcription inverse et réaction de PCR dégénérée selon la méthode publiée par Tuke et al, (1997) à partir d'ARN totaux extraits de coupes de biopsies de peau lésionnelles de patients atteints de psoriasis et clonage dans le vecteur pCR® 2.1 -Topo selon les conditions recommandées par le fabricant (TOPO™ TA-cloning® system ; Invitrogen, The Netherlands). Le clone séquence grâce au kit "DyeTerminator sequencing kit" (Perkin-Elmer, France) et un séquenceur AB1373 (Applied Biosystems, USA) a permis de définir une amorce spécifique de PsoRV, dénommé 58-1 3 (SEQ ID n° 6). Par une étude d'homologie entre MSRV1 et ERV9, numéro d'accès # AF009668 et # X57147XX (sur la base de données EMBL) respectivement, les auteurs de l'invention ont pu définir une amorce dénommée CONS SEQ ID n° 7. Le clone CONS-58-13 a été obtenu par transcription inverse et PCR à l'aide des amorces définies ci-dessous et identifiées comme les séquences SEQ ID n° 6 et SEQ ID n° 7, à partir d'ARNs totaux extraits de coupes de biopsies de peau lésionnelles de patients atteints de psoriasis et clonage dans le vecteur pCR® 2.1 -Topo selon les conditions recommandées par le fabricant (TOPO™ TA-cloning® system ; Invitrogen, The Netherlands). amorce 58-13 : SEQ ID n° 6 :Clone 58-1 3 was obtained by reverse transcription and degenerate PCR reaction according to the method published by Tuke et al, (1997) from total RNA extracted from biopsy sections of lesional skin of patients with psoriasis and cloning. in the vector pCR® 2.1 -Topo according to the conditions recommended by the manufacturer (TOPO ™ TA-cloning® system; Invitrogen, The Netherlands). The clone sequence using the "DyeTerminator sequencing kit" (Perkin-Elmer, France) and an AB1373 sequencer (Applied Biosystems, USA) made it possible to define a specific primer for PsoRV, called 58-1 3 (SEQ ID No. 6) . By a homology study between MSRV1 and ERV9, access number # AF009668 and # X57147XX (on the EMBL database) respectively, the authors of the invention were able to define a primer called CONS SEQ ID No. 7. The clone CONS-58-13 was obtained by reverse transcription and PCR using the primers defined below and identified as the sequences SEQ ID No. 6 and SEQ ID No. 7, from total RNAs extracted from sections of lesional skin biopsies of patients with psoriasis and cloning into the vector pCR® 2.1 -Topo according to the conditions recommended by the manufacturer (TOPO ™ TA-cloning® system; Invitrogen, The Netherlands). primer 58-13: SEQ ID n ° 6:
5'-GTA CGG ATG GAC AAA AGT G-3' amorce CONS : SEQ ID n° 7 :5'-GTA CGG ATG GAC AAA AGT G-3 'primer CONS: SEQ ID n ° 7:
5'-CAR CAG GAC TGA GGG TGC C-3'5'-CAR CAG GAC TGA GGG TGC C-3 '
EXEMPLE 2 :EXAMPLE 2:
Détection de l'expression de PsoRVl dans les biopsies de lésions de patients atteints de psoriasis.Detection of PsoRVl expression in biopsies of lesions of patients with psoriasis.
A. MéthodeA. Method
La recherche de l'expression de la séquence PsoRVl a été obtenue par les techniques de transcription inverse à partir d'ARNs totaux extraits de coupes de biopsies de peaux lésionnelles de patients atteints de psoriasis. La synthèse d'ADNc a été réalisée avec une amorce oligo dT16 et la transcriptase inverse (GIBCO-BRL) selon les conditions préconisées par le fournisseur. Une PCR a ensuite été réalisée en utilisant les amorces définies à partir de la séquence du clone CONS-58-13. Deux couples d'amorces ont été utilisés : - le premier couple dénommé WALE-PIVA, SEQ ID n° 8 et SEQThe search for the expression of the PsoRVl sequence was obtained by reverse transcription techniques from total RNAs extracted from biopsy sections of lesional skin of patients suffering from psoriasis. The cDNA synthesis was carried out with an oligo dT 16 primer and reverse transcriptase (GIBCO-BRL) according to the conditions recommended by the supplier. A PCR was then carried out using the primers defined from the sequence of the clone CONS-58-13. Two pairs of primers were used: - the first pair called WALE-PIVA, SEQ ID n ° 8 and SEQ
ID n° 9, respectivement ;ID No. 9, respectively;
- le deuxième couple dénommé WALE-WQG, SEQ ID n° 8 et SEQ ID n° 10, respectivement.- the second pair called WALE-WQG, SEQ ID No. 8 and SEQ ID No. 10, respectively.
Les échantillons sont ensuite analysés par migration électrophorètique sur gel d'agarose et détectés sous lumière UV après marquage du gel au bromure d'éthidium. Les échantillons sont considérés positifs après visualisation d'une amplification sur gel d'agarose. amorce 5' : SEQ ID n° 8The samples are then analyzed by electrophoretic migration on agarose gel and detected under UV light after labeling the gel with ethidium bromide. The samples are considered positive after visualization of an amplification on agarose gel. 5 'primer: SEQ ID # 8
5'-GAA GTT TGG GCA TTG GAA G-3" amorce 3' : SEQ ID n° 95'-GAA GTT TGG GCA TTG GAA G-3 "primer 3 ': SEQ ID n ° 9
5'-GGA ATT ACT GCC TCA TTG-3' amorce 3' : SEQ ID n° 105'-GGA ATT ACT GCC TCA TTG-3 'primer 3': SEQ ID n ° 10
5'-CTC CAC TGA CCT TTC RG-3' B. Résultats5'-CTC CAC TGA CCT TTC RG-3 ' B. Results
Le tableau 1 ci-après représente la fréquence de détection de l'expression de PsoRVl dans les biopsies de lésions de patients atteints de psoriasis, de lichen ou d'atopie ou encore de carcinome baso ou spinocellulaire, et dans les contrôles sujets sains. Table 1 below represents the frequency of detection of the expression of PsoRVl in biopsies of lesions of patients with psoriasis, lichen or atopy, or basal or squamous cell carcinoma, and in controls on healthy subjects.
Figure imgf000020_0001
Figure imgf000020_0001
Ces résultats suggèrent fortement que l'expression des séquences rétrovirales de PsoRV participe au développement des pathologies indiquées ci-dessus.These results strongly suggest that the expression of PsoRV retroviral sequences participates in the development of the pathologies indicated above.
EXEMPLE 3 :EXAMPLE 3:
Obtention du clone Env-PsoRV2a codant pour l'enveloppe du rétrovirus. Les clones Env-PsoRV2-5' et Env-PsoRV2-3' ont été obtenus par transcription inverse et réaction de PCR dégénérée à l'aide d'amorces conservées au sein de la famille HERV-K, identifiées par les séquences Env5' SEQ ID n°11 , LTR3' SEQ ID n°12, Env3' SEQ ID n°13 et LTR5' SEQ ID n°14, à partir d'ARN totaux extraits de coupes de biopsie de peaux lésionnelles de patients atteints de psoriasis. Les produits de PCR ont ensuite été clones et séquences selon les méthodes décrites dans l'exemple 1.Obtaining the Env-PsoRV2a clone coding for the envelope of the retrovirus. The clones Env-PsoRV2-5 'and Env-PsoRV2-3' were obtained by reverse transcription and degenerate PCR reaction using primers conserved within the HERV-K family, identified by the sequences Env5 'SEQ ID n ° 11, LTR3 'SEQ ID n ° 12, Env3' SEQ ID n ° 13 and LTR5 'SEQ ID n ° 14, from total RNA extracted from biopsy sections of lesional skin of patients with psoriasis. The PCR products were then cloned and sequenced according to the methods described in Example 1.
- Séquence Env5' n°11 :- Sequence Env5 'n ° 11:
5'-TTA CTG TGG CCT CAC ACC A-3'5'-TTA CTG TGG CCT CAC ACC A-3 '
- Séquence LTR3' n°12 :- LTR3 'sequence n ° 12:
5'-ATG CTG CCT TCA AGC ATC TG-3'5'-ATG CTG CCT TCA AGC ATC TG-3 '
- Séquence Env3' n°13 :- Env3 'sequence n ° 13:
5'-TCA CCA GCA GAA TAC GGT G-3'5'-TCA CCA GCA GAA TAC GGT G-3 '
- Séquence LTR5' n°14 :- Sequence LTR5 'n ° 14:
5'-CTT ACG AGA AAC ACC CAC AG-3' BIBLIOGRAPHIE5'-CTT ACG AGA AAC ACC CAC AG-3 ' BIBLIOGRAPHY
- Benoit et al., PNAS USA, 79, 917-921 (1982). - Dalen, A.B., Hellgren, L., Iversen, O.J. & Vincent, J. A virus-like particle associated with psoriasis. Acta Pathol. Microbiol. Immunol. Scand. 91 , 221-229 (1983).- Benoit et al., PNAS USA, 79, 917-921 (1982). - Dalen, A.B., Hellgren, L., Iversen, O.J. & Vincent, J. A virus-like particle associated with psoriasis. Acta Pathol. Microbiol. Immunol. Scand. 91, 221-229 (1983).
- Guilhou, J.-J., Meynadier, J. & Clôt, J. New concepts in the pathogenesis of psoriasis. Br. J. Dermatol. 95, 585-592 (1978). - Guilhou, J.-J., Vannereau, H., Theunynck, D., Bergoin, M. &- Guilhou, J.-J., Meynadier, J. & Clôt, J. New concepts in the pathogenesis of psoriasis. Br. J. Dermatol. 95, 585-592 (1978). - Guilhou, J.-J., Vannereau, H., Theunynck, D., Bergoin, M. &
Blanchard, J.-M. Virological studies on psoriatic lymphocytes. In «Psoriasis». Eds. Farber E.M., Cox A.J. and Jacobs P. H. publ.: Grune & Stratton Inc. 1982.Blanchard, J.-M. Virological studies on psoriatic lymphocytes. In "Psoriasis". Eds. Farber E.M., Cox A.J. and Jacobs P. H. publ .: Grune & Stratton Inc. 1982.
- Iversen, O.J. Isolation of virus-particles in urine fro a psoriatic patient. Acta Path. Microbiol. Immunol. Scand. 91 , 407-412 (1983). - Kôhler et Milstein, Nature, 256, 495-497, (1975).- Iversen, O.J. Isolation of virus-particles in urine fro a psoriatic patient. Acta Path. Microbiol. Immunol. Scand. 91, 407-412 (1983). - Kôhler and Milstein, Nature, 256, 495-497, (1975).
- Kevin J. Petty Chapitre X, "Short Protocols in Molecular Biology", 3eme Edition, Editions Aussubel et al, imprimerie John Willey and Son Corp- Kevin J. Petty Chapter X, "Short Protocols in Molecular Biology", 3 rd Edition, Editions Aussubel et al, printer John Willey and Son Corp
- Pyra et al, 1994 "Ultrasensitive retroviruses détection by reverse transcriptase assay based on product enhancement", PNAS, vol. 91 , 1544-- Pyra et al, 1994 "Ultrasensitive retroviruses detection by reverse transcriptase assay based on product enhancement", PNAS, vol. 91, 1544-
15481548
- Sambrook et al., Molecular cloning, a laboratory manual Spring Harbor Laboratory Press, 9.54-62 (1989)- Sambrook et al., Molecular cloning, a laboratory manual Spring Harbor Laboratory Press, 9.54-62 (1989)
- Tuke, P.W., Perron, H., Bedin, F., Beseme, F. & Garson, J.A. Development of a pan-retrovirus détection system for multiple sclerosis. Anal.- Tuke, P.W., Perron, H., Bedin, F., Beseme, F. & Garson, J.A. Development of a pan-retrovirus detection system for multiple sclerosis. Anal.
Neurol. Scand. 169, 16-21 (1997). Neurol. Scand. 169, 16-21 (1997).

Claims

REVENDICATIONS
1. Acide nucléique isolé, constitué d'une séquence choisie parmi SEQ ID n° 1 , SEQ ID n° 2 ou SEQ ID N° 15, ou la séquence des fragments allant du nucleotide 1 au nucleotide 291 sur SEQ ID n°1 , ou allant du nucleotide 207 au nucleotide 950 sur SEQ ID n°1 , ou allant du nucleotide 3 au nucleotide 260 sur SEQ ID n°2.1. Isolated nucleic acid, consisting of a sequence chosen from SEQ ID No.1, SEQ ID No.2 or SEQ ID No.15, or the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID No.1, or going from nucleotide 207 to nucleotide 950 on SEQ ID no 1, or going from nucleotide 3 to nucleotide 260 on SEQ ID no 2.
2. Acide nucléique isolé, constitué d'une séquence identique à au moins 80 % de la séquence SEQ ID n° 1 , SEQ ID n° 2 ou SEQ ID N° 15 ou de la séquence des fragments allant du nucleotide 1 au nucleotide 291 sur SEQ ID n°1 , ou allant du nucleotide 207 au nucleotide 950 sur SEQ ID n°1 , ou allant du nucleotide 3 au nucleotide 260 sur SEQ ID n°2.2. Isolated nucleic acid, consisting of a sequence identical to at least 80% of the sequence SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 15 or of the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID no.1, or going from nucleotide 207 to nucleotide 950 on SEQ ID no.1, or going from nucleotide 3 to nucleotide 260 on SEQ ID no.2.
3. Acide nucléique isolé, constitué d'une séquence identique à au moins 90 % de de la séquence SEQ ID n° 1 , SEQ ID n° 2 ou SEQ ID n° 15 ou de la séquence des fragments allant du nucleotide 1 au nucleotide 291 sur SEQ ID n°1 , ou allant du nucleotide 207 au nucleotide 950 sur SEQ ID n°1 , ou allant du nucleotide 3 au nucleotide 260 sur SEQ ID n°2.3. Isolated nucleic acid, consisting of a sequence identical to at least 90% of the sequence SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 15 or of the sequence of the fragments going from nucleotide 1 to nucleotide 291 on SEQ ID No 1, or going from nucleotide 207 to nucleotide 950 on SEQ ID No 1, or going from nucleotide 3 to Nucleotide 260 on SEQ ID No 2.
4. Acide nucléique isolé, dont la séquence code pour un polypeptide, comprenant la séquence d'acides aminés SEQ ID n° 3 à SEQ ID n°5, ou n° 16.4. Isolated nucleic acid, the sequence of which codes for a polypeptide, comprising the amino acid sequence SEQ ID No. 3 to SEQ ID No. 5, or No. 16.
5. Acide nucléique isolé, dont la séquence code pour un polypeptide constitué d'une séquence identique à au moins 80 % de la séquence SEQ ID n° 3 à 5, ou n° 16.5. Isolated nucleic acid, the sequence of which codes for a polypeptide consisting of a sequence identical to at least 80% of the sequence SEQ ID No. 3 to 5, or No. 16.
6. Acide nucléique isolé dont la séquence code pour un polypeptide constitué d'une séquence identique à au moins 90 % de la séquence SEQ ID n° 3 à 5, ou n° 16. 6. Isolated nucleic acid whose sequence codes for a polypeptide consisting of a sequence identical to at least 90% of the sequence SEQ ID No. 3 to 5, or No. 16.
7. Polypeptide isolé comprenant la séquence d'acides aminés SEQ ID n°3 à SEQ ID n°5, ou n° 16.7. Isolated polypeptide comprising the amino acid sequence SEQ ID No. 3 to SEQ ID No. 5, or No. 16.
8. Polypeptide isolé, constitué par une séquence identique à au moins 80% de la séquence SEQ ID n°3 à n°5, ou n° 16.8. Isolated polypeptide, consisting of a sequence identical to at least 80% of the sequence SEQ ID No. 3 to No. 5, or No. 16.
9. Polypeptide isolé, constitué par une séquence identique à au moins 90 % de la séquence SEQ ID n° 3 à n° 5, ou n° 16.9. Isolated polypeptide, consisting of a sequence identical to at least 90% of the sequence SEQ ID No. 3 to No. 5, or No. 16.
10. Vecteur de clonage et/ou d'expression contenant un acide nucléique selon l'une des revendications 1 à 6.10. Cloning and / or expression vector containing a nucleic acid according to one of claims 1 to 6.
11. Cellule hôte transfectée par un vecteur selon la revendication 10.11. Host cell transfected with a vector according to claim 10.
12. Utilisation d'un acide nucléique selon l'une des revendications 1 à 6 pour la fabrication de sondes nucléiques ou d'amorces oligonucléotidiques, capables d'hybrider spécifiquement, dans des conditions stringentes, avec tout ou partie dudit acide nucléique tel que défini dans l'une des revendications 1 à 6.12. Use of a nucleic acid according to one of claims 1 to 6 for the manufacture of nucleic probes or oligonucleotide primers, capable of specifically hybridizing, under stringent conditions, with all or part of said nucleic acid as defined in one of claims 1 to 6.
13. Anticorps dirigé contre le polypeptide tel que défini dans l'une des revendications 7 à 9.13. Antibody directed against the polypeptide as defined in one of claims 7 to 9.
14. Utilisation d'au moins un anticorps selon la revendication 13 pour la détection ou la purification d'un polypeptide tel que défini dans l'une des revendications 7 à 9 dans un échantillon biologique.14. Use of at least one antibody according to claim 13 for the detection or the purification of a polypeptide as defined in one of claims 7 to 9 in a biological sample.
15. Utilisation d'un acide nucléique ayant au moins 10 nucléotides, qui hybride spécifiquement avec l'une des séquences d'acide nucléique selon l'un des revendications 1 à 6 comme amorce pour l'amplification de tout ou partie d'une séquence rétrovirale associée à une affection cutanée. 15. Use of a nucleic acid having at least 10 nucleotides, which specifically hybridizes with one of the nucleic acid sequences according to one of claims 1 to 6 as a primer for the amplification of all or part of a sequence retroviral associated with a skin condition.
16. Utilisation d'un acide nucléique ayant au moins 10 nucléotides, qui hybride spécifiquement avec l'une des séquences d'acide nucléique selon l'un des revendications 1 à 6 comme sonde pour la détection d'une séquence rétrovirale associée à une affection cutanée.16. Use of a nucleic acid having at least 10 nucleotides, which specifically hybridizes with one of the nucleic acid sequences according to one of claims 1 to 6 as a probe for the detection of a retroviral sequence associated with a disease skin.
17. Procédé de détection in vitro de la présence d'une séquence nucléique rétrovirale associée à une affection cutanée chez un patient, comprenant les étapes consistant à : ai ) isoler l'ADN ou l'ARN d'un échantillon biologique ; b1 ) éventuellement produire de l'ADNc à partir dudit ARN ; d ) mettre en présence ledit ADN ou ADNc avec des acides nucléiques ayant au moins 10 nucléotides, qui hybride spécifiquement avec l'une des séquences d'acide nucléique selon l'un des revendications 1 à 6, comme amorces, dans des conditions permettant l'amplification spécifique d'une séquence telle que définie à l'une des revendications 1 à 6, d1 ) soumettre ledit ADN ou ADNc à une réaction d'amplification, e1 ) détecter les produits d'amplification, indicateurs de la présence d'une séquence rétrovirale associée à une affection cutanée, et/ou a2) la mise en contact d'un ADN ou ARN d'un échantillon biologique, avec un acide nucléique ayant au moins 10 nucléotides, qui hybride spécifiquement avec l'une des séquences d'acide nucléique selon l'un des revendications 1 à 6, comme sonde, marquée de manière détectable, dans des conditions permettant l'hybridation spécifique dudit ADN ou ARN avec ladite sonde ; b2) la détection des complexes d'hybridation formés, indicateurs de la présence d'une séquence rétrovirale associée à une affection cutanée.17. A method of in vitro detection of the presence of a retroviral nucleic acid sequence associated with a skin condition in a patient, comprising the steps of: ai) isolating DNA or RNA from a biological sample; b1) optionally producing cDNA from said RNA; d) bringing said DNA or cDNA into contact with nucleic acids having at least 10 nucleotides, which hybridizes specifically with one of the nucleic acid sequences according to one of claims 1 to 6, as primers, under conditions allowing the specific amplification of a sequence as defined in one of claims 1 to 6, d1) subjecting said DNA or cDNA to an amplification reaction, e1) detecting the amplification products, indicators of the presence of a retroviral sequence associated with a skin condition, and / or a2) bringing DNA or RNA from a biological sample into contact with a nucleic acid having at least 10 nucleotides, which specifically hybridizes with one of the sequences of Nucleic acid according to one of claims 1 to 6, as a probe, detectably labeled, under conditions allowing specific hybridization of said DNA or RNA with said probe; b2) the detection of the hybridization complexes formed, indicators of the presence of a retroviral sequence associated with a skin condition.
18. Procédé de diagnostic in vitro d'une affection cutanée, telle que notamment un psoriasis, un lichen ou un eczéma atopique, chez un patient, dans lequel on détecte, dans un échantillon biologique, la présence d'une séquence nucléique rétrovirale associée à l'une de ces pathologies, au moyen du procédé de la revendication 17.18. Method for in vitro diagnosis of a skin condition, such as in particular psoriasis, lichen or atopic eczema, in a patient, in which the presence, in a biological sample, is detected of a retroviral nucleic acid sequence associated with one of these pathologies, using the method of claim 17.
19. Composition pharmaceutique comprenant un acide nucléique selon l'une des revendications 1 à 6, en association avec un véhicule pharmaceutiquement acceptable.19. Pharmaceutical composition comprising a nucleic acid according to one of claims 1 to 6, in association with a pharmaceutically acceptable vehicle.
20. Composition pharmaceutique comprenant un polypeptide selon l'une des revendications 7 à 9, en association avec un véhicule pharmaceutiquement acceptable.20. Pharmaceutical composition comprising a polypeptide according to one of claims 7 to 9, in association with a pharmaceutically acceptable vehicle.
21. Composition pharmaceutique comprenant un anticorps selon la revendication 13, en association avec un véhicule pharmaceutiquement acceptable.21. A pharmaceutical composition comprising an antibody according to claim 13, in association with a pharmaceutically acceptable vehicle.
22. Utilisation d'une composition selon l'une des revendications 19 à 21 , pour la fabrication d'un médicament destiné à la prévention ou au traitement d'une affection cutanée, telle que notamment un psoriasis, un lichen ou un eczéma atopique.22. Use of a composition according to one of claims 19 to 21, for the manufacture of a medicament intended for the prevention or treatment of a skin condition, such as in particular psoriasis, lichen or atopic eczema.
23. Utilisation d'un acide nucléique selon l'une des revendications 4 à 6 ou d'un polypeptide selon l'une des revendications 7 à 9, pour le criblage de composés susceptibles d'inhiber l'activité dudit polypeptide.23. Use of a nucleic acid according to one of claims 4 to 6 or a polypeptide according to one of claims 7 to 9, for the screening of compounds capable of inhibiting the activity of said polypeptide.
24. Méthode in vitro de préparation d'un ensemble cellulaire destiné à une implantation chez un patient receveur, comprenant l'incorporation d'une séquence nucléotidique de l'une des revendications 1 à 6 dans des cellules en culture, telles que notamment des kératinocytes. 24. An in vitro method for preparing a cell assembly intended for implantation in a recipient patient, comprising incorporating a nucleotide sequence of one of claims 1 to 6 into cells in culture, such as in particular keratinocytes .
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