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• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3513—Protein; Peptide

Definitions

• the present invention relates to new
• compositions containing, at a high concentration and / or at a high degree of purity, a human cyclin A, said compositions possibly being useful in particular for preparing agents for detecting cyclin, such as in particular antisera or antibodies, monoclonal or not .
• the invention also relates to a process allowing the preparation of these compositions.
• the invention also relates to a sequence
• nucleotide encoding human cyclin A as well as expression vectors incorporating this sequence to express cyclin A and a process for the preparation of cyclin A and compositions containing it by expression of these vectors.
• the invention also relates to a method and agents for detecting or diagnosing cell proliferation.
• the invention relates to a method and agents for inhibiting cell proliferation.
• Chronic hepatitis B virus (HBV) infection is known to be a risk factor for HBV.
• HBV HBV in the vicinity of a cell gene has been demonstrated only in a tumor in which the viral DNA has interrupted the region coding for the beta receptor for retinoic acid (31, 32).
• the present invention has made it possible to establish that there is an individualized site for integrating the DNA of the HBV virus in the human genome (chromosome 4q27) and more specifically in a gene coding for a human cyclin A of which
• hepatic liver in an early state of tumor development without significant chromosomal rearrangement, exhibiting none of the histological features of chronic hepatitis or cirrhosis, in a clonal proliferation of a cell containing a single integration of HBV, absent in the adjacent hepatic tissue , so it is more that it is likely, by the resulting dysregulation of the function of cyclin A, that this insertion is
• cyclin A mRNA and protein
• cyclin A appears at the time of the S phase and of the G2 / M phase of the cell cycle and is therefore associated with cell division.
• cyclin A was maximum in acute myeloblastic hyperleukocytic leukemias with rapid doubling time and very low in primary liver cancer for which the doubling time of the tumor is slow.
• the inventors have also discovered that it is possible to block the synthesis of cyclin A in heparocytes using a human "anti-sense" cyclin A DNA.
• the DNA corresponding to the insertion locus was cloned and sequenced and expression vectors were constructed making it possible to produce the encoded cyclin A in large quantities and the subject of the invention is a method of preparation of human cyclin A by genetic expression as well as new compositions concentrated in human cyclin A with a high degree of purity.
• the subject of the invention is also the sequence
• nucleotide encoding human cyclin A and vectors containing and / or expressing the sequence encoding cyclin A.
• the subject of the invention is in particular the sequence marked No.: 1 on the attached list of sequences.
• This sequence can be associated with other usual sets such as promoters, initiation or stop signals or introns or other non-coding sequences at the 3 ′ and / or 5 ′ end. It also includes the variants obtained in particular by substitution of codons or nucleotides preserving the meaning of the code, or by substitutions, insertions or deletions and coding for an equivalent polypeptide and in particular preserving the antigenicity of the polypeptide. It also includes any fragment allowing the expression of a polypeptide retaining this
• sequence also extends to the human cyclin A gene, incorporating or not incorporating some or all of the elements of an operon, and devoid of natural flanking sequences, and to any fragment allowing the expression of a polypeptide retaining a cyclin A antigenicity
• the invention also relates to a method of
• detection allowing the diagnosis of cell proliferation, in which the level of human cyclin A in the cells concerned is determined, in particular using polyclonal antibodies, for example animal antisera induced by immunization with human cyclin A or antibodies polyclonal cells obtained by expression of the human cyclin A protein in bacterial systems, or human anticyclin A monoclonal antibodies.
• polyclonal antibodies for example animal antisera induced by immunization with human cyclin A or antibodies polyclonal cells obtained by expression of the human cyclin A protein in bacterial systems, or human anticyclin A monoclonal antibodies.
• the invention therefore also relates to such antibodies.
• the invention also relates to a method of
• cDNA also covers the relevant fragments of the cDNA sequence.
• this cDNA can be, in particular, either included in a retroviral vector (for example according to the techniques described by JA Wolff et al., Proc. Natl. Acad. Sci.84: 3344, 1987 or by DG Miller et al., Mol. Cell. Biol. 10: 4239, 1990), or carried by a desialilated protein capable of binding to a receptor carried by the target cell for the entry of the cDNA therein, the cDNA and the protein being in particular linked by a polylysine, (for example according to the techniques described by GY Wu & GH Wu, J. Biol. Chem. 263: 14621, 1988 and by GH Wu et al., J. Biol. Chem. 264: 16985, 1989), or included in a liposome.
• a retroviral vector for example according to the techniques described by JA Wolff et al., Proc. Natl. Acad. Sci.84: 3344, 1987 or by DG Miller
• the invention therefore also relates to the antisense cyclin A cDNA, the plasmids including this cDNA and the retroviral vectors incorporating it.
• a subject of the invention is also the compound consisting of the antisense cDNA and the desialilated protein, as well as the liposomes including the antisense cDNA.
• FIG. 1 represents
• the open box represents the 3.1 kb viral insert indicating the orientation of the virus (+);
• the restriction sites are: B, Bam HI; Bg, Bgl II; E, Eco RI; H, Hind III; P, Pst I; S, Sst I;
• A is a 2.2 kb Sst I / Bgl II fragment
• B is a 1.1 kb Eco RI / Bgl II fragment
• fragments X (0.5 kb Bgl II) and Y (0.7 kb Bam Hl / Pst I) contain the left and right recombination sites;
• FIG. 2 represents
• HCC early primary hepatocellular carcinoma
• the library was built by partial digestion of tumor DNA by Hind III and ligation into Hind III branches of phage L 47. A population of overlapping phage clones was isolated using the cloned HBV as a probe. Subcloning was done using Bluescript vectors
• the cDNA was isolated from a library constructed from oligo (dT) primed adult human liver mRNA (32).
• FIG. 1A shows the resulting restriction map of the HBV provirus and the flanking cell sequences in the tumor.
• FIG. 1B shows the subclones A, X, Y, B, lacking repetitive sequences, which were derived from the phage clones. Sequencing of fragments X and Y (FIG. 1B) of the provirus-host junction, with detailed restriction analysis of the insert
• virus-cell junctions are both located in the end regions of HBV (nucleotides 1840 and 1617 in the viral nucleus and the open reading frames X, respectively (1) ( Figure 1D).
• FIG. 1C The restriction map of the region where HBV integration takes place, in its native configuration, has been represented in FIG. 1C.
• a comparison of the restriction maps of FIGS. 1A and 1C shows that the viral integration in the tumor did not lead to major rearrangements in its vicinity.
• Hybridization of clone B by Northern blots of polyadenylated and non-poiyadenylated RNA from normal newborn liver made it possible to identify two polyadenylated molecules of transcribed RNA of 1.8 kb and 2.7 kb (FIG. 1E).
• the subsequent screening of a phage library of normal human liver cDNA allowed the isolation of 5 cDNA clones which, according to their restriction maps, represent the same RNA molecule.
• the protein deduced is a human cyclin A.
• the N-terminus of the protein does not show sensitive homology to other known proteins, including other cyclins, which is a characteristic common to all known cyclins.
• Fragment A ( Figure IB) was found to react by hybridization with the cDNA ( Figure 1). Fragments A and B were therefore partially sequenced and sequences were found from the 5 'and 3' ends of the cDNA respectively. The resulting orientation of the cyclin A gene is from left to right in FIG. 1. The study of the sequence then shows that cyclin A has several exons and, owing to the fact that the cellular sequences of the fragments of virus-host junction X, Y (FIG. 1B) are not found in the cDNA, it follows that the integration of the DNA of the HBV virus takes place in an intron of the gene.
• the cloned cDNA molecule is inserted into a bacterial or preferably eukaryotic expression vector, for example by cloning of the cDNA in the Bluescript cloning vector, insertion into the plasmid expression vector pET -3b, transfection of a host, for example bacterial, the ends of the sequence being determined by the restriction enzymes Eco RI and Sma I.
• a host for example bacterial
• the ends of the sequence being determined by the restriction enzymes Eco RI and Sma I.
• Cyclin A preparation can be obtained from bacterial culture by gel purification
• CDNA is used as a probe to study
• Normal rat hepatocytes can be maintained in primary culture for a period of about ten days keeping part of the differentiated functions of the hepatocyte. However, there is no proliferation of cells. It is possible to stimulate DNA synthesis by adding epidermal growth factor EGF, pyruvate and
• RNA and of protein cyclin A are located at the phase of synthesis of DNA (third day of the culture after stimulation) contrary to cyclin Bl which is detected from the first day of hepatocyte culture until the fifth day.
• cyclin A is not detected in hepatocytes whose DNA synthesis has not been stimulated (unlike cyclin B1).
• Cyclin A is not detected in a normal liver. - Cyclin A is detected (RNA and protein) at the time of the cellular DNA synthesis phase.
• cyclin A The accumulation of cyclin A is followed by a slight decrease in the level of cyclin A, then a re-ascent at the time of mitosis.
• cyclin B1 has an entirely different profile since it is detected in a normal liver and its level does not increase at the time of phase S. IV Detection of cyclin A on sections of tissue obtained by biopsy.
• BSA bovine serum albumin
• Tris-HCl Tris-HCl
• Reading allows a certain quantification of the level of cyclin A present in the tissue by counting the
• antibodies in particular ELISA and in particular ELISA
• the preparation of anticyclin A antibodies can be done in rabbits by injection of cyclin A obtained by gel purification in order to obtain a rabbit anti-serum.
• Detection with quantification of human cyclin A in the tissues can then be carried out by Western blot or immunohistochemistry.
• This detection can use the counting of labeled cell nuclei and the comparison of the counted rate with the rate of the same normal tissue.
• constructions are used to bring the antisense DNA inside the cell to be treated.
• a retroviral vector (JA Wolff et al. And DG Miller et al. already cited) including the anti-sense cDNA and capable of infecting the target cell and therefore of inserting the anti-cDNA sense in the genome of it.
• human cyclin A cDNA is inserted in the antisense position in a plasmid, in particular the commercial plasmid PKC4, with an appropriate promoter, for example the promoter of the SV40 virus.
• the plasmids which integrate the DNA well in the antisense position are selected using a restriction map before introducing the cDNA and its promoter into the retrovirus.
• the antisense cDNA is attached to a desialilated protein (G. Y. Wu & C.H. Wu, and C.H. Wu et al. already cited) via a polylysine. After binding to the target cell, the cDNA is introduced into the cytoplasm of the cell in which it is transcribed into antisense RNA which will block the synthesis of cyclin A by hybridization on the cellular RNA.
• a desialilated protein G. Y. Wu & C.H. Wu, and C.H. Wu et al. already cited
• Liposomes including antisense cDNA can also be used.
• CHARACTERISTICS 1293 base pairs peptide.
• PROPERTIES coding for human cyclin A. 1 CGGCAGCCAGCCTATTCTTTGGCCGGGTCGGTGCGAGTGGTCGGCTGGGCAGAGTGCAC60 GCTGCTTGGCGCCGCACGTGATCCCGCCGTCCACTCCCGGGAGCAGTGATGTTGGGCACA

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• 1990
  • 1990-02-12 FR FR9001596A patent/FR2658204A1/fr active Granted
• 1991
  • 1991-02-06 DE DE69133067T patent/DE69133067T2/de not_active Expired - Lifetime
  • 1991-02-06 EP EP91904077A patent/EP0471804B1/fr not_active Expired - Lifetime
  • 1991-02-06 AT AT91904077T patent/ATE220717T1/de not_active IP Right Cessation
  • 1991-02-06 WO PCT/FR1991/000089 patent/WO1991012324A1/fr not_active Ceased
  • 1991-02-06 ES ES91904077T patent/ES2181667T3/es not_active Expired - Lifetime
• 1995
  • 1995-06-05 US US08/460,895 patent/US5849508A/en not_active Expired - Fee Related
• 1998
  • 1998-12-15 US US09/210,889 patent/US6103887A/en not_active Expired - Fee Related

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