EP1220920A1 - Gene prv-1 et son utilisation - Google Patents

Gene prv-1 et son utilisation

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Publication number
EP1220920A1
EP1220920A1 EP00972665A EP00972665A EP1220920A1 EP 1220920 A1 EP1220920 A1 EP 1220920A1 EP 00972665 A EP00972665 A EP 00972665A EP 00972665 A EP00972665 A EP 00972665A EP 1220920 A1 EP1220920 A1 EP 1220920A1
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Prior art keywords
sequence
amino acids
nucleotides
polypeptide
prv
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EP00972665A
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German (de)
English (en)
Inventor
Heike Pahl
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Universitaetsklinikum Freiburg
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Universitaetsklinikum Freiburg
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • 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
    • 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
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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

Definitions

  • the invention relates to a nucleotide sequence which encodes the PRV-1 gene, recombinant DNA which contains this nucleotide sequence, vectors which contain the recombinant DNA and cells transformed therewith, and a PRV-1 polypeptide, antibodies against this polypeptide, process for Detection of the PRV-1 polypeptide and medicament containing the PRV-1 polypeptide or antibodies directed against the PRV-1 polypeptide.
  • Polycythemia rubra vera also known as polycythemia vera or P. vera
  • P. vera is a malignant hematological disease in which there is an increased formation of erythroid, granulocytic and megakaryocytic cells.
  • the disease is of clonal origin and arises from mutation of a single hematopoietic progenitor cell.
  • the incidence of P. vera is 4 to 6 per million inhabitants in Germany. If left untreated, the disease leads to death within 18 months. Treatment by bloodletting or chemotherapy extends the average survival to over 13 years.
  • P. vera is diagnosed using clinical criteria.
  • the clinical picture includes headache, pruritus, splenomegaly in two thirds of the patients, bleeding or thrombosis, high blood pressure in one third of the patients, gout caused by increased uric acid production and in some cases septic ulcers.
  • the most important laboratory finding is an increase in the values for hemoglobin, hematocrit, erythrocyte count and total erythrocyte volume as well as neutrophil granulocytosis or thrombocytosis in many cases. Since on the one hand most of the criteria are rather diffuse and on the other hand not all patients meet these criteria, it is often difficult to differentiate P.
  • PRV-1 gene Polycythemia rubra vera
  • An object of the invention is therefore a polynucleotide which codes for the PRV-1 gene and essentially comprises the sequence ID No. 1.
  • the polynucleotides of the present invention can be single or double stranded DNA or RNA. If it is RNA, it is clear to the person skilled in the art that "U” nucleotides are present instead of “T” nucleotides.
  • “Polynucleotide” means nucleic acids with 15 or more nucleotides.
  • the nucleotide sequence according to the invention is shown in FIG. 1.
  • the invention therefore relates to a polynucleotide which corresponds to the sequence in FIG. 1 and a polynucleotide whose nucleotide sequence has slight deviations (variant).
  • minor deviations mean those sequences in which a few, preferably no more than 50 and particularly preferably no more than 25 nucleotides can be exchanged, but the function of the gene encoded by the nucleotide sequence is not affected.
  • a base triplet coding for an amino acid can be replaced by another triplet which codes for the same amino acid.
  • less important areas can be slightly deleted and / or mutated.
  • the polynucleotide comprises nucleotides 36 to 1346 of sequence no. 1, that is to say the coding region of the PRV-1 gene. Further embodiments include nucleotides 36 to 1262 and 36 to 1238 of sequence No. 1. This area presumably codes for the active area of the PRV-1 polypeptide.
  • the polynucleotide of the invention may also include nucleotides 39 to 1346, 39 to 1262 or 39 to 1238 of sequence # 1 so that the codon encoding the start methionine is not included.
  • a preferred embodiment is a polynucleotide comprising nucleotides 99 to 1346, 99 to 1262 or 99 to 1238 of sequence # 1. The codons at the 5 'end which code for the signal peptide of the PRV-1 polypeptide are therefore not included.
  • the polynucleotide according to the invention can also be a fragment of the PRV-1 gene.
  • the fragment usually has more than 100 nucleotides, but preferably more than 300 nucleotides.
  • the fragments can also be used as primers or as probes, in particular for the PCR, in which case the fragments can be shortened according to the purpose.
  • primers are between 10 and 30 nucleotides in length and probes are between 15 and 50 nucleotides in length.
  • the PRV-1 gene is an endogenous gene, but is only expressed in a few organs in healthy individuals. It is usually expressed mainly in the hematopoietic organs, ie in the bone marrow and fetal liver, and weakly in the spleen, but not in the heart, muscle, pancreas or kidney. In patients who under P. vera suffer, this gene is very overexpressed, especially in the hematopoietic cells.
  • the PRV-1 gene codes for a protein which has the protein sequence shown in FIG. 2.
  • the signal peptide which is contained in the protein sequence of all surface molecules and is usually removed during processing of the protein, is separated by a hyphen.
  • the protein has the sequence ID No. 2.
  • a further aspect of the invention is therefore an essentially pure polypeptide of sequence No. 2 or a polypeptide of sequence No. 2 in which the signal peptide is not present (amino acids 22 to 437 of sequence No. 2). Further embodiments include amino acids 1 to 409, 22 to 409, 1 to 401 or 22 to 401 of sequence No. 2 (presumably active region of the protein).
  • the polypeptide according to the invention is preferably glycosylated, most preferably it is N-glycosylated. It can then be glycosylated on at least one of the amino acids Asn-46, Asn-189 and Asn-382 of the PRV-1 polypeptide (the amino acid numbers relate to sequence No. 2).
  • the invention also includes fragments of the polypeptides according to the invention which are N-glycosylated. The fragments are at least 50 amino acids long, preferably at least 100 amino acids, most preferably at least 150 amino acids.
  • the polypeptide can be O-glycosylated.
  • the PRV-1 polypeptide can, for example, have a glycosylphosphatidylinositol anchor due to its production. This is then bound to the amino acids which correspond to amino acids 407 to 409 of sequence ID no. 2.
  • a GPI anchor is used to anchor a protein to the outside of the cell membrane using a lipid.
  • GPI-linked proteins are also released into the medium.
  • shedding It has not yet been clarified whether this is a specific process, that is, such proteins are released from the membrane by enzymes in a controlled manner, or whether it is an unspecific loss of the anchor. It is therefore very likely that PRV-1 can be found both on the cell membrane and extracellularly.
  • the secreted, non-membrane-bound form is probably more important for the effect as a growth factor and growth inhibitor, since it can diffuse as a growth factor and reach other cells.
  • the gene codes for a surface receptor of the uPAR / Ly6 family. This family of receptors can produce mitogenic signals, i.e. Transmit signals that stimulate cell division. It is therefore believed that overexpression of the PRV-1 gene, including on the bone marrow cells of P. vera patients, contributes to hyperproliferation of these cells.
  • the polypeptide encoded by the PRV-1 gene is generated in a suitable manner from recombinant DNA, the recombinant DNA preferably having the nucleotide sequence ID No. 1 or at least the coding region of the PRV-1 Gene, ie nucleotides 36 to 1346 of sequence ID No. 1, or at least nucleotides 39 to 1262 or 39 to 1238, functionally linked to a promoter.
  • the recombinant DNA can also comprise only a fragment of sequence No. 1.
  • Another object of the invention is a vector which contains the recombinant DNA for the PRV-1 polypeptide or a fragment thereof, and a host cell transfected or transformed with this vector.
  • the host cells can be prokaryotic, for example bacteria such as E. coli. However, non-glycosylated polypeptides are expressed. Eukaryotic host cells which can post-translationally glycosylate and otherwise modify the expressed protein are therefore preferred. Examples of eukaryotic host cells are insect cells such as Sf9 cells for expression after infection with recombinant baculoviruses, mammalian cells such as 293 cells, COS cells, CHO cells, HeLa cells. These examples are not exhaustive. Yeast cells are also possible as host cells.
  • glycosylation pattern can differ depending on the host cell.
  • the biological activity of the expression product can thus also vary. Host cells which glycosylate the expression product in such a way that the biological activity of the protein is retained are particularly preferred.
  • Another aspect of the invention is a method for producing a polypeptide according to the invention.
  • a DNA coding for the polypeptide according to the invention is brought to expression in a host cell. Depending on whether that expressed polypeptide is secreted from the host cell into the culture medium or remains in the cell, the culture medium or the cells are used for the further recovery of the polypeptide.
  • the polypeptide according to the invention is then enriched or purified by methods known in the art, for example chromatographic methods. Methods of protein purification are described, for example, in Scopes, R., Protein Purification: Principles and Practice (3rd edition), Springer Verlag (1994).
  • the method according to the invention comprises a step in which glycosylated polypeptide is enriched or purified.
  • This step can take place before the polypeptide according to the invention has been essentially purified or after it has already been essentially purified. In the latter case, the glycosylated portion of the purified polypeptide is then separated off and recovered. In the most preferred embodiment of the method, specifically N-glycosylated polypeptide is obtained. In a further embodiment of the method, polypeptide is obtained which is glycosylated on at least one of the amino acids Asn-46, Asn-189 and Asn-382 of sequence No. 2.
  • the PRV-1 polypeptide obtained from granulocytes or recombinantly produced can be used both for the diagnosis of polycythemia vera and for the treatment of the disease.
  • an "antisense” RNA molecule that is an RNA that is complementary to the PRV RNA. Since the PRV-1 RNA initially has the sequence 5 '-AAAAGCAGAAAGAGATTACCAGCC-3' (Seq. ID-No. 3), the required antisense RNA against this sequence would have the following nucleotide sequence: 5'-GGCTGGTAATCTCTTTCTGCTTTT-3 '(Seq. ID-No. 4). This antisense RNA is inserted into a vector and introduced into P. vera cells.
  • This RNA is introduced, for example, via transfection, the one used for the transfection Vector is preferably designed so that it is specifically introduced into the P. vera cells.
  • the expression of the antisense RNA means that the PRV-1 mRNA can no longer be translated into a polypeptide. No PRV-1 protein is then produced in cells treated in this way.
  • Another object of the invention is therefore a method for the detection of P. vera, which is characterized in that the PRV-1 polypeptide or an epitope thereof is detected and the extent of expression is determined.
  • the detection is suitably carried out with an immunoassay using antibodies which are directed against the PRV-1 receptor.
  • the known variants of immunoassays are suitable as test methods, in which specific antibodies are used for the PRV-1 poly ⁇ eptide together with other labeled antibodies, which can be immobilized or in solution.
  • the marking can take place in a manner known per se, e.g. with radioactive isotopes, through fluorescence or luminescence, with enzymes, through color-forming reactions or other groups suitable for determination.
  • ELISA tests are particularly preferred.
  • the antibodies required for the specific detection of the PRV-1 receptor can also be produced in a manner known per se. Both monoclonal and polyclonal antibodies are suitable, the use of monoclonal antibodies being preferred.
  • Protein derived peptides can be used.
  • the polyclonal antibodies are usually generated by immunizing a suitable host (rabbit) with the PRV-1 polypeptide, optionally bound to an immunological carrier (adjuvant), and eliciting an immune response.
  • Monoclonal antibodies can be generated in a manner known per se using the hybridoma technique.
  • the antibodies can be purified by affinity purification. The production and purification of antibodies are described, for example, in "Antibodies: A Laboratory Manual” by Harlow and Lane, Cold Spring Harbor Laboratory Press.
  • polyclonal or monoclonal antibodies directed against PRV-1 can also be used for the therapy of the disease.
  • the PRV-1 receptor can be detected using an RT-PCR method.
  • RNA is first isolated from the PRV-1 overexpressing cells, usually granulocytes. Then reverse transcription is carried out in a manner known per se using an RT primer.
  • the RT primer is preferably a primer with the following nucleotide sequence (SEQ ID No. 7)
  • This DNA is then amplified in a PCR reaction in a manner known per se.
  • the following two primers are preferably used for the amplification cycles.
  • Antisense primer SEQ ID-No. 9
  • the PCR signal is only positive in those cases in which the PRV-1 gene is also expressed. As stated above, this is only the case if the patient has P. vera. PRV expression does not occur in the granulocytes in healthy patients. The absence of an RT-PCR signal indicates that there is no P. vera.
  • the quantification in the RT-PCR method is preferably carried out using TaqMan e technology. In addition to primers, a "sample” is also required for this quantification.
  • the preferred sequence of the "probe” is 5 '-TTCTTGTTGAACCACACCAGACAAATCGG-3'
  • a blotting method preferably a Northern blot
  • the RNA is isolated from granulocytes and then blotted, e.g. Northern blot, examined for the expression of PRV-1.
  • the cDNA sequence of SEQ ID No. 1 or a section of the sequence can be used as a probe.
  • Hybridization only occurs if the granulocytes come from a patient with P. vera, since only then is there an expression on the granulocytes. The absence of hybridization indicates that the person from whom the granulocytes are derived has no P. vera.
  • a fragment of the gene can also be used for Northern blot hybridization.
  • One such fragment is usually more than 100 bases long, preferably more than 300 bases long.
  • various different fragments of the gene can be produced which can be used as probes in the Northern blot. If the fragments originate from the cDNA, they are present as double strands, which have to be separated into the single strands for hybridization. Suitable examples are the Bam HI-Pstl fragment from base pair 420 to base pair 831 or the Pstl-Pstl fragment ent from base pair 831 to base pair 1900.
  • the detection of PRV-1 mRNA and thus the PRV-1 expression can also be carried out by first transversely transcribing the mRNA in an RT-PCR reaction and then amplifying the cDNA and then amplifying the DNA with a probe in a hybridization process is detected.
  • the invention therefore furthermore relates to a medicament which, in addition to conventional carriers, contains antibodies directed against the PRV-1 receptor.
  • the PRV-1 polypeptide has hematopoietic activity.
  • the PRV-1- Polypeptide is able to stimulate certain hematopoietic progenitor cells to form erythroid colonies.
  • the N-glycosylated polypeptides of PRV-1 in particular have this function.
  • the N-glycosylated PRV-1 polypeptides and fragments thereof which have the growth factor activity are preferred.
  • Another aspect of the invention is therefore a medicament which, in addition to a pharmaceutically acceptable carrier, contains the PRV-1 polypeptide or a biologically active fragment thereof. It is preferably a glycosylated PRV-1 polypeptide, more preferably an N-glycosylated PRV-1 polypeptide or a biologically active fragment thereof.
  • the invention also relates to medicaments which contain at least one polynucleotide according to the invention.
  • the present invention further relates to the use of PRV-1 polypeptide or a biologically active fragment thereof or a biologically active variant thereof as a growth factor in vivo and ex vivo.
  • the PRV-1 polypeptide or a biologically active fragment thereof or a biologically active variant thereof can be used to treat all pan-cytopenias and pan-cytopathies in the bone marrow and in the circulation (change in the cellular components of the peripheral blood and the bone marrow).
  • the polypeptides of the present invention can be used, for example, for the treatment of anemia with kidney failure, chemotherapy or whole body radiation, for the treatment of neutropenia and thrombocytopenia under chemotherapy or whole body radiation, for the ex vivo treatment of peripheral or bone marrow stem cells for expansion (reproduction) and retransfusion in the Patients, and for the treatment of sepsis, "systemic inflammatory response syndrome" (SIRS) or regional inflammatory response.
  • SIRS systemic inflammatory response syndrome
  • the polypeptides of the present invention or medicaments containing them can be applied in various ways.
  • the dosage forms include intravenous, intramuscular, subcutaneous, intraperitoneal, oral, transdermal and transmucosal administration.
  • the polynucleotides according to the invention can also be used for the treatment of pan-cytopenias and pan-cytopathies.
  • the aim is the expression of a PRV-1 polypeptide or a functional fragment thereof in cells of the patient concerned.
  • gene therapy procedures are used.
  • Cells from the patient can be isolated and transfected with a polynucleotide according to the invention (ex vivo manipulation) in order to then be returned to the patient.
  • Methods are also conceivable in which the polynucleotides according to the invention reach the target cells by viral transfer. Expression of the introduced nucleic acids then leads to hematopoietic activity.
  • the PRV-1 polypeptide in higher concentration has an inhibitory effect on the growth of cells.
  • PRV-1 protein practically completely prevented the formation of erythroid and granulocytic / monocytic colonies.
  • This effect is similar to the effect of interferon- ⁇ , which is used therapeutically in chronic myeloid leukemia (CML) and in P. vera.
  • CML chronic myeloid leukemia
  • a body's inhibitory substance has great advantages over a chemical cytostatic like
  • the present invention provides a hematopoiesis-inhibiting substance, the inhibitory activity being concentration-dependent.
  • Another aspect of the invention is therefore the use of a PRV-1 polypeptide described in this application for inhibiting the growth of cells, in particular the use as a cytostatic.
  • the polypeptide is preferably used to inhibit the growth of hematopoietic cells.
  • the invention also relates to the use of a polypeptide according to the invention for the manufacture of a medicament for the treatment of proliferative diseases.
  • diseases are in particular myeloproliferative diseases, P. vera, essential thrombocythemia, myelofibrosis, CML and all leukemias and lymphomas as well as solid tumors.
  • Another aspect of the invention is the use of a polynucleotide described in this application, a biologically active fragment or a biologically active variant thereof to inhibit the growth of cells.
  • the polynucleotide can be incorporated into a suitable vector and transfected into suitable target cells. After expression of the PRV-1 polypeptide or a biologically active fragment thereof or a biologically active variant thereof in an appropriate concentration, the growth-inhibiting effect comes into play.
  • the polynucleotide can also be incorporated into a viral vector, after which corresponding target cells are infected virally, which leads to the expression of PRV-1.
  • the invention also relates to the use of a polynucleotide of this application for the manufacture of a medicament for the treatment of proliferative diseases such as e.g. the myeloproliferative diseases, P. vera, essential thrombocythemia, myelofibrosis, CML as well as all leukaemias and lymphomas as well as solid tumors.
  • proliferative diseases such as e.g. the myeloproliferative diseases, P. vera, essential thrombocythemia, myelofibrosis, CML as well as all leukaemias and lymphomas as well as solid tumors.
  • kits for the detection of either polycythemia vera or disorders of the hematopoietic system contain a polynucleotide according to the invention and / or a polypeptide according to the invention and / or one or more antibodies according to the invention.
  • the kit can also contain a container or compositions that are used for Carrying out detection reactions are included. Examples of such compositions are buffer solutions, reagents for blocking membranes, hybridization solutions, secondary antibodies,
  • the kit is preferably used to carry out PCR reactions, RT-PCR, Northern blots, Southern blots, Western blots, ELISA, RIA or similar reactions.
  • the approach separated into two phases.
  • the upper bright phase was removed and centrifuged for 10 minutes at 1800 g and RT.
  • the cells were centrifuged at 1800 g and RT for 10 minutes.
  • the cell pellet contained 95-99% pure granulocytes.
  • PRV-1 has growth factor activity
  • Embryos were removed from a pregnant mouse on day 13.5 after fertilization.
  • the fetal livers were removed.
  • the cells contained therein were stained with antibodies and enriched for certain cells by column chromatography and depleted for other cell types.
  • the result is a cell mixture that is enriched for certain hematopoietic progenitor cells (so-called colony forming units-erythroid, CFU-E).
  • CFU-E colony forming units-erythroid
  • 293 T cells are one established human embryonic kidney cell line. 293 T cells are stably transfected with several genes of a retrovirus. If these 293-T cells are transfected with two plasmids called pOS and pKAT, the 293-T cells produce a retrovirus that can infect murine fetal liver cells. If the 293 T cells are transfected with an empty pOS vector and pKAT, a "wild type" retrovirus is produced which only expresses retroviral proteins.
  • a human gene has been cloned into the pOS vector, for example PRV-1
  • a retrovirus is produced which, when it has infected cells, expresses this protein.
  • the retrovirus is secreted by the 293 T cells into the cell culture medium.
  • the cell culture medium of the transfected 293 T cells which contains the retrovirus, is harvested and filtered once through a 0.45 ⁇ m filter.
  • these cells are mixed with the filtered cell culture medium which contains the retrovirus and centrifuged for 2 hours at 1800 rpm, 20 ° C. with the addition of polybrene.
  • the transfected fetal liver cells are then cultivated in a medium (Methocult, from Cell Systems) which, in addition to the usual salts and amino acids, fetal calf serum, 0.0001-0.4 IU / ml erythropoietin (EPO) and methyl cellulose (0, 8%) contains.
  • a medium Metalhocult, from Cell Systems
  • the EPO requires the CFU-E to form hematopoietic colonies.
  • the methyl cellulose makes the medium jelly-like, and it is possible to fix individual cells in this jelly so that, unlike in a liquid medium, they cannot move. It can therefore be observed whether or not a hematopoietic colony is formed from a single cell.
  • CFU-It form erythroid colonies, i.e. colonies that contain red blood cells and their progenitor cells.
  • GFP a non-hematopoietically active protein.
  • Approach 4 Cells transfected with pOS-PRV-1 (vector + gene according to the invention).
  • Table 1 The results of three tests carried out as described are shown. The numbers indicate the number of colonies.
  • the "packaging cell line”, 293-T produces a retrovirus not only after transfection with the pOS and pKAT vectors.
  • the 293 T cells also synthesize the protein encoded by the gene cloned in pOS, in this case PRV-1. Is the gene product a soluble protein, it is secreted into the medium surrounding the "packaging cell line", 293-T. If the 293 T cells are transfected only with the pOS vector, without pKAT, no retroviruses are produced.
  • the cell culture medium then contains only the soluble protein produced by the cells.
  • Medium from pOS-PRV-1 transfected cells, without retrovirus, is mixed with CFU-Es, and plated out in the methyl cellulose medium and the colonies formed are counted.
  • Table 2 Solubility of PRV-1. The numbers indicate the number of colonies.
  • PRV-1 also has inhibitory, cytostatic effects.
  • peripheral blood cells Since a small number of progenitor cells circulate in peripheral blood even in healthy people, hematopoietic colonies can be grown from peripheral blood cells in a suitable medium (methyl cellulose).
  • a suitable medium methyl cellulose.
  • 40 ml of peripheral venous blood was taken from a healthy donor (with the presentation of heparin or EDTA as an anti-coagulant).
  • 15 ml of Ficoll / Hypaque were added to the blood and centrifuged at 1600 rpm without brake for 40 minutes. This creates a density gradient that separates the blood into its cellular components.
  • the so-called "mononuclear cells”, among which the stem cells are located, can be found after centrifugation on the interphase between serum and Ficoll. This interphase was removed, washed in PBS (isotonic saline). This results in purified mononuclear cells in which there are approximately 0.1% hematopoietic stem cells.
  • the mononuclear cells were taken up in a particularly rich medium (IMDM) containing 3% FCS (fetal calf serum). This 3% FCS / IMDM also contained the modifications, which means that it was either PRV-1 or not.
  • IMDM particularly rich medium
  • FCS fetal calf serum
  • IMDM mononuclear cells in IMDM were added at a density of 7 ⁇ 10 5 cells / ml to a commercially available medium from Stem Cell Technologies (Methocult), which contains IMDM and 30% FCS, 1% BSA (Bovine Serum Albumin), mercaptoethanol,
  • a cell line was also constructed that expresses a very high amount of PRV-1.
  • the PRV-1 which is produced by these cells, is modified in such a way that it no longer has a lipid anchor.
  • the expression product consists of amino acids 1-401 of the sequence SEQ ID NO: 2, so amino acids 402-437 are missing.
  • This modified PRV-1 is therefore not, like wild-type PRV-1, built into the cell membrane by means of a lipid anchor, but is completely secreted from the cells.
  • the cell line was as in example
  • the growth factor PRV-1 is N-glycosylated
  • Granulocytes were isolated from a patient with P. vera and protein extracts were prepared from these cells using the standard protocol. These protein extracts were treated according to the protocol of the "N-Glycosidase F Deglycosylation Kit” from Boehringer Mannheim. Specifically, this means that the protein extracts were mixed with a "denaturing buffer", heated for 3 minutes at 95 ° C. and then either only with “reaction buffer” or with “reaction buffer” plus N-glycosidase were added. The mixture was incubated at 37 ° C. overnight and the proteins were analyzed on a PAGE gel electrophoresis with subsequent Western blot. The PRV-1 protein was raised with an antibody against a protein with the amino acid sequence ID-No. 5 detected.

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Abstract

L'invention concerne une séquence nucléotidique qui est codée pour la protéine PRV-1 et qui contient principalement la séquence ID N° 1. L'invention concerne également un procédé permettant de détecter ce gène de l'ARNm codé par ce gène et du polypeptide codé par ce gène.
EP00972665A 1999-09-30 2000-09-29 Gene prv-1 et son utilisation Withdrawn EP1220920A1 (fr)

Applications Claiming Priority (3)

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DE19947010 1999-09-30
DE19947010A DE19947010A1 (de) 1999-09-30 1999-09-30 Das Gen PRV-1 und dessen Verwendung
PCT/EP2000/009594 WO2001023554A1 (fr) 1999-09-30 2000-09-29 Gene prv-1 et son utilisation

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EP1220920A1 true EP1220920A1 (fr) 2002-07-10

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BG (1) BG106554A (fr)
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CA (1) CA2387702A1 (fr)
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EA (1) EA200200286A1 (fr)
EE (1) EE200200168A (fr)
HR (1) HRP20020269A2 (fr)
HU (1) HUP0203080A2 (fr)
IL (1) IL148770A0 (fr)
MX (1) MXPA02002800A (fr)
NO (1) NO20021498L (fr)
PL (1) PL354184A1 (fr)
SK (1) SK4262002A3 (fr)
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ZA (1) ZA200202379B (fr)

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IL158599A0 (en) * 2003-10-26 2004-05-12 Yeda Res & Dev Methods of modulating hematopoiesis
TW201400132A (zh) 2012-05-21 2014-01-01 Genentech Inc 改善血腦屏障運送之安全性之方法
US20210147807A1 (en) * 2017-06-14 2021-05-20 Helmholtz Zentrum Munchen - Deutsches Forschungszentrum Fur Gesundheit Und Umwelt (Gmbh) Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells

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WO2000036102A2 (fr) * 1998-12-16 2000-06-22 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant pour ceux-ci
WO1998050552A1 (fr) * 1997-05-06 1998-11-12 Zymogenetics, Inc. Nouveaux antigenes tumoraux
CA2328895A1 (fr) * 1998-06-02 1999-12-09 Genentech, Inc. Proteines membranaires et acides nucleiques codant ces proteines
DE19849044A1 (de) * 1998-10-23 2000-04-27 Univ Ludwigs Albert Das Gen PRV-1 und dessen Verwendung

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DE19947010A1 (de) 2001-04-05
KR20020047191A (ko) 2002-06-21
WO2001023554A1 (fr) 2001-04-05
NO20021498D0 (no) 2002-03-26
HUP0203080A2 (hu) 2002-12-28
EA200200286A1 (ru) 2002-12-26
HRP20020269A2 (en) 2003-06-30
BR0014444A (pt) 2002-06-11
CZ20021094A3 (cs) 2002-06-12
EE200200168A (et) 2003-04-15
CN1377408A (zh) 2002-10-30
MXPA02002800A (es) 2002-07-22
ZA200202379B (en) 2003-10-29
JP2003510077A (ja) 2003-03-18
BG106554A (bg) 2003-01-31
IL148770A0 (en) 2002-09-12
CA2387702A1 (fr) 2001-04-05
SK4262002A3 (en) 2002-09-10
PL354184A1 (en) 2003-12-29
AU1132401A (en) 2001-04-30
NO20021498L (no) 2002-05-23

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