WO1986006096A1 - Facteur de von willebrand - Google Patents

Facteur de von willebrand Download PDF

Info

Publication number
WO1986006096A1
WO1986006096A1 PCT/US1986/000760 US8600760W WO8606096A1 WO 1986006096 A1 WO1986006096 A1 WO 1986006096A1 US 8600760 W US8600760 W US 8600760W WO 8606096 A1 WO8606096 A1 WO 8606096A1
Authority
WO
WIPO (PCT)
Prior art keywords
vwf
dna
human
cell
sequence
Prior art date
Application number
PCT/US1986/000760
Other languages
English (en)
Inventor
David Ginsburg
Stuart H. Orkin
Randal J. Kaufman
Original Assignee
The Children's Medical Center Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Children's Medical Center Corporation filed Critical The Children's Medical Center Corporation
Publication of WO1986006096A1 publication Critical patent/WO1986006096A1/fr
Priority to US07/559,509 priority Critical patent/US8597910B1/en

Links

Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • VWF Von Willebrand Factor
  • Factor VIIIR Factor VIIIR
  • Factor VIII Blood coagulation in mammals involves the interaction of a number of protein factors and tissue components.
  • One coagulation factor complex is called Factor VIII and is composed of at least two distinct proteins: Factor VIIIC (antihemophili ⁇ factor, the protein that corrects the coagulation disorder known as hemophilia A), and VWF.
  • VWF is a protein that binds to platelets and is an essential component in platelet-vessel wall interaction during the clotting process.
  • Zimmerman et al. Progress in Hematology Vol. XIII, "Factor VIII/Von Willebrand Factor” (Grune & Stratton 1983). Diminished or abnormal VWF activity can result in Von Willebrand's Disease (VWD) , a relatively common and complex hereditary bleeding disorder.
  • VWD Von Willebrand's Disease
  • the Factor VIII complex obtained as a cryoprecipitate from donor blood, is administered as a therapy for VWD.
  • Mitra U.S. Patent 4,386,068.
  • cDNA's specific for other coagulation proteins, e.g., Factor VIIIC have been cloned and expressed in host systems [Wood et al (1984) Nature 312: 330-3371].
  • VWF is particulary difficult to analyze and produce because it is very large and there has been very little, if any, sequence data available heretofore. Moreover, cells that produce substantial amounts of VWF, e.g., endothelial cells and megakaryocytes, are difficult to grow in culture. Summary of the Invention
  • One aspect of the invention generally features producing VWF using an expression vector that includes: 1) a DNA sequence encoding a functional mammalian VWF protein; and 2) regulatory DNA capable of effecting expression of that DNA sequence in a host cell transformed with the vector.
  • a functional mammalian VWF protein we mean a protein that corresponds sufficiently to a naturally occurring mammalian VWF protein to have or to be processed to have the function of a naturally occurring Von Willebrand factor; processing may include gly ⁇ osylation and/or assembly into multimers.
  • a host cell we mean any suitable host cell such as a mammalian cell. At least some of the expression vector is exogenous to the VWF-encoding DNA sequence, meaning that it does not naturally occur in the same molecule with that sequence.
  • the VWF is human VWF
  • the host cell for producing the functional VWF is a eukaryotic cell, most preferably a mammalian cell.
  • a second aspect of the invention generally features analyzing a mammalian DNA sample using a probe comprising DNA encoding a VWF protein, or a fragment thereof, labeled with a detectable label.
  • the probe is contacted with the sample to determine whether it hybridizes with the sample.
  • the mammalian DNA that is to be analyzed is fragmented using restriction enzyme digestion, the DNA fragments are separated on the basis of size, the fragments are contacted with the above described probe, and the relative lengths of the DNA fragments that hybridize to the probe are determined.
  • both the probe and the DNA being analyzed are human DNA.
  • the invention thus provides a relatively plentiful and pure source of VWF for treating bleeding disorders, for example, by administering: VWF to those with VWD; VWF in a stable complex with factor VIIIC to those with hemophilia A; or VWF to those with bleeding disorders associated with renal failure.
  • the invention also provides diagnostic and research tools for evaluating VWF genes and defects in them using labled probes comprising the VWF gene or fragments of it.
  • a mammalian DNA sample can be analyzed using a DNA probe to detect a restriction fragment length polymorphism (RFLP) specifically associated with that VWF-related gene.
  • RFLP restriction fragment length polymorphism
  • Restriction fragment length polymorphism means an identifiable DNA sequence that is associated with the VWF-related gene and is conserved together with the VWF-related gene.
  • RFLP's can be used to determine the pattern of inheritance of VWF genes, e.g. in a fetus at risk for one of the various forms of VWD.
  • FIG. 1A represents a restriction map of VWF DNA.
  • Fig. IB represents a restriction map of the 3' portion of VWF cDNA derived from a plasmid designated pVWd and the 3' end of the overlapping clone pVWE6.
  • Fig. 2 is a diagram of the chromosomal localization for the human VWF gene as determined by in situ hybridization.
  • Section A describes how to make functional human VWF by first assembling cDNA fragments to yield a cDNA coding for a functional human VWF protein and then expressing that cDNA in a host cell.
  • Section B describes the use of cDNA encoding VWF protein to analyze sample DNA, e.g., by detecting restriction fragment length polymorphisms (RFLP's) that are associated with the human gene.
  • An assay can then be performed to determine the source of a VWF gene in a human DNA sample-e.g. which allele of each parent was inherited.
  • RFLP's restriction fragment length polymorphisms
  • A. Functional VWF Protein The process of making functional VWF protein involves:
  • VWF mRNA As a source of VWF mRNA, a primary culture of human umbilical vein endothelial cells (HUVEC) is grown and passaged in cell culture in Medium 199 with 20% fetal bovine serum in the presence of bovine endothelial cell growth factor and fibronectin according to the method of Maciag et. al. [J. Cell Biol. 91:420-426 (1981), J. Cell Biol. 94.:511-520 (1982)]. Growth is markedly enhanced by the addition of heparin as described by Thornton et al. [Science 222: 623-625 (1983)].
  • both the cultured cells and the conditioned medium are tested for the presence of VWF using anti-VWF antibody obtained by standard techniques.
  • Standard immunofluorescense and ELISA assay, respectively, can be used for this purpose. After four additional passages, cells are harvested and total RNA prepared in guanidine HC1 by standard techniques.
  • Poly-A + mRNA is isolated from total endothelial cell RNA by oligo-dT cellulose column chromatography.
  • Two cDNA pools for the preparation of two different cDNA libraries are synthesized from the mRNA using standard techniques. For the first cDNA pool, oligo-dT is used as primer for the first strand synthesis, whereas for the second cDNA pool strand.
  • the cDNA pools are made blunt-ended by treatment with T4 DNA polymerase and ligated to EcoRI linkers with T4 DNA ligase after protection of internal EcoRI sites by treatment with E. coli methylase.
  • the linker-ligated cDNA's are then digested with an excess of EcoRI restriction enzyme and seperated from free linkers by passage over a Sepharose CL4B column.
  • the phage vector selected for carrying the VWF cDNA into a bacterial host is lambda gtll, a derivative of bacteriophage lambda which contains a bacterial gene for beta-galactosidase with a single EcoRI cloning site located near its 3' end, corresponding to the C-terminal portion of the beta-galactosidase protein.
  • cDNA molecules are inserted into this site to construct a cDNA library.
  • a fusion protein By infecting an appropriate strain of bacteria with this phage using known techniques, a fusion protein will be produced containing most of beta-galactosidase at its amino terminus and a peptide fragment of the protein of interest at the carboxy-terminus. If this cDNA-encoded peptide contains one of the antigeni ⁇ determinants of VWF, it is detected by screening with anti-VWF antibody. Large numbers of phage particles can be grown on a bacterial plate and the protein products transferred to a nitrocellulose filter and screened with a specific antibody to identify the location of the recombinant plaque producing the protein of interest.
  • the VWF cDNA is ligated into EcoRI digested, phosphatase treated lambda gtll vector DNA and two libraries containing between 3-4 x 10 6 recombinant clones each are plated and amplified.
  • Nonrecombinant background as assessed by growth on ITPG/XGal plates is approximately 30%.
  • Recombinant clones are obtained having cDNA inserts ranging in size from approximately 1 to 3 kilobases (kb) in length.
  • Affinity purified rabbit heteroantiserum prepared against human factor VIII-VWF is obtained using standard methods.
  • the antiserum is passed over gelatin-sepharose, and adsorbed and eluted from a column of VWF-sepharose.
  • Recombinant clones from the above lambda-gtll endothelial cell cDNA libraries are screened as phage plaques in E. coli host strain Y1090 with this antibody at a 1:1000 dilution. Potential positive plaques are purified, replated and rescreened. For example, one primary filter screened with anti-human VWF antibody showed a positive plaque designated LVWd. As a positive control, purified VWF protein can be spotted onto the filter and can be detected at amounts between 100 and 0.1 nanograms (ng) total protein.
  • Positive plaques from the rescreening are purified and phage DNA prepared by standard methods. Purification and characterization of the cDNA insert in the above-described LVWd plague are described below.
  • the 553 bp cDNA insert of LVWd is purified by agarose gel ele ⁇ trophoresis following EcoRI digestion and used as a probe to examine Northern blots of total mRNA from endothelial cells prepared as described above. Northern blot analysis was performed on total cell RNA from HPB-ALL (a T-cell line), endothelial cells (HUVEC) , fibroblasts, and Hela cells, with the LVWd cDNA insert as the hybridization probe. The LVWd cDNA probe hybridized with a single mRNA band between 8 and 10 kb in length. This mRNA species is large enough to code for a protein on the order of 250K dalton in molecular weight.
  • RNA's from the controls i.e., human fibroblasts, Hela cells, or a human T-cell line (HPB-ALL).
  • RNA's from the controls i.e., human fibroblasts, Hela cells, or a human T-cell line (HPB-ALL).
  • HPB-ALL human T-cell line
  • the cDNA insert of clone LVWd corresponds to a segment of an mRNA molecule that is present only in endothelial cells and that is large enough to code for VWF.
  • the clone contains a polypeptide epitope which reacts with affinity purified anti-VWF antibody.
  • Fig. 1A is a restriction map of cDNA coding for a VWF protein.
  • Fig. 1B represents a restriction fragment map and sequencing strategy for DNA from the 3' portion of VWF cDNA derived from pVWd and the 3' end of the overlapping clone pVWE6 (see Fig. 1A).
  • the DNA segment designated pVWD is the above-described cDNA fragment of the plasmid pVWD.
  • Solid lines and arrows in Fig. 1B indicate regions sequenced by the method of Maxam and Gilbert [Meth. Enzymol.
  • Table 1 shows the DNA sequence of the cDNA fragment of Fig. 1B.
  • the cDNA sequence contains a single large open reading frame encoding 193 amino acids followed by a single in the same reading frame and orientation as the beta-galatosidase gene with which it was fused, consistent with expression of a fusion protein product.
  • the predicted amino acid sequence for 193 amino acid residues at the carboxy-terminus of VWF is also shown using the standard single letter amino acid code, the six nucleotides at the beginning and at the end of the DNA sequence correspond to the synthetic EcoRi linker introduced by the cloning procedure.
  • the single termination codon is marked by a diamond. Arrowheads indicate two potential N-glycosylation sites.
  • Fig. 2 represents the hybridization patterns which indicate the localization of the VWF gene to the short arm of chromosome 12 (12p).
  • an insert from a positive plaque such as the 553 base pair insert of LVWd is used as a probe to rescreen the above-described HUVEC libraries, and to produce the restriction map of VWF cDNA shown in Fig. 1A.
  • the cDNA insert is purified in low melt agarose (Bethesda Research Labs) following EcoRI digestion.
  • a 217 bp EcoRI/PstI fragment from the 5' end of pVWE2 is used to rescreen the HUVEC cDNA library and a third series of overlapping VWF cDNA clones is identified, one of which, pVWG8b, is shown in Fig. 1A.
  • a fourth series of overlapping clones is obtained, including pVWH33 and pVWH5 which, together with the sequence shown in Table 1, span DNA sufficient to encode an entire monomer of human VWF protein.
  • cDNA clones pVWH33, pVWH5 and pVWE6, which span 9kb pairs of DNA and encompass the entire protein coding region of VWF, were selected to construct full length cDNA. Nucleotide sequence of the ends of these clones confirmed that together they include the translational start, protein coding and translational stop sequences.
  • the full length cDNA was constructed by standard techniques using fragments derived from individual clones. A fragment of pVWH33 from the left hand EcoRI site to the unique BamHI site was linked to a pVWH5 fragment from the BamHI site to the Sac I site. This was then linked to a fragment from pVWE6 from the Sac I site to the right hand EcoRI site. The full-length cDNA was then inserted as an EcoRI fragment into the EcoRI site of the expression vector pMT2 in which transcription occurs under the control of the adenovirus major late promoter.
  • pMT2 is a derivative of the mammalian cell expression vector p91023(B) (Wong et al., 1985, Science 228;810) in which the tetracycline resistance marker is substituted for the ampicillin resistance marker.
  • the functional elements of the VWF expression plasmid have been previously described (Kaufman, 1985, Proc. Natl. Acad. Sci. USA82:689).
  • pMT2-VWF contains the SV40 origin and enhancer element; the adenovirus major late promoter with the first, second and two thirds of the third tripartite leader; an intron from the 5'splice site from the first late leader of adenovirus and 3' splice site from an immunoglobulin gene (Kaufman & Sharp '82); the VWF cDNA; DHFR coding region, and SV40 early polyadenylation site; the adenovirus VA genes in a derivative of pBR322 containing the Col El origin of replication and ampicillin resistance.
  • Plasmid DNA was prepared by twice banding to equilibrium on CsCl gradients.
  • the above-described cDNA encoding a VWF protein can be inserted into a suitable vector and expressed in any one of a number of mammalian expression systems known to the art, for example using the general method described by Wood et al. (1984) Nature 312;330-337.
  • Host systems can be selected for appropriate post translational processing of the VWF gene product, and enable efficient recovery of VWF.
  • Active VWF has thus been expressed in COS monkey cells and in CHO cells, for example, as described below. Pure VWF produced in this way will be useful in the treatment of VWD, and patients with chronic renal failure whose abnormal bleeding times are corrected by crude cryoprecipitate. Pure VWF can also be used to carry, stabilize, and improve the therapeutic efficacy of factor VIII:C.
  • the SV40-transformed COS monkey cells (clone M6) have been described (Horowitz et al . , 1983 , J. Mol. Appl. Genet. 2 : 147-149).
  • DNA transfections using pMT2 and pMT2-VWF were performed as described (Kaufman, 1985, Pro ⁇ . Natl. Acad. Sci. USA 82:689 ) by the. DEAE-dextran procedure with the addition of a chloroquin treatment (Sompayrac and Danna, 1981, Proc. Natl. Acad. Sci. 78:75757578; Luthman and Magnusson, 1983, Nuc. Acids Res. 11:12951308).
  • Transfected cells were fed with DMEM (Dulbecco's Modified Eagle's Media) with 10% total bovine serum for 48 hr. Then the media was removed, the cells rinsed, and serum-free DMEM applied (4 ml per 3x10 6 cells) for measurement for VWF using an inhibition ELISA assay in which purified VWF was adsorbed onto the surface of microtiter wells followed by anti-VWF antibody. The ability of test material to displace anti-VWF antibody from the immobilized antigen was tested using peroxidase conjugated anti-rabbit IgG as the indicator substance.
  • DMEM Dulbecco's Modified Eagle's Media
  • serum-free DMEM applied (4 ml per 3x10 6 cells) for measurement for VWF using an inhibition ELISA assay in which purified VWF was adsorbed onto the surface of microtiter wells followed by anti-VWF antibody.
  • a transfection of COS cells was performed as above and 72 hours post-transfection, the media was replaced with fresh cysteine-free media containing 35 S-cysteine. After an additional 1 to 5 hours of incubation, the media was removed and cell extracts were prepared as described (Kaufman and Sharp, 1982, J. Mol. Biol. 159:601). The cell extracts and media were then used for studies of VWF processing and multimer assembly.
  • VWF was immunoprecipated by incubation with rabbit anti-human VWF antibody, followed by protein-A sepharose. The immunoprecipated material was washed in a buffer containing 0.1% SDS and Nonidet P40 to minimize non-specific adsorption of other proteins to the immune complex.
  • the precipitated proteins were then analyzed by 4 to 6% SDS-PAGE in the presence and absence of lmM DTT.
  • Recombinant cell lysates contained a band migrating with an apparent molecular weight of 260 kd.
  • Cell media contained a mixture of 260 kd and 220 kd species.
  • Immunopre ⁇ iptates derived from COS cells or media transfected with non-recombinant plasmid did not contain these two bands.
  • Analysis of non-reduced species by 4% SDS-PAGE showed a series of four or five very high molecular weight proteins varying from 1 to 3 or 4 million daltons.
  • COS cells were transfected as above and 48 hr. post- transfection were rinsed and fed with serum free media. Two hundred ml of serum free COS cell media was collected after incubation with COS cells for 24 hours. It contained between 50 and 200 ng/ml VWF by ELISA. The serum free media was concentrated by dialysis against 50% Ficoll to a concentration of 2.2 ug/ml VWF protein for use in the competitive binding assays.
  • This recombinant VWF was used in varying concentrations as a competing ligand against purified, radiolabelled human VWF multimers in assays as described by Luscalzo, J. and
  • one particularly useful approach to obtain high level expression is to select for cells that contain a high degree of amplification of the heterologous VWF gene.
  • One amplifiable marker which is available for this approach is the dihydrofolate reductase gene for which cells harbouring increased gene copies can be selected by propagation in increasing concentrations of methotrexate (MTX) (Kaufman and Sharp, 1982, J. Mol. Biol. 159: 601).
  • MTX methotrexate
  • This approach can be used to select and amplify the VWF gene in a variety of different cell types and has been used to obtain expression of active, full-length human VWF in Chinese hamster ovary cells.
  • the VWF expression plasmid pMT2-VWF and the DHFR expression plasmid pAdD26SV (A) 3 were introduced into DHFR deficient CHO DUKX-BII cells by calcium-phosphate coprecipitation and transfection.
  • DHFR+ transformants were selected for growth in alpha media with dialyzed fetal calf serum and subsequently selected for amplification by growth in increasing concentrations of MTX (sequential steps in 0.02, 0.2, 1.0, and 5.0 uM) as described (Kaufman et al., 1985, Mol. Cell. Biol. 5:1750).
  • XMTVWF-C1 One transformant, designated XMTVWF-C1, was isolated in alpha media and propagated in MTX as described above.
  • VWF expression increased with increasing levels of MTX resistance.
  • the VWF derived from the CHO cells was assayed by a direct ELISA assay using a rabbit anti-human VWF antibody (Calbiochem) immobilized on microtiter plates and a secondary antibody conjugated to horse raddish peroxidase (Dacco). There was minimal activity in the media from the original CHO cells (less than 5 ng/ml). Values were determined by comparison to a standard derived from normal human pooled plasma (1 unit/ml) which was assumed to contain 10ug/ml of VWF.
  • VWF expression has also been verified by 35 S-cysteine labeling of the cells and analysis of the conditioned media and cell extracts by immunopre ⁇ ipitation with rabbit anti-human-VWF antisera (Calbiochem) and electrophoresis of SDS-polyacrylamide gels as described above for the VWF derived from transfected COS cells.
  • the VWF DNA sequence was derived from the same overlapping cDNA clones which were used in the construction of the full-length expressed clone pMT2-VWF.
  • the entire sequence was determined on both strands using the Sanger dideoxy method on single-stranded M13 subclones (Sanger, F. Nicklen., S. and Coulson, A.R. , 1977, Proc. Natl. Acad.- Sci. U.S.A. 74: 5463-5467; Messing, J., 1983, J. Methods Enzymol. 101:30-78) .
  • Sub-clones for sequencing were generated by exonuclease digestion of the inserts of cDNA clones pVWH33, pVWH5, and pVWE2, using nuclease Bal31 or T4 DNA polymerase. Gaps were completed by subcloning appropriate restriction fragments from the same clones into M13 mp10 and mpll.
  • the sequence of 8588 base pairs is shown in Table 2. It contains continuous open reading frame encoding a polypeptide of 2815 amino acids.
  • Cys rich regions are arranged as a series of 6 repeats of a basic 400 amino acid unit.
  • the first repeat block begins almost immediatly after the signal peptide.
  • the second repeat is truncated just preceding this duplication contains the cleavage site between the pro-piece and mature VWF.
  • the sequence is interrupted by the triplicated cys-poor "A domain" repeat, after which the Cys rich repeats resume encoding the C-terminal end of the molecule.
  • the fifth and sixth repeats are incomplete, but include the region of the short "B domain” repeats of Sadler et al. Those authors' "C domain" repeats follow in two copies.
  • DNA sequence polymorphisms are neutral variations in DNA sequence present throughout the genome, which can often be detected by restriction enzyme digestion and blot hybridization analysis. By neutral we mean that the variations per se are not themselves responsible for any phenotypic trait. However, the value of polymorphisms is that they are linked to or associated with adjacent portions of the genome, and therefore they can be used as markers of those portions of the genome.
  • DNA sequence polymorphisms Two types have been described. One type involves single nucleotide changes, or small insertions or deletions, which result in the presence or absence of a particular restriction enzyme recognition site. In another type of polymorphism, a large segment of DNA of unknown function varies widely in length among individuals. Both types of sequence differences are inherited in Mendelian fashion.
  • RFLP's linked to the VWF gene are identified in genomic DNA from individuals examined by cutting sample DNA with a series of restriction enzymes. The resulting restriction length fragments are segregated by molecular weight.
  • Hybridization with a radiolabeled VWF cDNA probe e.g., the cDNA insert from clone pVWE6 yields (e.g., using Southern blot techniques) a unique band pattern.
  • RFLP's are detected by the above procedure using restriction enzymes TaqI and Sa ⁇ l and the pVWE6 probe. Specifically, peripheral blood specimens are collected in 10% acid-citrate-dextrose.
  • High molecular weight DNA is prepared by standard techniques either from dextran sedimented leukocytes or isolated nuclei separated by centrifugation following Triton X-100 solubulization. From 2 to 16 g of DNA are digested to completion with the restriction enzymes TaqI and Sad. DNA fragments are then fra ⁇ tioned by electrophoresis in 0.6 to 1.0% agarose gels and transferred to nitrocellulose filters. The probes constructed from pVWE6 are labeled and hybridized with the DNA on the above-described nitrocelloulose filters. The hybridized filters are washed and examined by autoradiography. Once identified RFLP's can be used as described above to assay a sample of human DNA and determine the source of a VWF gene in that sample. For example, DNA of parents and other family members can be screened, e.g., by Southern blotting, with such a probe, and then fetal DNA can be screened to determine the inheritance pattern of its VWF alleles.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Treating Waste Gases (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le facteur de Von Villebrand (VWF) est produit au moyen d'un vecteur d'expression qui comporte: premièrement une séquence d'ADN codant une protéine fonctionnelle du VWF; et deuxièmement un ADN régulateur capable de réaliser l'expression de cette séquence d'ADN dans une cellule hôte transformée avec le vecteur. Des polymorphismes de la longueur d'un fragment de restriction (RFLP's) associés avec le gène VWF sont identifiés et utilisés dans une sonde pour déterminer la source d'un gène VWF dans un échantillon d'ADN. Le gène du VWF est localisé au niveau du bras court du chromosome humain 12 (12p).
PCT/US1986/000760 1985-04-11 1986-04-10 Facteur de von willebrand WO1986006096A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US07/559,509 US8597910B1 (en) 1985-04-11 1990-07-23 DNA encoding Von Willebrand Factor (VWF) and methods and cells for producing VFW, and VFW produced by the DNA, methods and cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US72210885A 1985-04-11 1985-04-11
US722,108 1985-04-11

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US72210885A Continuation-In-Part 1985-04-11 1985-04-11

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US06882983 A-371-Of-International 1986-04-10
US07/559,509 Continuation US8597910B1 (en) 1985-04-11 1990-07-23 DNA encoding Von Willebrand Factor (VWF) and methods and cells for producing VFW, and VFW produced by the DNA, methods and cells

Publications (1)

Publication Number Publication Date
WO1986006096A1 true WO1986006096A1 (fr) 1986-10-23

Family

ID=24900540

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1986/000760 WO1986006096A1 (fr) 1985-04-11 1986-04-10 Facteur de von willebrand

Country Status (4)

Country Link
EP (1) EP0218692A4 (fr)
JP (1) JPS62502589A (fr)
AU (1) AU591671B2 (fr)
WO (1) WO1986006096A1 (fr)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0222003A1 (fr) * 1985-05-16 1987-05-20 Rhone-Poulenc Rorer Pharmaceuticals Inc. CLONAGE MOLECULAIRE D'ADNc DE FACTEUR HUMAIN VIIR (FACTEUR DE VON WILLEBRAND)
US5198349A (en) * 1986-01-03 1993-03-30 Genetics Institute, Inc. Method for producing factor VIII:C and analogs
WO1994005800A1 (fr) * 1992-09-03 1994-03-17 The Regents Of The University Of California Facteur influant sur le tissu dorsal et compositions
US5821124A (en) * 1992-09-03 1998-10-13 Regeneron Pharmaceuticals, Inc. Hybridoma cell lines and antibodies that bind noggin
US5837488A (en) * 1990-03-02 1998-11-17 Bio-Technology General Corp. Cloning and production of human von Willebrand Factor GP1b binding domain polypeptides and methods of using same
US6008193A (en) * 1990-03-02 1999-12-28 Bio-Technology General Corp. Methods of using human von Willebrand factor GPIb binding domain polypeptides
US7884075B2 (en) 2004-12-27 2011-02-08 Baxter International Inc. Polymer-factor VIII-von Willebrand factor-conjugates
WO2011017414A2 (fr) 2009-08-04 2011-02-10 Baxter International Inc. Souris transgénique dépourvue du fviii endogène et du vwf en tant que modèle de l'hémophilie a
WO2011022657A1 (fr) 2009-08-20 2011-02-24 Baxter International Inc. Purification du vwf pour une meilleure élimination des virus à enveloppe non lipidique
US8053561B2 (en) 2006-03-31 2011-11-08 Baxter International Inc. Pegylated factor VIII
WO2012006594A1 (fr) 2010-07-08 2012-01-12 Baxter International Inc. Procédé de production d'adamts13 recombinant en culture cellulaire
WO2012171031A1 (fr) 2011-06-10 2012-12-13 Baxter International Inc. Traitement d'une maladie de la coagulation par l'administration du fvw recombinant
EP2601932A1 (fr) 2008-10-21 2013-06-12 Baxter International Inc. Formulations de FVW recombinant lyophilisé
US8575102B2 (en) 2008-08-01 2013-11-05 Nektar Therapeutics Conjugates having a releasable linkage
US8597910B1 (en) 1985-04-11 2013-12-03 Children's Medical Center Corporation DNA encoding Von Willebrand Factor (VWF) and methods and cells for producing VFW, and VFW produced by the DNA, methods and cells
US8791247B2 (en) * 2008-02-29 2014-07-29 Korea University Industrial & Academic Collaboration Foundation Recombinant expression vector system for variants of coagulation factor VIII and von willebrand factor
WO2019010497A1 (fr) 2017-07-07 2019-01-10 Baxalta Incorporated Traitement de saignements gastro-intestinaux chez des patients atteints d'une forme grave de la maladie de von willebrand par l'administration de vwf recombiné
WO2019010496A1 (fr) 2017-07-07 2019-01-10 Baxalta Incorporated Traitement de patients atteints d'une forme grave de la maladie de von willebrand subissant une intervention chirurgicale non urgente par l'administration de vwf recombiné
WO2019183290A1 (fr) 2018-03-21 2019-09-26 Baxalta Incorporated Séparation de vwf et de propeptide de vwf par des procédés chromatographiques
WO2020160460A1 (fr) 2019-02-01 2020-08-06 Baxalta Incorporated Procédés de traitement prophylactique utilisant vwf recombinant (rvwf)
WO2021050718A1 (fr) 2019-09-11 2021-03-18 Baxalta Incorporated Méthodes de traitement liés à des complexes du facteur de von willebrand et du complément c1q
US11020458B2 (en) 2006-03-31 2021-06-01 Takeda Pharmaceutical Company Limited Factor VIII polymer conjugates
WO2021158777A1 (fr) 2020-02-04 2021-08-12 Baxalta Incorporated Traitement de la ménorragie chez des patients atteints de maladie de von willebrand sévère par administration de vwf recombinant
US11191837B2 (en) 2007-12-28 2021-12-07 Takeda Pharmaceutical Company Limited Recombinant VWF formulations
US11197916B2 (en) 2007-12-28 2021-12-14 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
US12121561B1 (en) * 2024-02-17 2024-10-22 King Faisal University Composition for treating or preventing insulin resistance

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4386068A (en) * 1980-02-26 1983-05-31 Cutter Laboratories, Inc. Antihemophilic factor concentrate and method for preparation
EP0128018A2 (fr) * 1983-05-31 1984-12-12 Orgenics Ltd. Sonde moléculaire génétique et méthode pour sa préparation, technique d'analyse et trousse utilisant cette sonde moléculaire génétique
WO1985001961A1 (fr) * 1983-10-28 1985-05-09 Genetics Institute Production du facteur viii et des produits relatifs

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI852866L (fi) * 1984-07-27 1986-01-28 Meloy Lab Rekombinantfaktor viii-r.
NL8500961A (nl) * 1985-04-01 1986-11-03 Stichting Vrienden Van De Stic Cdna-codering voor de humane von willebrand-factor, plasmiden met een dergelijke cdna-codering respektievelijk fragmenten ervan, alsmede micro-organismen, welke dergelijke plasmiden bevatten.
WO1986006745A1 (fr) * 1985-05-16 1986-11-20 Meloy Laboratories, Inc. CLONAGE MOLECULAIRE D'ADNc DE FACTEUR HUMAIN VIIR (FACTEUR DE VON WILLEBRAND)
DE3684017D1 (de) * 1985-11-05 1992-04-02 Genetics Inst Vielfach amplifizierbare vektoren fuer hohe expression exogener dns.
IL83192A (en) * 1986-07-18 1992-11-15 Gist Brocades Nv Method for the preparation of proteins with factor viii activity by microbial host cells;expression vectors,host cells,antibodies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4386068A (en) * 1980-02-26 1983-05-31 Cutter Laboratories, Inc. Antihemophilic factor concentrate and method for preparation
EP0128018A2 (fr) * 1983-05-31 1984-12-12 Orgenics Ltd. Sonde moléculaire génétique et méthode pour sa préparation, technique d'analyse et trousse utilisant cette sonde moléculaire génétique
WO1985001961A1 (fr) * 1983-10-28 1985-05-09 Genetics Institute Production du facteur viii et des produits relatifs

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
Cell, Volume 41, issued May 1985, (Cambridge, Massachusetts, USA), (D.C. LYNCH et al), "Molecular Cloning of cDNA for Human von Willebrand Factor: Authentication by a New Method", see pages 49-56. *
E.B. BROWN ed., Progress in Hematology, Volume XIII, 1983, GRUNE & STRATTON (New York, New York, USA), (T.S. ZIMMERMAN et al.), "Factor VIII/von Willebrand Factor", see pages 279-309, especially 279-285. *
Federation Proceedings, Volume 44, 5 March 1985, (Bethesda Maryland, USA), (J.E. SADLER et al.), "Cloning and Characterization of a cDNA for Human von Willebrand Factor", see pages 1069, Abstract 3950. *
Nature. Volume 312, issued 22 November 1984, (London, England), (W.I. WOOD et al), "Expression of Active Human Factor VIII from Recombinant DNA Clones." see pages 330-337, especially 334 & 335. *
Nucleic Acids Research, Volume 13, issued July 1985, (Oxford, England), (C.L. VERWEIJ et al)., "Construction of cDNA Coding for Human von Willebrand Factor Using Antibidy Probes for Colony-Screening and Mapping of the Chromosomal Gene", see pages 4699-4717. *
Nucleic Acids Research, Volume 13, issued November 1985, (Oxford, England), (C.L. VERWEIJ et al), "RFLP for a Human von Willebrand Factor (vWF) cDNA Clone. pvWF1100", see page 8289. *
Proceedings National Academy Sciences, USA, Volume 81, issued August 1984 (Washington D.C., USA), (S. TIMMONS et al), "ADP-Dependent Common Receptor Mechanism for Binding of von Willebrand Factor and Fibrinogen to Human Platelets", see pages 4935-4939, especially 4935 and 4936. *
Proceedings National Academy Sciences, USA, Volume 82, issued Ocotober, 1985, (Washington, D.C. USA), (J.E. SADLER et al), "Cloning and Characterization of two cDNAs Coding for Human von Willebrand Factor", see pages 6394-6398. *
Science, Volume 228, issued 21 June 1985, (Washington, D.C., USA), (D. GINSBURG et al)., "Human von Willebrand Factor (vWF): Isolation of Complementary DNA (cDNA) Clones and Chromosomal Localization", see pages 1401-1406. *
See also references of EP0218692A4 *
Thromb. Haemostas, Volume 50, issued 1983, (Stuttgart, West Germany), (V. CHAN et al), "Cell-Free Synthesis of Factor VIII Related Protein", see pages 835-837 *

Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8597910B1 (en) 1985-04-11 2013-12-03 Children's Medical Center Corporation DNA encoding Von Willebrand Factor (VWF) and methods and cells for producing VFW, and VFW produced by the DNA, methods and cells
EP0222003A1 (fr) * 1985-05-16 1987-05-20 Rhone-Poulenc Rorer Pharmaceuticals Inc. CLONAGE MOLECULAIRE D'ADNc DE FACTEUR HUMAIN VIIR (FACTEUR DE VON WILLEBRAND)
EP0222003A4 (fr) * 1985-05-16 1989-03-23 Meloy Lab CLONAGE MOLECULAIRE D'ADNc DE FACTEUR HUMAIN VIIR (FACTEUR DE VON WILLEBRAND).
US5198349A (en) * 1986-01-03 1993-03-30 Genetics Institute, Inc. Method for producing factor VIII:C and analogs
US5849702A (en) * 1990-03-02 1998-12-15 Bio-Technology General Corp. Cloning and production of human von Willebrand factor GPIB binding domain polypeptides and methods of using same
US5837488A (en) * 1990-03-02 1998-11-17 Bio-Technology General Corp. Cloning and production of human von Willebrand Factor GP1b binding domain polypeptides and methods of using same
US5849536A (en) * 1990-03-02 1998-12-15 Bio-Technology General Corp. Cloning and production of human von willebrand factor GPIb binding domain polypeptides and methods of using same
US6008193A (en) * 1990-03-02 1999-12-28 Bio-Technology General Corp. Methods of using human von Willebrand factor GPIb binding domain polypeptides
US5821124A (en) * 1992-09-03 1998-10-13 Regeneron Pharmaceuticals, Inc. Hybridoma cell lines and antibodies that bind noggin
US5843775A (en) * 1992-09-03 1998-12-01 Regeneron Pharmaceuticals, Inc. Human dorsal tissue affecting factor (noggin) and nucleic acids encoding same
US5670481A (en) * 1992-09-03 1997-09-23 The Regents Of The University Of California Dorsal tissue affecting factor (noggin) and compositions comprising same
WO1994005800A1 (fr) * 1992-09-03 1994-03-17 The Regents Of The University Of California Facteur influant sur le tissu dorsal et compositions
US8357779B2 (en) 2004-12-27 2013-01-22 Baxter International Inc. Polymer-von Willebrand factor-conjugates
US7884075B2 (en) 2004-12-27 2011-02-08 Baxter International Inc. Polymer-factor VIII-von Willebrand factor-conjugates
US8835388B2 (en) 2004-12-27 2014-09-16 Baxter International Inc. Polymer von Willebrand factor-conjugates
US8076463B2 (en) 2004-12-27 2011-12-13 Baxter International, Inc. Polymer-von Willebrand factor-conjugates
US8053561B2 (en) 2006-03-31 2011-11-08 Baxter International Inc. Pegylated factor VIII
US11020458B2 (en) 2006-03-31 2021-06-01 Takeda Pharmaceutical Company Limited Factor VIII polymer conjugates
US11191813B2 (en) 2007-12-28 2021-12-07 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
US11191837B2 (en) 2007-12-28 2021-12-07 Takeda Pharmaceutical Company Limited Recombinant VWF formulations
EP3936116A1 (fr) 2007-12-28 2022-01-12 Takeda Pharmaceutical Company Limited Formulations comprenant de vwf recombinant
US11197916B2 (en) 2007-12-28 2021-12-14 Takeda Pharmaceutical Company Limited Lyophilized recombinant VWF formulations
US9249210B2 (en) 2008-02-29 2016-02-02 Korea University Industrial & Academic Collaborative Foundation Recombinant expression vector system for variants of coagulation factor VIII and von Willebrand factor
US8791247B2 (en) * 2008-02-29 2014-07-29 Korea University Industrial & Academic Collaboration Foundation Recombinant expression vector system for variants of coagulation factor VIII and von willebrand factor
US8575102B2 (en) 2008-08-01 2013-11-05 Nektar Therapeutics Conjugates having a releasable linkage
US10232022B2 (en) 2008-10-21 2019-03-19 Baxalta Incorporated Lyophilized recombinant VWF formulations
EP2601932A1 (fr) 2008-10-21 2013-06-12 Baxter International Inc. Formulations de FVW recombinant lyophilisé
WO2011017414A2 (fr) 2009-08-04 2011-02-10 Baxter International Inc. Souris transgénique dépourvue du fviii endogène et du vwf en tant que modèle de l'hémophilie a
WO2011022657A1 (fr) 2009-08-20 2011-02-24 Baxter International Inc. Purification du vwf pour une meilleure élimination des virus à enveloppe non lipidique
US9315560B2 (en) 2009-08-20 2016-04-19 Baxalta Incorporated Purification of VWF for increased removal of non-lipid enveloped viruses
US9409971B2 (en) 2010-07-08 2016-08-09 Baxalta Incorporated Method of producing recombinant high molecular weight vWF in cell culture
EP3909977A1 (fr) 2010-07-08 2021-11-17 Takeda Pharmaceutical Company Limited Procédé de production de recombinant adamts13 dans une culture cellulaire
US10100099B2 (en) 2010-07-08 2018-10-16 Baxalta Incorporated Method of producing recombinant high molecular weight vWF in cell culture
EP3392271A1 (fr) 2010-07-08 2018-10-24 Baxalta GmbH Procédé de production de recombinant adamts13 dans une culture cellulaire
EP3064508A1 (fr) 2010-07-08 2016-09-07 Baxalta GmbH Procédé de production de vwf de poids moléculaire élevé recombinant dans une culture cellulaire
WO2012006591A1 (fr) 2010-07-08 2012-01-12 Baxter International Inc. Méthode de production de vwf recombinant de masse moléculaire élevée en culture cellulaire
WO2012006594A1 (fr) 2010-07-08 2012-01-12 Baxter International Inc. Procédé de production d'adamts13 recombinant en culture cellulaire
US9458222B2 (en) 2010-07-08 2016-10-04 Baxalta Incorporated Method of producing recombinant ADAMTS13 in cell culture
US9834591B2 (en) 2010-07-08 2017-12-05 Baxalta Incorporated Method of producing recombinant high molecular weight vWF in cell culture
EP3626736A1 (fr) 2010-07-08 2020-03-25 Baxalta GmbH Procédé de production de vwf de poids moléculaire élevé recombinant dans une culture cellulaire
US11780904B2 (en) 2010-07-08 2023-10-10 Takeda Pharmaceutical Company Limited Method of producing recombinant high molecular weight vWF in cell culture
US8852888B2 (en) 2010-07-08 2014-10-07 Baxter International Inc. Method of producing recombinant high molecular weight vWF in cell culture
US10822394B2 (en) 2010-07-08 2020-11-03 Baxalta GmbH Method of producing recombinant high molecular weight vWF in cell culture
EP3412305A1 (fr) 2011-06-10 2018-12-12 Baxalta GmbH Traitement d'une maladie de la coagulation par l'administration du fvw recombinant
EP3858375A1 (fr) 2011-06-10 2021-08-04 Takeda Pharmaceutical Company Limited Traitement d'une maladie de la coagulation par l'administration du fvw recombinant
WO2012171031A1 (fr) 2011-06-10 2012-12-13 Baxter International Inc. Traitement d'une maladie de la coagulation par l'administration du fvw recombinant
WO2019010496A1 (fr) 2017-07-07 2019-01-10 Baxalta Incorporated Traitement de patients atteints d'une forme grave de la maladie de von willebrand subissant une intervention chirurgicale non urgente par l'administration de vwf recombiné
US12016904B2 (en) 2017-07-07 2024-06-25 Takeda Pharmaceutical Company Limited Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF
US11529395B2 (en) 2017-07-07 2022-12-20 Takeda Pharmaceutical Company Limited Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF
US10905746B2 (en) 2017-07-07 2021-02-02 Baxalta GmbH Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF
EP4424366A2 (fr) 2017-07-07 2024-09-04 Takeda Pharmaceutical Company Limited Traitement du saignement gastro-intestinal chez des patients atteints d'une maladie grave de von willebrand par administration de vwf recombiné
US10632176B2 (en) 2017-07-07 2020-04-28 Baxalta Incorporated Treatment of gastrointestinal bleeding in patients with severe von Willebrand disease by administration of recombinant VWF
WO2019010497A1 (fr) 2017-07-07 2019-01-10 Baxalta Incorporated Traitement de saignements gastro-intestinaux chez des patients atteints d'une forme grave de la maladie de von willebrand par l'administration de vwf recombiné
WO2019183290A1 (fr) 2018-03-21 2019-09-26 Baxalta Incorporated Séparation de vwf et de propeptide de vwf par des procédés chromatographiques
US10934340B2 (en) 2018-03-21 2021-03-02 Baxalta Incorporated Separation of VWF and VWF propeptide by chromatographic methods
WO2020160460A1 (fr) 2019-02-01 2020-08-06 Baxalta Incorporated Procédés de traitement prophylactique utilisant vwf recombinant (rvwf)
WO2021050718A1 (fr) 2019-09-11 2021-03-18 Baxalta Incorporated Méthodes de traitement liés à des complexes du facteur de von willebrand et du complément c1q
WO2021158777A1 (fr) 2020-02-04 2021-08-12 Baxalta Incorporated Traitement de la ménorragie chez des patients atteints de maladie de von willebrand sévère par administration de vwf recombinant
US12121561B1 (en) * 2024-02-17 2024-10-22 King Faisal University Composition for treating or preventing insulin resistance

Also Published As

Publication number Publication date
EP0218692A4 (fr) 1988-03-22
EP0218692A1 (fr) 1987-04-22
AU5776186A (en) 1986-11-05
JPS62502589A (ja) 1987-10-08
AU591671B2 (en) 1989-12-14

Similar Documents

Publication Publication Date Title
AU591671B2 (en) Von willebrand factor
EP0160457B1 (fr) Facteur VIII humain, compositions le contenant, méthodes et matières utilisables pour sa production
US5618788A (en) Preparation of functional human factor VIII and pharmaceutical treatment therewith
KR970002915B1 (ko) 제ⅷ인자 활성을 가지는 신규한 단백질 : 유전공학-제조 세포를 사용하는 그의 제조방법 및 그것을 함유하는 의약 조성물
Verweij et al. Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene
FI107539B (fi) Menetelmä yhdistelmä-humaani-hyytymistekijä VIII:n johdannaisen valmistamiseksi ja menetelmässä käytettävä DNA-jakso, vektori ja isäntäsolu
EP0232112B1 (fr) Complexe de protéines recombinantes ayant l'activité du facteur VIII:C humain, sa production et son emploi
US5045455A (en) Factor VIII:C cDNA cloning and expression
CA1339356C (fr) Recepteur de facteur de croissance derive de plaquettes humaines
Kroner et al. Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets
EP0294910B1 (fr) Protéines à activité de facteur VIII, procédé pour leur production utilisant des cellules modifiées par génie génétique et compositions pharmaceutiques les contenant
EP0627923B1 (fr) Domaines therapeutiques du facteur von willebrand
US7138505B1 (en) Factor VIII:C nucleic acid molecules
Mohr et al. A single point mutation results in A allele-specific exon skipping in the bovine αs1-casein mRNA
US8597910B1 (en) DNA encoding Von Willebrand Factor (VWF) and methods and cells for producing VFW, and VFW produced by the DNA, methods and cells
US6110737A (en) Human platelet-derived growth factor receptor, type A
WO1993000357A1 (fr) Polypeptides therapeutiques bases sur le facteur de von villebrand
AU604055B2 (en) Human B-cell differentiation factor and process of producing said factor
WO1992017192A1 (fr) Fragments therapeutiques du facteur willebrand
WO1992006999A1 (fr) Fragments therapeutiques du factor de von willebrand
AU1809788A (en) Novel proteins with factor VIII activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LU NL SE

CR1 Correction of entry in section i

Free format text: IN PAT.BUL.23/86,UNDER PUBLISHED AFTER "WITH AMENDED CLAIMS" ADD "AND STATEMENT"

WWE Wipo information: entry into national phase

Ref document number: 1986902714

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1986902714

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1986902714

Country of ref document: EP