EP1846549A1 - Heparinähnliche moleküle produzierende mastzellinien aus schwein - Google Patents

Heparinähnliche moleküle produzierende mastzellinien aus schwein

Info

Publication number
EP1846549A1
EP1846549A1 EP05809321A EP05809321A EP1846549A1 EP 1846549 A1 EP1846549 A1 EP 1846549A1 EP 05809321 A EP05809321 A EP 05809321A EP 05809321 A EP05809321 A EP 05809321A EP 1846549 A1 EP1846549 A1 EP 1846549A1
Authority
EP
European Patent Office
Prior art keywords
culture
heparin
cells
porcine
line
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP05809321A
Other languages
English (en)
French (fr)
Inventor
Jean-Marc Guillaume
Sandrine Perez
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
Aventis Pharma SA
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 Rhone Poulenc Rorer SA, Aventis Pharma SA filed Critical Rhone Poulenc Rorer SA
Publication of EP1846549A1 publication Critical patent/EP1846549A1/de
Withdrawn legal-status Critical Current

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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present application relates to porcine mast cell lines whose growth is independent of growth factors. It also relates to a method of producing bioactive heparin-like molecules comprising culturing these mast cell lines.
  • Mast cells are cells of the immune system, derived from hematopoietic precursors, which are involved in the inflammatory response, especially in the phenomena of allergy and hypersensitivity. They are located in the connective tissue, especially in the skin, intestinal and respiratory mucosa.
  • the mast cells are in the form of rounded cells with a diameter of about 5 to 25 ⁇ m and have a rounded, single, central or eccentric nucleus. They are also characterized by the presence of numerous meta-chromatic cytoplasmic granulations. These granules contain various molecular species having a pro ⁇ inflammatory activity such as histamine, serotonin, proteoglycans such as heparin or chondroitin sulfate, enzymes, cytokines and chemoattractant factors of eosinophils and neutrophils. These species are released during mast cell activation.
  • mediators such as leukotrienes, prostaglandins, PAF (platelet activating factor), interleukins (IL4, IL5, IL6, IL10, IL12 and IL13) takes place.
  • mediators such as leukotrienes, prostaglandins, PAF (platelet activating factor), interleukins (IL4, IL5, IL6, IL10, IL12 and IL13) takes place.
  • cytokines TGF beta, IFN gamma, GM-CSF
  • chemokines MCP-1, IL8, RANTES
  • Ashraf et al (Veterinary Parasitology, 29, 134-158, 1988) isolated pig mast cells from intestinal mucosa without maintaining amplifiable cultures. In addition, the characterization of isolated mast cells reveals the absence of heparin. Razin et al (J. Biol Chem., 257, 7229-7236, 1982) describe obtaining mouse mast cells using culture media containing IL3.
  • SCF growth factors
  • the SCF induces, following its fixation on its ckit receptor, various cellular responses such as proliferation, (differentiation, etc.).
  • the study of murine and human mastocytomas has identified mutations or deletions of the c-kit gene responsible for the constitutive activation of the c-kit receptor (ie without fixation of the SCF).
  • the expression of the c-kit gene mutated at V814 in immature mast cells IC2 induces the transformation of these cells, namely the acquisition of a growth independent of SCF and of a tumorigenic potential (Pia et al, Blood, 87). (8), 3117-3123, 1996).
  • Heparin belongs to the family of glycosaminoglycans (GAG), which groups linear polysaccharides containing a repetition of a disaccharide sequence consisting of an amino sugar (D-glucosamine or galactosamine) and a uronic acid (D-glucuronic acid). or L-iduronic).
  • GAG glycosaminoglycans
  • heparin which belongs, with heparan sulfate, to the sub-family of glucosaminoglycans
  • the amino sugar is D-glucosamine.
  • Uronic acid is either glucuronic acid (GIc) or iduronic acid (Ido).
  • Glucosamine can be N-acetylated, N-sulfated and O-sulfated.
  • heparin refers to highly sulphated polysaccharides in which more than 80% of the glucosamine residues are N-sulphated and the number of O-sulphate is greater than that of the N-sulphates.
  • the sulfate / carboxylate ratio is generally greater than 2 for heparin.
  • the structure of heparin is in fact very heterogeneous, and there are chains that can contain very different ratios.
  • heparin is synthesized as a proteoglycan mainly by mast cells.
  • the first step in heparin synthesis is the formation of the serglycine protein core, consisting of a repetition of serine and glycine residues.
  • the elongation of the heparin chain is done by adding a tetrasaccharide, then by successive additions of glucosamines and uronic acids, regularly alternated.
  • the proteoglycan thus formed undergoes numerous sequential transformations: N-deacetylation, N-sulfation, epimerization of D-glucuronic acid, and O-sulfation. However, this complete maturation takes place only on part of the proteoglycan, which generates a great structural variability of the heparin, responsible for its heterogeneity.
  • the polysaccharide chains are then cleaved from serglycine by an endoglucuronidase. These chains then have a molecular weight between 5000 and 30,000 Da. They form complexes with basic proteases and are thus stored in the mast cell granules. Heparin is excreted only during degranulation mastocytosis.
  • the present invention relates to cultures or lines of porcine mast cells characterized in that they produce heparin-type molecules comprising tetrasaccharides lia They glu.
  • these cultures or lines produce detectable amounts of tetrasaccharides lia They glu.
  • these cultures or lines produce heparin-like molecules containing tetrasaccharides which are present in amounts of at least 0.3%, 0.5%, 1% or 1.5% of the total saccharides.
  • these porcine mast cell cultures or lines produce heparin-like molecules having an anti-Xa activity greater than at least 50 IU / mg, preferably greater than 75 IU / mg, at 100 IU / mg or at 150 IU / mg.
  • these porcine mast cell cultures or lines produce heparin-like molecules having an anti-IIa activity of at least 50 IU / mg, preferably greater than 75 IU / mg, at 100 IU / mg or at 150 IU / mg.
  • heparin-type molecules comprising at least 30% of Is disaccharides (preferably at least 40%, more preferably at least 50%).
  • the lines according to the present invention are modified so as to overexpress an enzyme acting on the sulfation of heparin-like molecules.
  • said lines comprise an exogenous nucleic acid encoding an enzyme acting on the sulfation of heparin-like molecules.
  • the stable overexpression of an enzyme acting on the sulfation of heparin-like molecules makes it possible to increase the sulfation of the heparinic compounds, and consequently their biological activity.
  • the enzyme acting on the sulfation of heparin-like molecules is a 3-OST and particularly advantageously a 3OST1.
  • the enzyme acting on the sulfation of the heparin-type molecules is preferably the porcine 3OST1 having the sequence SEQ ID No. 6, and encoded by the sequence SEQ ID N5.
  • genes of other species coding for the expression of 3-0 sulfatase activity and having an identity of at least 80%, 90%, 95% or 99% identity.
  • nucleotides with a nucleic acid of sequence SEQ ID No. 5 Nucleic acids hybridizing under conditions of high stringency with a nucleic acid of sequence SEQ ID NO: 5 can also be used.
  • the porcine mast cell lines are modified so as to express a c-kit protein having a mutation or a deletion which makes it possible to overcome the SCF.
  • said lines comprise an exogenous nucleic acid encoding a c-kit protein having a mutation or a deletion.
  • the c-kit having a mutation or a deletion is advantageously a c-kit protein having a mutation at a position equivalent to that of the murine G559 c-kit.
  • He can be a protein c-kit with a mutation in a position equivalent to that of the c-kit G559 Wall nj j t e]
  • the murine G559 c-kit carrying a point mutation, preferentially that responsible for the modification of the glycine valine, having the sequence SEQ ID No. 2, and encoded by the sequence SEQ ID N 0 I II may also be the porcine G559 c-kit having the sequence SEQ ID No. 4, and encoded by the sequence SEQ ID No. 3.
  • nucleotide nucleic acid sequence SEQ ID No. 3 can be used. Nucleic acids hybridizing under conditions of high stringency with a nucleic acid of sequence SEQ ID No. 3 can also be used.
  • the Applicant has observed a heparin profile of the cells expressing the improved murine G559 c-kit compared to the cells not expressing the murine G559 c-kit, ie a different constitution of the heparin-like molecules produced by these cells.
  • c-kit protein having a point mutation responsible for the modification of aspartic acid to valine 814 or 816 respectively in mice and in humans.
  • the present invention relates to one of the porcine mast cell lines deposited with the Collection of Microorganism Cultures of the Institut Pasteur on October 17, 2001 under No. I-2734, I-2735 or I-2736 modified to express a c-kit protein with a mutation or deletion.
  • these lines comprise an exogenous nucleic acid encoding an enzyme acting on the sulfation of heparin-like molecules.
  • culture here refers, in general, to a cell or a set of cells cultured in vitro. A culture developed directly from a cell or tissue sample taken from an animal is referred to as "primary culture".
  • line is used from the moment when at least one passage, and generally several consecutive passages in subculture have been successfully performed, and designates any culture that is derived (SCHAEFFER, In Vitro Cellular and
  • nucleic acid is used to refer to a DNA or an RNA.
  • nucleotide or amino acid sequences within the meaning of the present invention, can be determined by comparing two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide sequence or polypeptide in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) so as to obtain an optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which an identical nucleobase or amino acid residue is observed for the two (nucleic or peptide) sequences compared and then dividing the number of positions at which there is identity between the two bases. or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity.
  • the optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms contained in the package of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.
  • the percentage of sequence identity can be carried out using the BLAST software (BLAST versions 1.4.9 of March 1996, BLAST 2.0.4 of February 1998 and BLAST 2.0.6 of September 1998), using exclusively the default parameters (S. F Altschul et al., J. Mol Biol., 1990 215: 403-410, S. F Altschul et al, Nucleic Acids Res 1997 25: 3389-3402).
  • the data used may be peptide or nucleic, any combination being possible.
  • hybridization conditions described above are suitable for hybridization under conditions of high stringency, of a nucleic acid molecule of variable length from 20 nucleotides to several hundred nucleotides.
  • hybridization conditions described above can be adapted as a function of the length of the nucleic acid whose hybridization is sought or of the type of marking chosen, according to techniques known to those skilled in the art.
  • the suitable hybridization conditions may for example be adapted to the teaching in the book of Hames and HIGGlNS (1985 ⁇ NucIeic acid hybridization: a practical approach, Hames and Higgins Ed., IRL Press, Oxford) or in the work of F.AUSUBEL et al (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY).
  • the present application is further related to a method for producing heparin-like molecules comprising culturing porcine mast cell cultures or lines as described above.
  • the lines according to the present invention are preferably cultured in a defined culture medium (MEM ⁇ / DMEM, RPMI, IMDM, etc.).
  • a defined culture medium MEM ⁇ / DMEM, RPMI, IMDM, etc.
  • the media can also be supplemented with bovine serum at a concentration of between 0.5% and 20% (v / v).
  • bovine serum in the culture media can be replaced by the use of a serum-free culture medium such as AIMV (INVITROGEN) so as to reduce the protein concentration of the medium and the risks associated with the use thereof.
  • AIMV AIMV
  • compounds of animal origin KAMBE et al., J. Immunol Methods, 240, 101-10, 2000).
  • Mast cells can be cultured using the techniques developed for the mass culture of eukaryotic cells, as described for example by Griffiths et al. (Animal CeII Biology, Eds Spier and Griffiths, Academy Press, London, vol.3, 179-220, 1986). Bioreactors with a capacity greater than several m 3 can be used as described by PHILIPS et al. (Large Scale Mammalian CeII Culture, Eds Feder and Tolbert, Academy Press, Orlando, USA, 1985), or by MIZRAHI (Process Biochem, August, 9-12, 1983).
  • the culture can also be carried out in suspension or on a microcarrier according to the technique described by VAN WEZEL (Nature, 216, 64-65, 1967). It is also possible to use batch culture systems, which are frequently used for eukaryotic cell cultures, because of their greater simplicity of implementation on an industrial scale (VOGEL and TODARO, Fermentation and Biochemical Engineering Handbook, 2 ⁇ d edition, Noys Publication, Westwood, New Jersey, USA, 1997). The cell densities obtained with these systems are generally between 10 6 and 5 x 10 6 cells / ml.
  • the productivity of the batch cultures can be advantageously increased by removing a portion of the bioreactor cells (70% to 90%) for the GAG extraction and heparin isolation operations and retaining the remaining cells within the same bioreactor to initiate a new culture.
  • the optimum parameters of the cell growth phase can be distinguished, from those allowing a greater accumulation of GAGs and heparin within the cells.
  • Continuous perfusion type culture systems with or without cell retention, can also be used (VELEZ et al., J. Immunol Methods, 102 (2), 275-278, 1987, CHAUBARD et al., Gen. Eng. News, 20, 18-48, 2000).
  • perfused culture systems that allow the cells to be retained inside the reactor, and that result in growth and production that is greater than that obtainable in batch.
  • the retention can be carried out by means of spin-filter, hollow fiber or solid matrix retention systems (WANG et al., Cytotechnology, 9, 41-49, 1992, VELEZ et al., J. Immunol Methods, 102 (2), 275-278, 1987).
  • the cell densities obtained are generally between 10 7 and 5 ⁇ 10 7 cells / ml.
  • the bio-reactor culture makes it possible, through the use of online measurement sensors, to better control the physico-chemical parameters of cell growth: pH, pO2, Red / Ox, growth substrates such as vitamins, amino acids, carbon substrates (eg glucose, fructose, galactose), metabolites such as lactate or ammonia, etc.
  • the cells are harvested and separated from the culture medium, generally by centrifugation or filtration.
  • centrifugation systems can be used, for example those described by VOGEL and TODARO (Fermentation and Biochemical Engineering Handbook, 2 ⁇ d Edition, Noys Publication, Westwood, New Jersey, USA).
  • the tangential microfiltration can be carried out using membranes whose porosity is less than the mean diameter of the cells (5 to 20 ⁇ m) while allowing the passage of the other compounds in solution / suspension. .
  • the speed of the tangential flow and the pressure applied to the membrane will be chosen so as to generate little shear force (Reynolds number less than 5000 sec -1 ) in order to reduce membrane clogging and preserve the integrity of the cells during the separation operation.
  • membranes can be used, for example, spiral membranes (AMICON, MILLIPORE), flat membranes or hollow fibers (AMICON, MILLIPORE, SARTORIUS, PALL, GF).
  • membranes whose porosity, charge or grafting makes it possible to carry out a separation and a first purification vis-à-vis any contaminants that may be present in the culture medium, such as cellular proteins, DNA , viruses or other macromolecules.
  • the heparin would have been released from the intracellular content, by degranulation or lysis of all or part of the mast cells, and is present in the culture medium at the time of the separation step.
  • the separation of the cells is combined with an ultrafiltration step on one or more membranes whose arrangement and porosity makes it possible to concentrate the heparin and to separate it from the other species present in the medium, by
  • the cut-off threshold of the membranes is preferably between 1000 and 5 kDa.
  • Membrane systems similar to those used for micro-filtration may be used, for example, spiral membranes, flat membranes or hollow fibers. It is advantageous to use membranes which make it possible to carry out a separation and a purification of heparin, because of their charge properties or the grafting of ligands having an affinity for heparin (for example antibody, ATIII, lectin).
  • the recovery of heparin from the mast cells can also be done after degranulation or lysis of the cells.
  • Degranulation can be caused by the binding of specific ligands to the receptors present on the surface of mast cells, for example the attachment of allergen-type agents (such as Fc fragment of IgE or analogs of this fragment) to the receptors.
  • allergen-type agents such as Fc fragment of IgE or analogs of this fragment
  • IgE mast cells Other agents may also induce degranulation of mast cells.
  • These agents can be classified into several categories such as cytotoxic agents, enzymes, polysaccharides, lectins, anaphylatoxins, basic compounds (compound 48/80, substance P, etc.), calcium (ionophore A23187, ionomycin, etc. ). [D. Lagunoff and T. W. Martin. 1983. Agents that release histamine from mast cells. Ann. Rev. Pharmacol. Toxicol., 23: 331-51].
  • the use of degranulation agent can be carried out repeatedly on the same cells maintained in culture. In this mode of production productivity is significantly increased by simplifying the harvesting process from the supernatant and by keeping the cells in culture.
  • mast cell degranulation can be induced, for example, by treatment of 2.10 6 cells / ml of mast cells with the ionophore A23187 at concentrations of between 1 and 100 ⁇ g / ml and reaction times. action varying from 1 minute to 4 hours.
  • the lysis of mast cells can be induced, for example, by osmotic shock using hypotonic or hypertonic solutions, by thermal shock (freezing / thawing), by mechanical shock (for example sonication or pressure variation), by action of agents.
  • thermal shock for example sonication or pressure variation
  • mechanical shock for example sonication or pressure variation
  • enzymatic lysis papain, trypsin, ...) or by combination of two or more of these methods.
  • the cell lysate can be subjected to one or more enzymatic digestions (pronase, trypsin, papain, etc.);
  • the heparin-protein bonds can be hydrolysed in an alkaline medium, in the presence of sulphates or chlorides;
  • the present invention also relates to heparin preparations that can be obtained from mast cell cultures by implementing a method according to the invention.
  • heparin preparations according to the invention which have biological properties comparable to those of the heparin preparations obtained in the prior art from animal tissues, can be used in all usual heparin applications.
  • Figure 1 Chemical structures of isaccharides Is, They NIs and IVs corresponding to sulfated N disaccharides of heparin, homologous disaccharides acetylated the IIa, IHa and.lva as well as tetrasaccharides IIa glu and Ha IVs glu.
  • Figures 2 molecular analysis of cultures MCpcDNA3.1 / Hygro and
  • Figure 2B RT-PCR performed in absence (-) or in the presence (+) of reverse transcriptase on 1 RNA extracted from control cultures MCpcDNA3.1 / Hygro (1) or cultures MCpBXL2015 (2) (C: control plasmid DNA pBXL2015).
  • Figure 3A Growth of MCpcDNA3.1 / Hygro Control Cells and Cells
  • FIG. 3B Comparison of the growth of MCpcDNA3.1 / Hygro control cells in the presence of SCF with MCpBXL2015 cells in medium without SCF.
  • Figure 5 Growth curve of the mother PM125 culture and clones PMC125 pBXL2033.
  • Figure 6 Study of the stability of proteoglycan production over time by clone PMC125 pBXL2033 19-D4. The mucus, extracted and analyzed under the same conditions as the cells is placed as a control.
  • the present invention is illustrated by the following implementation examples. These examples are purely illustrative and should not be considered as limiting.
  • Mast cells can be engineered by the introduction of an exogenous nucleic acid using, for example, transfection, electroporation, nucleoporation or infection techniques which will result in transient or stable expression of the nucleic acid.
  • ⁇ DN can be integrated into the cellular genome or maintained as an episome.
  • Stably transfected cells can be obtained using the nucleoporation method described below, by applying, 24 to 72 hours after the nucleoporation, a selection pressure (hygromycin, geneticin, blasticidine, puromycin or zeocin).
  • a selection pressure hygromycin, geneticin, blasticidine, puromycin or zeocin.
  • the resistance to the selection agent is conferred by the integration of the plasmid carrying the gene of interest and the resistance gene.
  • This method is used preferentially because it makes it possible to target the DNA directly in the nucleus.
  • mast cells in the exponential phase, preferably after 3 or 4 days of culture, are centrifuged at 1000 rpm for 5 minutes and taken up in 100 ⁇ l of nucleofection solution (Amaxa, Kit 8351). 2 to 4 ⁇ g of pcDNA3.1-eGFP, a plasmid encoding GFP, are then added to the cell suspension. The cells are then transferred to the electroporation cuvette and subjected to electric shock by the use of a specific program (such as U14, U23 and U28 AMAXA).
  • a specific program such as U14, U23 and U28 AMAXA
  • the cells are then transferred into 2 ml of complete medium preheated to 37 ° C. and then incubated at 37 ° C., 5% CO 2.
  • the cells are harvested for fixation with paraformaldehyde (Prolabo) 1%. For this, the entire culture is centrifuged for 5 min at 1000 rpm. After removal of the supernatant, the cells are washed in 4 ml of 1x PBS and centrifuged again. The cell pellet is then taken up in 1 ml of paraformaldehyde 1%. The cells thus fixed are then analyzed by cytometer (Cytomics FC 500, Beckman Coulter)
  • the nucleoporation conditions described above make it possible to transfect porcine mast cells with a transfection efficiency of between 30% and 50% while obtaining a good cell viability, greater than 50%.
  • recombinant and deleted retroviral vectors can be used for replication.
  • pseudo-typed vectors can be used with the envelope glycoprotein of the vesicular stomatitis virus (VSV-G) which allows the production of pantropic retro viral vectors capable of infecting porcine cells.
  • VSV-G vesicular stomatitis virus
  • the retroviral vector carrying the gene of interest to be expressed or integrated into the porcine mastocyte is initially produced with the aid of the packaging cell such as GP-293 (Clontech protocol ref PT 3132-1), which possesses the genetic elements for the production of the vector (gag and pol) with the exception of the gene for the production of the pseudo-typed envelope protein (env-VSV-G).
  • the packaging cells are cotransfected with the plasmid encoding the VSV-G envelope gene and a retroviral plasmid encoding the gene of interest under control of a promoter with or without selection gene.
  • the GP-293 cells are cultured 48 hours to 72 hours before transfection in order to be in the exponential phase of growth.
  • the culture medium is changed by fresh medium (15-20 ml per 10 6 cells), then 1 to 2 ml of solution containing the mixture of plasmid VSV-G (5 to 20 ⁇ g per 10 6 cells) and the plasmid carrying the gene of interest (10 to 30 ⁇ g per 10 6 cells) in pH7 calcium phosphate buffer are added dropwise into the culture medium (1 to 2 ml (Promega)
  • the cells are then incubated for 16-24 hours at 37 ° C or preferably at 32-35 ° C.
  • the culture medium is changed again with fresh medium.
  • the cells are incubated for an additional 48 hours at 32-35 ° C.
  • the culture supernatant containing the neo-formed retro-viral vectors is harvested.
  • Part of the packaging cell infection supernatant is aliquoted and frozen at -80 ° C., the other part is mixed with the mast cell culture medium in exponential growth growth phase.
  • mast cells in culture are centrifuged and resuspended in a medium containing 50% fresh medium and 50% infection supernatant.
  • Mast cells are incubated for 24 hours at 32-35 0 C, then the medium is changed again with fresh medium.
  • cytofluorimetry 48 to 72 hours after infection of the mast cells, samples are taken for cytofluorimetry analysis in the case of the control of infection with the fluorescent reporter gene GFP (Green Fluorescent Protein) or by PCR for infections with the gene of interest .
  • GFP Green Fluorescent Protein
  • the populations of transfected cells are then selected by adding in the culture medium the cytotoxic agent (Hygromycin, puromycin, G418, Zeocyne) for which only the mast cells transfected with the retro-viral vector carrying the gene of interest and the gene of resistance continue to grow.
  • the cytotoxic agent Hygromycin, puromycin, G418, Zeocyne
  • the selection agent can be removed from the culture medium while maintaining the expression of the gene of interest.
  • the retroviral vector produced in this way does not replicate in the host mast cell and therefore there is no production of replicative vectors.
  • the mast cells were transformed with the mutated c-kit gene.
  • the murine c-kit gene carrying a point mutation responsible for the modification of valine 559 to glycine was used.
  • the mast cells used for transfection are derived from a culture of porcine mast cells obtained from fetal liver as described in patent application WO 03/035853. This 1-2735 mast cell line was maintained in culture for 929 days in the presence of cytokines.
  • Mast cells are cultured in MEM ⁇ medium supplemented with 15% fetal calf serum (Hyclone), 2 mM glutamine (Invitrogen), 10 nM pGE2 (Sigma), 2 ng / ml rpIL-3 (Biotransplant) and 80 ng / ml rpSCF (Biotransplant).
  • This culture medium is called complete medium 1.
  • the cells are incubated at 37 ° C. under 5% CO 2 and inoculated at 2.10 5 C / ml every 3-4 days.
  • the murine plasmid pEF-BOS-ckit G559 was obtained by cloning the XbaI fragment of the murine ckit gene mutated in the plasmid pEF-BOS (described by Mizushima and Nagata, Nucleic Acid).
  • the c-kit gene carries a point mutation responsible for the modification of valine 559 to glycine (gene noted c-kit G559 ). This mutation is analogous to the mutants c-kit G556 in pigs and c-kit G560 in humans.
  • the murine plasmid pEF-BOS-ckit G559 was digested with XbaI to obtain mutated murine cK12 G559 cDNA.
  • the cDNA fragment was cloned into the plasmid pcDNA3.1 (Invitrogen) at the XbaI site.
  • the plasmid obtained, after complete sequencing of the cDNA, was named pBXL2008.
  • Plasmid pBXL2008 was digested with XbaI to obtain the mutated murine ckit G559 cDNA.
  • the cDNA fragment was cloned into the plasmid pcDNA3.1 / Hygro (Invitrogen) at the XbaI site.
  • the resulting plasmid is called pBXL2015.
  • the cDNA fragment comprises the sequence SEQ ID NO :
  • the DNA preparations for the transfections were carried out with the kit "Endofree plasmid maxi” (Quiagen). Transfection-selection of a hygromycin resistant population
  • transfection medium MEM ⁇ supplemented with 2 mM glutamine, 10 nM PGE2, 80 ng / ml rpSCF and 2 ng / ml rplL3
  • 30 ⁇ g of pcDNA3.1 / Hygro or pBXL2015 are then added to the cell suspension.
  • the cells are then transferred to the electroporation cuvette and subjected to an electric shock of 960 ⁇ F, 340 V (Gene Pulser, Biorad).
  • the cells After electroporation, the cells are transferred into 5 ml of complete medium 1 preheated to 37 ° C. and then incubated at 37 ° C., 5% CO 2. 48 hours after transfection, the cells are digitized, centrifuged and inoculated at 2 ⁇ 10 5 C / ml in selection medium (complete medium 1 supplemented with hygromycin
  • a hygromycin-resistant cell population is obtained 16 days after application of the hygromycin selection.
  • Cells transfected stably with the plasmid pcDNA3.1 / Hygro are named MCpcDNA3.1 / Hygro; those stably transfected with plasmid pBXL2015 are named MCpBXL2015.
  • the cell cultures are maintained in culture in the selection medium.
  • Detection of the murine ckit G559 gene in MCpBXL2015 cells The detection of the murine ckit G559 gene was carried out by PCR from genomic DNA extracted from the MCpcDNA3.1 / Hygro and MCpBXL2015 cultures, using primers making it possible to specifically amplify the gene. murine without amplifying the endogenous porcine gene.
  • the genomic DNA was extracted from 5 ⁇ 10 6 cells from the MCpcDNA3.1 / Hygro and MCpBXL2015 cultures according to the Quiagen Dneasy Tissue kit protocol.
  • the detection of the expression of the ckit G559 gene was carried out by RT-PCR.
  • RNA was extracted from 10 7 cells from the MCpcDNA3.1 / Hygro and MCpBXL2015 cultures according to the Quiagen Rneasy mini kit protocol. 30 ⁇ g of RNA was then digested with 20 U of DNase I for 1 hour at 37 ° C. and then purified according to the "Cîeanup" protocol of the Quiagen Rneasy mini kit.
  • RNAs 2 .mu.l of these RNAs was reverse transcripted into cDNA with the AMV RT reverse transcriptase (Roche) using, as primer, the oligonucleotide of sequence SEQ ID No. 9 5'-gac cac gcg tat cga tgt cga ctt ttt ttt ttt ttt ttv -3 '.
  • the PCR was performed on 1 .mu.l of cDNA under the conditions described in the previous paragraph.
  • the functionality of the ckit G559 was evaluated by analyzing the ability of cells to grow in a medium lacking SCF.
  • the control MCpcDNA3.1 / Hygro cells are not able to grow in such a medium: there is a stop growth followed by a large cell death (Figure 3A).
  • MCpBXL2015 cells there is an initial period of about three weeks during which growth is low ( Figure 3A). This latency period can be explained by the fact that not all cells, despite their resistance to hygromycin, express a functional G559 ckit murine receptor, which translates into a disability. to grow in the absence of SCF. After this latency phase, a population of MCpBXL2015 cells able to grow in the absence of SCF appears. This subpopulation was amplified after counting and seeding at 2.10 5 C / ml every 3-4 days for at least 11 weeks. Under these conditions, after seeding at 2.10 5 C / ml, the maximum cell density reached is between 4 to 10.10 5 cells / ml with an exponential phase doubling time of between 24 and 48 hours.
  • MCpBXL2015 cells in the absence of SCF, is identical to that of the control cells, MCpcDNA3.1 / Hygro, in medium supplemented with SCF (FIG. 3B).
  • the expression of the murine ckit G559 gene thus makes it possible to dispense with the SCF while maintaining the same level of growth.
  • the samples are treated as follows: Proteolysis: The cell samples, 2x10 6 cells, are centrifuged and rinsed twice in PBS buffer. Each pellet is taken up in 100 ⁇ l of distilled water added with 10 ⁇ l of alkalase (Novozymes) and then heated for 5 hours at 60 ° C. with stirring.
  • Proteolysis The cell samples, 2x10 6 cells, are centrifuged and rinsed twice in PBS buffer. Each pellet is taken up in 100 ⁇ l of distilled water added with 10 ⁇ l of alkalase (Novozymes) and then heated for 5 hours at 60 ° C. with stirring.
  • proteolysis step makes it possible to release the intracellular content and to dissociate the protein-polysaccharide bonds.
  • GAGs GlycoaminoGlycans
  • the GAGs are depolymerized with a mixture of heparinases I, 11 and III (Grampian enzymes). Each heparinase solution is adjusted to 0.5 IU / ml in phosphate buffer. Heparinase solution I, II, III is prepared by mixing 1/3 volume to volume of each heparinase solution. For 100 ⁇ l of sample to be analyzed, add
  • the samples are then analyzed by HPLC on Spherisorb SAX 5 ⁇ m, 250x3mm, Thermohypersil column.
  • 50 ⁇ l of sample are injected by analysis, the buffer of the mobile phase is composed of 2.5 mM Sodium Di Hydrogen phosphate (Na 2 HPO 4, Prolabo) whose pH is adjusted to 2.9 with ortho-phosphoric acid (H 3 PO 4, Prolabo ).
  • the elution of the constituent disaccharides of the GAGs extracted from the cell samples is carried out in a gradient of 0 to 100% in 50 minutes of 2.5 mM Na 2 HPO 4 buffer and 1 M of perchlorate (NaClO 4, Prolabo).
  • the disaccharides are detected by their retention time and compared to a standard heparin sample (Aventis) in UV at 234 nM.
  • the MCpBXL2015 cells were cloned by the limiting dilution technique in medium without SCF. For this, 10 96-well plates were inoculated with 0.3 cells / 100 ⁇ l / well. Only clones with satisfactory 96-well plate growth were retained and amplified. Once at confluence, the clones were recovered and then seeded in a 48-well plate with 500 ⁇ l of medium and then in 6-well plate with 2 ml of medium and finally in F25 with 5 ml of medium. At the end of this amplification, nine clones were obtained and amplified after counting and seeding at 2.10 5 C / ml every 3-4 days.
  • the final choice of the clone was made after an analysis of the growth and composition of mast cell proteoglycans.
  • the clone MC pBXL2015 6-G4 was retained. This clone was frozen under the name of PMC 125.
  • the mast cells used are derived from the PMC125 culture described in Example 3.
  • the mast cells are cultured in MEM ⁇ medium supplemented with 15% fetal calf serum (Hyclone), 2 mM glutamine (Invitrogen), 2 ng / ml rplL-3 (Biotransplant). This culture medium is called culture medium 2.
  • the cells are incubated at 37 ° C. under 5% CO 2 and passed every 3-4 days.
  • the plasmid HS3ST1-pE-IRES-neo2- coding for porcine 3-OST1 has been described in patent application PCT / FR04 / 00902 filed in the name of AVENTIS PHARMA SA. As a reminder, the plasmid HS3ST1-pE-IRES-neo2 was obtained as following. Isolation and sequencing of the 3 'coding sequence of the porcine 3-OST gene
  • the partial sequence of the porcine gene encoding 3-OST is available in an EST library (GenBank accession number BF075483). Alignment of this sequence with the human sequence shows that about 650 bp of the 3 'coding region is missing.
  • the missing portion of the porcine 3-OST gene was identified by combining RT-PCR and 3'-RACE using as RNA source porcine liver RNA isolated according to the Trizol kit protocol (Invitrogen)
  • RNA in cDNA was carried out following the protocol of the First Strand Synthesis Kit Sytem (Invitrogen), using as primer a mixture of the oligonucleotides BS02 and BS03 of respective sequences SEQ ID No. 5 ' GCA GCA GCC ACG TCG GG-3 'and SEQ ID No. 11 5'-TCA GTG YCA GTC RAA TGT TC-3'.
  • the sequence of this fragment was used to generate 2 primers BS21 and BS22 to isolate, by 3'-RACE, the entire 3 'region.
  • the 3 'region of the gene encoding 3-OST was then amplified by 2 successive PCRs.
  • the first PCR was performed on 2 .mu.l of cDNA, obtained previously, with the BS21 sense primer of sequence SEQ ID No. 5'-GCA CCC CCA CGA GAT CGA C-3 'and a CDSIII antisense primer.
  • 30 thermal cycles were applied (10 sec of denaturation at 94 ° C., 30 sec of hybridization at 60 ° C. and 120 sec of elongation at 68 ° C.).
  • the second PCR was then carried out under the same conditions as the first PCR with 1 ⁇ l of product obtained from the first PCR using the BS22 sense primer of SEQ ID No. 16 5'-CAA ACT CCT CAA TAA ACT GCA CG-3 'sequence and the CDSIlI antisense primer.
  • RNA 2 ⁇ g were reverse transcribed into cDNA according to the protocol of the First Strand Synthesis Sytem kit of Invitrogen, using as primer a dT 24 oligonucleotide.
  • the gene encoding 3-OST was then amplified by two-step PCR.
  • the first PCR allowed to amplify the gene including a portion of the 3 'non-coding sequence of the gene, the second PCR then allowed to amplify the coding sequence using primers compatible with the Gateway system (Invitrogen)
  • the first PCR was carried out on 2 .mu.l of cDNA with a BS10 sense primer of the 5'-AGG CCC GTG ACA CCC ATG AGT-3 'sequence hybridizing specifically in the 5' noncoding region of the porcine 3-OST gene and a 5'-CAC CTA GTG TAC ACCACA ATT TAC-3 'anti-sense sequence BS30 primer specifically hybridizing 3' at the level of the RTU.
  • 35 thermal cycles were applied (10 sec of denaturation at 98 ° C., 30 sec of hybridization at 64 ° C. and 150 sec of elongation at 68 ° C.)
  • a second PCR is then carried out on 1 .mu.l of the PCR product in order to specifically amplify the coding phase.
  • the PCR program used is identical to that used for the first PCR.
  • the 1 kb PCR product was then cloned according to the procedure of the Gateway cloning technology kit, Invitrogen in the pE-IRES-neo2 episomal vector.
  • the porcine gene sequence was verified by sequencing.
  • the nucleotide sequence obtained is the sequence SEQ ID No. 5.
  • the deduced protein sequence is the sequence SEQ ID No. 6.
  • the porcine 3OST1 gene was amplified by PCR from plasmid HS3ST1-pE-IRES-neo2 in the presence of a C21755 sense primer of sequence SEQ ID No. 19 5'-CGC GGA TCC CAG CAT GGC CGC GCT GCT CC -3 and C21756 antisense primer of sequence SEQ ID NO: 5 CTA GTC TAG ATT AGT GCC AGT CAA ATG TTC -3 'according to Advantage 2GC PCR kit protocol (Clontech). After the 25 thermal cycles (30 sec of denaturation at 94 ° C., 45 sec of hybridization at 55 ° C.
  • the PCR reaction is purified according to the Quiaquick PCR kit protocol. Quiagen purification. The entire PCR product is then digested for 4 hours at 37 ° C. with BamHI and XbaI (Biolabs). The digestion product is then purified, after migration on 1% TBEIx agarose gel, according to the protocol of Quiaquick gel extraction Quiagen kit. The PCR product thus digested is cloned into the BamHI-Xba I open pcDNA3.1 / Hygro vector.
  • Plasmid pBXL2032 was digested with XbaI-BamHI to obtain 3OST1 porcine cDNA.
  • the cDNA fragment was cloned into the plasmid pcDNA3.1 (Invitrogen) opened by XbaI-BamHI.
  • the plasmid thus obtained was named pBXL2033.
  • the cells are digitized, centrifuged and inoculated at 2 ⁇ 10 5 C / ml in selection medium (culture medium 2 supplemented with geneticin (Invitrogen) at a concentration of 0.8 mg / ml).
  • selection medium culture medium 2 supplemented with geneticin (Invitrogen) at a concentration of 0.8 mg / ml.
  • a population resistant to geneticin was obtained 20 days after application of the selection.
  • PMC125 cells stably transfected with the plasmid pcDNA3.1 are named PMC125pcDNA3.1; those stably transfected with plasmid pBXL2033 are named PMC125pBXL2033.
  • the cell cultures are maintained in culture in the selection medium. Characterization, in HPLC, of proteoglycans contained in cultures
  • 3-OST1 The functionality of 3-OST1 was assessed by analyzing the level of sulfation of the proteoglycans produced by the control mast cells PMC125 pcDNA3.1 and mast cells PMC125 pBXL2033. The level of sulfation can be monitored by quantifying the amount of sulfalating developer llallas tetrasaccharide.
  • proteoglycans produced by the PMC125pcDNA3.1 control cells do not contain detectable amounts of llallsglu tetrasaccharide (Table 3).
  • proteoglycans synthesized by PMC125pBXL2033 cells contain 0.9% llallsglu indicative of a significant increase in the level of sulfation in these cultures.
  • the reaction takes place in three stages:
  • the amount of Paranitroaniline released is inversely proportional to the amount of heparin.
  • the anti-Xa or Anti-lla quantity is measured relative to a calibration line established with the standard SPIM (Standard International Heparin).
  • SPIM Standard International Heparin
  • the sensitivity of the method is 0.006 IU / ml.
  • the biological activity is expressed in IU / mg taking into account the quantification of disaccharides obtained by HPLC.
  • the biological activity of samples identical to those analyzed in HPLC 1 was determined.
  • proteoglycans synthesized by the PMC125pcDNA3.1 control cells do not exhibit any detectable biological activity.
  • the analysis of the proteoglycan activity produced by PMC125pBXL2033 cells reveals the presence of anti-Xa and anti-11a biological activity of between 60 and 80 IU / mg (Table 3). It is noted that at this stage of the culture, that is to say four days after seeding, the ratio between the two activities is close to 1, which is characteristic of the heparin resulting from extraction from the intestinal mucosa of pork.
  • PMC125 pBXL2033 cells were cloned by the limiting dilution technique. For this, 20 96-well plates were inoculated with 0.3 cells / 100 ⁇ l / well. Only clones with satisfactory 96-well plate growth were retained and amplified. Once at confluence, the clones were recovered and then seeded in a 24-well plate with 0.5 or 1 ml, in a 12-well plate with 2 ml of medium and then in a 6-well plate with 4 ml of medium and finally in F25 with 10 ml. of middle. At the end of this amplification, 156 clones were obtained and amplified after counting and inoculation at 2.10 5 C / ml every 3-4 days.
  • a first analysis of the synthesized proteoglycans was performed by HPLC to identify clones exhibiting sulfation activity. At the end of this first study, only 25 of the 156 clones tested were positive in HPLC. These clones were then analyzed for biological activity of the synthesized proteoglycans. At the end of this second analysis, only nineteen clones were retained. The nineteen clones selected at the end of the two analyzes produce proteoglycans with an increased level of sulfation.
  • Table 1 Analysis, by HPLC, of proteoglycans synthesized by MCpcDNA3.1 / Hygro in the presence of SCF and by MCpBXL2015 in a SCF-deficient medium.
  • Table 2 Analysis, by HPLC, of the proteoglycans synthesized by the uncloned MCPBXL2015 parent culture and the various clones obtained by limiting dilution, in a SCF-deficient medium.
  • Table 3 Analysis of the structure and biological activity of proteoglycans synthesized by cultures PMC125pcDNA3.1 and PMC125pBXL2033.
  • Table 5 Analysis of the biological activity of the proteoglycans synthesized by clones PMC125pBXL2033.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
EP05809321A 2004-10-12 2005-10-10 Heparinähnliche moleküle produzierende mastzellinien aus schwein Withdrawn EP1846549A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR0410763A FR2876386B1 (fr) 2004-10-12 2004-10-12 Lignees de mastocytes porcins produisant des molecules de type heparine
PCT/FR2005/002488 WO2006040463A1 (fr) 2004-10-12 2005-10-10 Lignees de mastocytes porcins produisant des molecules de type heparine

Publications (1)

Publication Number Publication Date
EP1846549A1 true EP1846549A1 (de) 2007-10-24

Family

ID=34950891

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05809321A Withdrawn EP1846549A1 (de) 2004-10-12 2005-10-10 Heparinähnliche moleküle produzierende mastzellinien aus schwein

Country Status (4)

Country Link
EP (1) EP1846549A1 (de)
JP (1) JP2008515417A (de)
FR (1) FR2876386B1 (de)
WO (1) WO2006040463A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011078357A (ja) * 2009-10-07 2011-04-21 Kochi Univ ビタミンｂ６の分別定量方法およびビタミンｂ６の分別定量用キット
CN114502739A (zh) * 2019-10-02 2022-05-13 国立大学法人九州大学 肝素样物质的制造方法、重组细胞及其制造方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014418A1 (en) * 1989-05-19 1990-11-29 The Uab Research Foundation Heparin-producing murine mastocytoma cell lines
FR2831186B1 (fr) * 2001-10-22 2004-06-18 Aventis Pharma Sa Production d'heparine a partir de cultures de mastocytes
FR2853663B1 (fr) * 2003-04-14 2007-08-31 Aventis Pharma Sa Procede d'obtention de lignees de mastocytes a partir de tissus de porcs et procede de production de molecules de type heparine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006040463A1 *

Also Published As

Publication number Publication date
WO2006040463A1 (fr) 2006-04-20
FR2876386B1 (fr) 2007-04-06
FR2876386A1 (fr) 2006-04-14
JP2008515417A (ja) 2008-05-15

Similar Documents

Publication Publication Date Title
US20080064095A1 (en) Method for obtaining mastocyte lines from pig tissues and for producing heparin-type modules
CA3224970A1 (en) Compositions and methods for efficient genome editing
JP2023072010A5 (de)
AU2019261438B2 (en) Expression of FOXP3 in edited CD34+ cells
AU2020223733A1 (en) Compositions and methods for the treatment of hemoglobinopathies
JP2020518256A (ja) Ｃｒｉｓｐｒ／ｃｐｆ１を用いる、ｔ細胞における遺伝子編集のための組成物および方法
CN114007655A (zh) 用于细胞疗法的环状rna
KR20200067190A (ko) A형 혈우병을 위한 유전자 편집용 조성물 및 방법
WO2024178144A1 (en) Methods and compositions for editing nucleotide sequences
EP0441695A1 (de) Verfahren zur Herstellung von humanem Faktor VIII und dessen Analogen
EP2167675B1 (de) Verfahren zur enzymatischen Spaltung von aus Algen gewonnenen Polysacchariden
KR20040084885A (ko) 형질도입된 조혈 선조 세포의 제조 방법
WO2006040463A1 (fr) Lignees de mastocytes porcins produisant des molecules de type heparine
WO2023215724A1 (en) Methods for reprogramming and gene editing cells
EP1438415A2 (de) Verfahren zur herstellung von heparin durch mastzellen
CA3213153A1 (en) Human macrophages resistant to tumor-induced repolarization
CN119855599A (zh) 用于细胞疗法的同种异体人巨噬细胞
WO2023157899A1 (ja) 組換えヘパリン様物質、及びその製造方法
KR20230158102A (ko) 인간 식작용 세포의 생체외 증식
JPH02200189A (ja) ヒトインターロイキン２活性をもつポリペプチドをコートする遺伝子
WO2025038881A1 (en) Prime editing of single base mutations in sickle cell disease
US20260071213A1 (en) Compositions targeting hbg1 and hbg2 and methods of use thereof
EP0789756A1 (de) Verfahren zur zersetzung von eukariotischen zellen in kultur
WO2025194133A1 (en) Prime editing of single base mutations in alpha-1 antitrypsin deficiency
WO2024163679A1 (en) Genome editing compositions and methods for treatment of cystic fibrosis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20070903

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20071129

DAX Request for extension of the european patent (deleted)
RIN1 Information on inventor provided before grant (corrected)

Inventor name: PEREZ, SANDRINEC

Inventor name: GUILLAUME, JEAN-MARC

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080410