JP2008515417A - Porcine mast cell line producing heparin-type molecules - Google Patents

Abstract

  The present invention relates to a porcine mast cell line whose proliferation is independent of growth factors. The present invention also relates to a method for producing a biologically active heparin-type molecule comprising culturing the mast cell line.

Description

  The present application relates to a porcine mast cell line whose growth is growth factor independent. The invention also relates to a method for producing bioactive heparin-type molecules, including culturing these mast cell lines.

  Mast cells are cells of the immune system derived from hematopoietic progenitors that are involved in inflammatory responses, particularly allergies and hypersensitivity phenomena. Mast cells are present in connective tissue (especially in the skin) and in the intestinal and respiratory mucosa.

  Mast cells are in the form of round cells with a diameter between about 5 and 25 μm and have a single, central or off-centered round nucleus. Mast cells are also characterized by the presence of numerous metachromatic cytoplasmic granule formations.

  These granules contain various molecular species with pro-inflammatory activity such as proteoglycans such as histamine, serotonin, heparin or chondroitin sulfate, enzymes, cytokines and factors that chemically attract eosinophils and neutrophils . These species are released during mast cell activation.

  After activation, a second response is initiated, during which leukotrienes, prostaglandins, PAF (platelet activating factor), interleukins (IL4, IL5, IL6, IL10, IL12 and IL13), cytokines (TGR-β , Γ-IFN, GM-CSF) and chemokines (MCP-1, IL8, RANTES) and the like. All of these species are involved in initiating inflammatory processes and initiating specific T lymphocyte dependent immune responses.

  Mast cell cultures have already been obtained in humans, mice and pigs. In pigs, the porcine mast cell line has been established by culturing stem cells obtained from fetal liver. These were deposited under the name of WO 03 under the name of the patent number 53 of the Institute National de la Recherche Agronomique (INRA) [French National Agricultural Research Institute] and Ecole National Veterinaire de Maisons Alffort (ENVA) [Maison Alfort French National Veterinary School] 03. The theme of the issue. On October 17, 2001, under the numbers I-2734, I-2735 and I-2736, respectively, three strains were found in the “Collection de Cultures de Microorganisms” [French Culture Collection in France] on Pasteur Institute. Deposited.

  These cells that are spontaneously immortalized have been maintained in culture for over 170 generations in the presence of growth factors such as rpSCF and rpIL3. Under these conditions, cells can synthesize heparin-type compounds. These mast cell lines produce significant amounts of heparin-type molecules.

  Patent application WO 03/035886, whose subject is a method for producing heparin from these strains, is described in Aventis Pharma S .; A. Deposited under the name of

  In pigs, Emery et al. (Experimental Hematology, 24, 927-935, 1996) maintained cell cultures obtained from bone marrow for 7 weeks. However, the resulting culture is a mixture of various cell types and appears not to be a uniform culture or strain of mast cells. In addition, these cultures contain non-differentiated cells in a uniform mast cell culture.

  Ashraf et al. (Veterinary Parasitology; 29, 134-158, 1988) isolated porcine mast cells from the supermucosa without maintaining an amplifiable culture. Furthermore, the characterization of isolated mast cells reveals the absence of heparin.

  Razin et al. (J. Biol. Chem., 257, 7229-7236, 1982) describe obtaining murine mast cells using a medium containing IL3.

  Wang et al. (Circ. Res., 84, 74-83, 1999) describe the isolation of serous mast cells obtained from rat pleura and abdominal cavity. Various molecular species are produced only when mast cells are co-cultured with rat aortic smooth muscle cells. Application WO 99/26983 describes similar work but is very inaccurate for applications to other species.

  In mice, cell lines have been established (Montgomery et al, Proc. Natl. Acad. Sci. USA, 89, 11327-11331, 1992 and application WO 90/14418), but are established from mast cell tumors. is there. These tumors are extremely rare in pigs.

  In humans, it has proven difficult to obtain mast cell cultures. This was initially possible by using a co-culture system with fibroblasts (Ishizaka et al, Current Opinion in Immunology, 5, 937-943, 1993). Subsequently, other authors succeeded in obtaining mast cells from intestinal cells and maintaining these cells in culture for about 6 months in the presence of SCF (Bischoff et al, J. Immunol. 159, 5560-5567, 1997).

  The authors of these various publications, however, only reported low production of heparin-type compounds when making measurements.

  Many mast cell cultures require growth factors such as SCF for optimal growth. SCF altered cellular responses such as proliferation and differentiation following binding to the receptor ckit. The study of mouse and human mastocytomas can identify mutations or deletions in the c-kit gene that are required for constitutive activation of the c-kit receptor (ie, without SCF binding). It has become possible. Expression of the V814-mutated c-kit gene in IC2 immortal mast cells induces transformation of these cells (ie, acquisition of SCF-independent growth and tumorigenic potential) (Pia et al , Blood, 87 (8), 3117-3123, 1996).

  Heparin belongs to the glycosaminoglycan (GAG) family and contains a disaccharide sequence repeat composed of amino sugar (D-glucosamine or galactosamine) and uronic acid (D-glucuronic acid or L-iduronic acid). Contains chain polysaccharides.

  In the case of heparin belonging to the glucosaminoglycan subfamily together with heparin sulfate, the amino sugar is D-glucosamine. Uronic acid is either glucuronic acid (Glc) or iduronic acid (Ido). Glucosamine can be N-acetylated, N-sulfated or O-sulfated.

  Conventionally, the term “heparin” is a highly sulfated polysaccharide in which more than 80% of the glucosamine residues are N-sulfated and the number of O-sulfates is greater than the number of N-sulfates. Represents. The sulfate / carboxylate ratio is generally greater than 2 for heparin. In practice, however, the structure of heparin is heterogeneous and there are chains that can contain very different proportions.

  Like all GAGs, heparin is synthesized in the form of proteoglycans substantially by mast cells.

  The first step in heparin synthesis is the formation of a serglycine protein core consisting of repeating serine and glycine residues. The heparin chain is extended by the addition of a tetrasaccharide and then by the sequential addition of glucosamine and uronic acid, which alternate uniformly.

  The proteoglycans thus formed undergo a number of sequential transformations (N-deacetylation, N-sulfation, D-glucuronic acid epimerization and O-sulfation). However, this complete mutation only occurs for a portion of the proteoglycan and produces a large structural variability in heparin that causes its heterogeneity. Next, the polysaccharide chain is cleaved from serglycine by endoglucuronidase. These chains then have a molecular weight between 5000 and 30000 Da. They are stored in mast cell granules because they form complexes with basic proteases. Heparin is excreted only during degranulation of mast cells.

  Heparin plays an important biological role, especially in hemostasis, and is very widely used in therapy, particularly as an anticoagulant and thrombosuppressant.

  Many of the heparins used are isolated from porcine intestinal mucosa (heparin is extracted from porcine intestinal mucosa by proteolysis) and then purified on anion exchange resins (various of heparin For a review on the preparation methods, see “Duclos, << L'Heparine: fabrication, structure, properties, analyze >> [heparin; production, structure, properties, analysis] publisher Masson, Paris, 1984).”

  Analysis of the disaccharide composition of porcine heparin after depolymerization with heparinase I, II, and III, and chromatography, makes it possible to distinguish heparin from other glycosaminoglycans. In particular, eight major disaccharides are distinguished (FIG. 1).

HPLC analysis also makes it possible to detect the presence of IIa IIs glu and IIa IVs glu tetrasaccharides (underlined are symbols representing 3-O-sulfation of disaccharides). The presence of these tetrasaccharides directly correlates with the presence of the ATIII affinity site. Indeed, 3-O-sulfation of the ATIII affinity site results in incomplete digestion of this site and thus the appearance of IIa IIs glu and IIa IVs glu tetrasaccharides. Biologically active heparin can be obtained by 3-O-sulfation of the ATIII affinity site.

  The above study allows the isolation of production strains. However, these need to be improved in order to enable their industrial use. In particular, it is desirable to make the growth of these strains independent of growth factors and to improve the biological activity of the molecules produced.

  Applicants have surprisingly been able to improve these two aspects by transforming a mast cell line with a mutant c-kit protein, and through overexpression of 3OST1 protein, an organism suitable for industrial production. It was shown that production of heparin-type molecules showing activity can be obtained.

The subject of the present invention is a porcine mast cell culture or strain producing a heparin-type molecule comprising the IIa IIs glu tetrasaccharide. Advantageously, these cultures or strains produce a detectable amount of IIa IIs glu tetrasaccharide. Preferably, these cultures or strains produce heparin-type molecules containing IIa IIs glu tetrasaccharide in an amount of at least 0.3%, 0.5%, 1% or 1.5% of all sugars. .

  According to an advantageous embodiment, these porcine mast cell cultures or strains are heparin-type molecules exhibiting anti-Xa activity of at least more than 50 IU / mg, preferably more than 75 IU / mg, more than 100 IU / mg or more than 150 IU / mg. Produce.

  According to another advantageous embodiment, these porcine mast cell cultures or strains are heparin exhibiting anti-IIa activity of at least more than 50 IU / mg, preferably more than 75 IU / mg, more than 100 IU / mg or more than 150 IU / mg. Producing type molecules.

  According to yet another advantageous embodiment, such a culture or strain produces a heparin-type molecule comprising at least 30% (preferably at least 40%, more preferably at least 50%) of the Is disaccharide.

  According to a preferred embodiment, the strain of the invention is modified to overexpress an enzyme that acts on sulfation of heparin-type molecules.

  Advantageously, said strain comprises a foreign nucleic acid encoding an enzyme that acts on sulfation of heparin-type molecules.

  Stable overexpression of enzymes that act on sulfation of heparin-type molecules can increase 3-O-sulfation of heparin compounds, and thus increase biological activity.

  Advantageously, the enzyme acting on the sulfation of heparin-type molecules is 3-OST, particularly preferably 3-OST1.

  The enzyme that acts on sulfation of heparin-type molecules is preferably porcine 3-OST1 having the sequence SEQ ID NO: 6 and encoded by the sequence SEQ ID NO: 5.

  Alternatively, using a gene from another species that encodes the expression of 3-O-sulfatase activity and exhibits at least 80%, 90%, 95% or 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 5 Is possible. Nucleic acids that hybridize under high stringency conditions to the nucleic acid sequence of SEQ ID NO: 5 may also be used.

Advantageously, the porcine mast cell line can be modified to express a c-kit protein exhibiting mutations or deletions, allowing for the absence of SCF. A foreign nucleic acid encoding a c-kit protein exhibiting loss.

A c-kit that exhibits a mutation or deletion is advantageously a c-kit protein that exhibits a mutation at a position equivalent to that of mouse c-kit ^G559 . Thus, this can be a c-kit protein that exhibits a mutation at a position equivalent to that of mouse c-kit ^G559 , such as porcine c-kit ^G556 or human c-kit ^G560 protein.

Preferably, mouse c-kit ^G559 having SEQ ID NO: 2 and having a point mutation responsible for modification of valine to glycine encoded by SEQ ID NO: 1.

It can also be porcine c-kit ^G559 having the sequence of SEQ ID NO: 4 and encoded by the sequence of SEQ ID NO: 3.

  For interspecies conservation of the c-kit gene, a mouse, human or bovine gene or any other gene exhibiting at least 80%, 90%, 95% or 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 3 It is possible to use. Nucleic acids that hybridize under high stringency conditions to the nucleic acid sequence of SEQ ID NO: 3 may also be used.

Surprisingly, the Applicant has in a cell expressing the murine c-kit ^G559, observed that compared to cells that do not express the murine c-kit ^G559 heparin profile is improved (Ie, different configurations of heparin-type molecules produced by these cells).

  It is also possible to use c-kit proteins that exhibit point mutations that cause modification of aspartic acid to valine 814 or 816 in mice and humans, respectively. Finally, it is also possible to use the mouse c-kit gene from which amino acids TQLPYDH 573 to 579 have been deleted. In pigs this deletion is equivalent to amino acids 570 to 576 and in humans it is equivalent to 574 to 580.

  More specifically, the present invention was founded on October 17, 2001 in Pasteur Institute at “Collection de Cultures de Microorganisms [French National Collection of Microorganisms Cultures], number I-2734, I-2735 or I-735. It relates to one of the porcine mast cell lines deposited at 2736 and modified to express c-kit proteins exhibiting mutations or deletions.

  Preferably, these strains contain a foreign nucleic acid encoding an enzyme that acts on sulfation of heparin-type molecules.

  As used herein, the term “culture” generally refers to a cell or group of cells cultured in vitro. A culture grown directly from a cell or tissue sample taken from an animal is referred to as a “primary culture”.

  The term “strain” is used once in at least one passage, generally all successive cultures from which several successive passages are carried out in succession in a subculture. (Schaeffer, In Vitro Cellular and Developmental Biology, 26, 91-101, 1990).

  The term “nucleic acid” is used to denote DNA or RNA. The nucleic acid is advantageously complementary DNA or genomic DNA.

  In the present invention, “percent identity” between two nucleotide or amino acid sequences can be determined by comparing two sequences optimally aligned through a window of comparison.

  A portion of the nucleotide or polypeptide sequence in the window of comparison may therefore be added or deleted relative to a reference sequence (not including additions or deletions) to obtain an optimal alignment of the two sequences. (Eg, a gap).

  The percent determines the number of positions where the same nucleobase or amino acid residue is observed for the two (nucleic acid or peptide) sequences being compared, and then by the total number of positions in the comparison window, Calculated by dividing the number of positions where identity exists between two bases or amino acid residues and then multiplying the result by 100 to obtain percent sequence identity.

  Optimal alignment of the comparative sequences is included in a package derived from the algorithm used in the computer derived from "Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin" It is possible.

  By way of example, percent sequence identity is exclusively obtained using BLAST software (from BLAST versions 1.4.9 1996, BLAST 2.0.4 from February 1998 and BLAST 2.0.6 September 1998). It can be calculated using 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). Blast uses the algorithm of Altschul et al. To search for sequences that are similar / homologous to a reference “request” sequence. The request sequences and databases used can be peptide-based or nucleic acid-based, and any combination is possible.

  In the present invention, the expression “high stringency hybridization conditions” means the following conditions.

1-membrane prehybridization and-mixing: salmon sperm DNA (10 mg / mL) 40 μL + human placenta DNA (10 mg / mL) 40 μL
-After denaturation at 96 ° C for 5 minutes, the mixture is placed in ice.

  Remove 2 × SSC and pour 4 mL of formamide mixture into the hybridization tube containing the membrane.

  -Add a mixture of two denatured DNAs.

  -Incubate at 42 ° C for 5-6 hours with rotation.

2- Labeled probe competition:
Add 10-50 μL of Cot I DNA to the labeled and purified probe according to the repeat volume.

  -Denaturate at 95 ° C for 7 to 10 minutes.

  Incubate at 65 ° C. for 2 to 5 hours.

3-Hybridization:
-Remove the prehybridization mixture.

  Mix 40 μL of salmon sperm DNA (10 mg / mL) + 40 μL of human placenta DNA (10 mg / mL); denature at 96 ° C. for 5 minutes, then place in ice.

  -Add 4 ml of formamide mixture (mixture of two DNA and denatured and labeled probe / CotI DNA) to the hybridization tube.

  Incubate for 15-20 hours at 42 ° C. with rotation.

4-washing:
-Wash once at ambient temperature in 2x SSC for rinsing-2x SSC at 65 ° C and 0.1% SDS at ambient temperature, 2 times 5 min-1x SSC at 65 ° C at 65 ° C and 0 1% SDS, 2 times 15 minutes.
Wrap the membrane in Saran wrap and expose.

  The hybridization conditions are suitable for hybridizing nucleic acid molecules of variable length from 20 nucleotides to several hundred nucleotides under high stringency conditions.

  It goes without saying that the hybridization conditions can be adjusted according to the length of diffusion desired to be hybridized or according to the type of label selected, according to techniques known to those skilled in the art.

  Suitable hybridization conditions are described in, for example, the book by Hames and Higgins (1985, “Nucleic acid hybridization: a practical approach”, Hames and Higgins Ed., IRL Press, Oxford) or F. It can be adjusted according to the teachings contained in the book by Ausaubel et al. (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY).

  The application also relates to methods for producing heparin-type molecules, including culture of porcine mast cell cultures or strains as described above.

  The strain of the present invention is preferably cultured in a defined medium (MEMα / DMEM, RPMI, IMDM, etc.).

  The medium may be supplemented with bovine serum at concentrations of 0.5% and 20% (v / v).

  In order to reduce the risks associated with the protein concentration of the medium and the use of compounds of animal origin, the addition of bovine serum to the medium can be replaced with the use of a serum-free medium such as AIMV (Invitrogen) (Kambe et al , J. Immunol. Methods, 240, 101-10, 2000).

A mast cell is a true cell as described, for example, in “Griffiths et al. (Animal CeII Biology, Eds. Spier and Griffiths, Academic Press, London, vol. 3, 179-220, 1986)”. It is possible to culture using techniques developed for mass culture. “Phillips et al. (Large Scale Mammalian Cell Culture, Eds. Feder and Tolbert, Academic Press, Orlando, USA, 1985)” or “Mizrahi (Process 9 eth). Bioreactors with a capacity of more than a few m ³ , as has been done, can be used.

  Culturing can also be performed in suspension or on a microsupport according to the technique described by Van Wezel (Nature, 216, 64-65, 1967).

For use on an industrial scale it is more simple, it is also possible to use a batch culture system that is used commonly for eukaryotic cell culture (Vogel and Todaro, Fermentation and Biochemical Engineering Handbook, 2 nd edition Noyes Publication, Westwood, New Jersey, U.S.A., 1997). The cell density obtained using these systems is generally between 10 ⁶ and 5 × 10 ⁶ cells / mL.

  The productivity of batch culture is that for GAG extraction and heparin isolation operations, some of the cells are removed from the bioreactor (70% to 90%) and at the same time in the same bioreactor to initiate a new culture. Can be advantageously increased by retaining the remaining cells. In this repeated batch culture mode, the optimal parameters of the cell growth phase can also be distinguished from them, allowing more accumulation of GAG and heparin in the cell.

  A continuous fusion feeding culture system 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).

  In the context of the present invention, in particular, a fusion-fed culture system may be used that allows the cells to be retained in the reactor and results in greater growth and production than can be obtained in batch culture. . Retention can be performed using a spin filter, hollow filter or solid matrix type retention system (Wang et al., Cytotechnology, 9, 41-49, 1992; Velez et al., J. Immunol. Methods, 102 (2), 275-278, 1987).

The resulting cell density is generally between 10 ⁷ and 5 × 10 ⁷ cells / mL. By culturing in a bioreactor, through the use of on-line measuring sensors, physicochemical parameters of cell growth: pH, pO ₂ , Red / Ox, vitamins, amino acids, carbon based substrates (eg glucose, fructose, galactose ), Allowing better control of growth substances such as metabolites such as lactate or aqueous ammonia.

  Following treatment with sodium butyrate, it can be envisaged to quantitatively and qualitatively increase the heparin-type molecular content of mast cells (Nakamura et al (Biochim. Biophys. Acta. 627, 60-70, 1980)).

  After 3 to 14 days of culture, preferably after 3 to 5 days, the cells are collected and separated from the medium, generally by centrifugation or filtration.

It is possible to use various centrifugal systems, for example, Vogel and Todaro (Fermentation and Biochemical Engineering Handbook, 2 nd Edition, Noyes Publication, Westwood, New Jersey, U.S.A.) as described by Can be mentioned.

  Alternatively, or in combination with centrifugation, separation by tangential microfiltration using membranes with a porosity less than the average cell diameter (5-20 μm) while simultaneously passing other compounds in the solution / suspension Can be implemented. The speed and pressure of the tangential flow imparted through the membrane seems to generate almost shear forces (Reynolds number less than 5000 / sec) to reduce membrane clogging and preserve cell integrity during the separation operation. Selected.

  Various membranes can be used, for example spiral membranes (Amicon, Millipore), flat membranes or hollow fibers (Amicon, Millipore, Sartorius, Pall, GF).

  It is also possible to select a porosity, charge or graft membrane that allows separation and first purification to be performed on contaminants that may be present in the medium, such as proteins, DNA, viruses or other microorganisms. It is.

  If heparin is released from the intracellular contents by degranulation or lysis of all or part of the mast cells, and heparin is present in the medium at the time of the separation step, the use of a membrane with a smaller porosity may also be used. Can be envisaged. In this case, cell separation is combined with a process consisting of ultrafiltration through one or more membranes, and its tissue and porosity allows for heparin concentration, according to size and molecular weight, and in some cases, Allows separation from other species present in the medium according to charge or biological properties.

  In the context of this embodiment, the membrane cutoff threshold is preferably between 1000 and 5 kDa. Membrane systems similar to those used for microfiltration can be used, for example spiral membranes, flat membranes or hollow fibers. Because of the charge and grafting properties of ligands that show affinity for heparin, membranes that allow the separation and purification of heparin can be used advantageously (eg, antibodies, ATIII, lectins).

  In general, however, cell harvesting and production methods that can keep heparin in the intracellular contents can be preferably used.

  Heparin can also be recovered from mast cells after cell degranulation or lysis.

  Degranulation is the binding of specific ligands to receptors present on the surface of mast cells (eg, binding of allergen type factors to mast cell IgE receptors (such as IgE Fc fragments or analogs of this fragment)). Can be caused by.

  Other factors can also induce degranulation of mast cells. These factors can be separated into several categories: cytotoxic factors, enzymes, polysaccharides, lectins, anaphylatoxins, basic compounds (compound 48/80, substance P, etc.), calcium (A23187 ionophore, ionomycin, etc.) It is possible [D. Lagunoff and T. W. Martin. 1983. Agents that release hishistamine from cells. Ann. Rev. Pharmacol. Toxicol. , 23: 331-51]. The degranulation factor can be used repeatedly on the same cells maintained during culture. In this production method, productivity is significantly increased by maintaining the cells in culture by simplifying the method of collecting from the supernatant.

In the case of the A23187 ionophore, degranulation of mast cells, eg, 2.10 ⁶ mast cells / mL with an A23187 ionophore at a concentration between 1 and 100 μg / mL with a duration of action of 1 min to 4 hours. It can be induced by processing.

Mast cell lysis can be achieved by chemical factors (NaOH, THESIT, for example, by osmotic shock using hypotonic or hypertonic solutions, by thermal shock (freeze / thaw), by mechanical shock (eg sonication or pressure fluctuation). ^TM , NP40 ^™ , TWEEN 20 ^™ , BRIJ-58 ^™ , TRITON X ^™ -100, etc.) or by enzymatic lysis (papain, trypsin, etc.) or a combination of two or more of these methods It is.

  Obtained from animal tissue to extract and purify heparin from cell lysates, to separate polysaccharide chains from the core of serglycine, and to separate heparin chains from other GAGs present in the extraction medium. Methods similar to those used in the context of the extraction and purification of heparin that are known per se and described in common works such as the manual by Duclos (above) may be used.

As a non-limiting example, to separate heparin from nucleic acids and from cellular proteins, solubilize heparin, i.e., to cleave the binding to serglycine core.
The cell lysate can be subjected to one or more enzymatic digestions (pronase, trypsin, papain, etc.);
The heparin-protein binding can be hydrolyzed in alkaline medium in the presence of sulfate or chloride;
-It is also possible to carry out the treatment (for example with trichloroacetic acid under cold conditions) in acid medium in order to destroy nucleic acids and proteins derived from the cells, including GAG-protein The use of an ionic solution that allows the interaction to be dissociated is added.

  After enzymatic hydrolysis, it is also possible to carry out extraction with guanidine and to purify the solubilized heparin. For example, it can be precipitated with potassium acetate, quaternary ammonium, acetone and the like.

  These purification steps can advantageously be added to them or can be replaced by one or more chromatography steps, in particular anion exchange chromatography steps.

  The subject of the invention is also a heparin preparation obtainable from a mast cell culture using the method of the invention.

  The heparin preparation according to the present invention, which has biological properties comparable to those of the heparin preparations obtained from animal tissue in the prior art, can be used in any of the usual applications for heparin It is.

  The invention is illustrated by the following examples. These examples are given purely for illustrative purposes and should not be considered limiting.

  Example

Example 1
Genetic modification of mast cells Mast cells can be produced, for example, by introducing foreign nucleic acids using transfection, electroporation, nucleoporation or infection techniques that result in transient or stable expression of the introduced nucleic acid. It can be genetically modified. In the case of stable expression, the DNA can be integrated into the cell genome or can be maintained episomally.

1 Transfection by nuclear drilling and electroporation Stablely transfected cells can be treated by applying selective pressure (hygromycin, geneticin, blasticidin, puromycin or zeocin) 24 to 72 hours after nuclear drilling. It can be obtained using the described nuclear drilling method. Resistance to the selection factor is conferred by integration of a plasmid having the gene of interest and the resistance gene.

Nuclear perforation This method is preferably used because it allows direct induction of DNA into the nucleus.

After exponential growth phase, preferably 3 or 4 days in culture, 1 to 2 × 10 ⁶ mast cells are centrifuged at 1000 rpm for 5 minutes and collected in 100 μL of nuclear drilling solution (Amaxa, kit 8351). 2-4 μg of pcDNA3.1-eGFP (plasmid encoding GFP) is then added to the cell suspension. The cells are then transferred into an electroporation cuvette and subjected to electric shock using specific programs (such as U14, U23 and U28 AMAXA).

Cells are then transferred into 2 mL of complete medium pre-heated to 37 ° C. and then incubated at 37 ° C., 5% CO ₂ .

  Cells are harvested for fixation with 1% paraformaldehyde (Prolabo) 24-48 hours after transfection. For this, the whole culture is centrifuged at 1000 rpm for 5 minutes. After removal of the supernatant, the cells are washed in 4 mL of 1 × PBS and then centrifuged again. The cell pellet is then taken up in 1 mL of 1% paraformaldehyde. The cells thus fixed are then analyzed in a hemocytometer (Cytomics FC 500, Beckman Coulter).

  The nuclear perforation conditions allow for the transfection of porcine mast cells with a transfection efficiency of between 30 and 50% while at the same time obtaining an excellent cell viability of more than 50%.

Electroporation 1-5 × 10 ⁶ cells in exponential growth phase are contacted with 1-30 μg of DNA. Cells transferred into 4 mm electroporation cuvettes are incubated in ice for 5 min before electroporation (Gene Pulser II, Biorad) with a voltage between 150 V and 400 V using a capacitance of 500 or 960 μF To do. After electroporation, the cells are incubated again in ice for 5 minutes and finally transferred into 5 mL of complete medium and incubated at 37 ° C., 5% CO ₂ .

  The method of selecting cells stably incorporating the transgene uses the same technique as described above, using the resistance to the selection factor conferred by plasmid integration.

2 Transfection with viral vectors, use of pan-affinity retroviral vectors As an alternative to the methods of transfection by electroporation and nuclear perforation, replication defective recombinant retroviral vectors can be used. For example, a vector pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) that allows the production of a pan-affinity retroviral vector capable of infecting porcine cells may be used.

  In this transfection method, GP-293 (Clontech protocol ref) containing genetic elements (gag and pol) for producing a vector except for a gene for production of pseudotyped envelope protein (env-VSV-G). Using a packaging cell such as PT 31312-1), a retroviral vector carrying a gene of interest to be expressed in or incorporated into porcine mast cells is first generated.

  At the time of production of the retroviral vector, a packaging cell containing a plasmid encoding a VSV-G envelope gene and a retroviral plasmid encoding a gene of interest under the control of a promoter with or without a selection gene Co-transfect with.

In practice, GP-293 cells are placed in culture for 48 to 72 hours prior to transfection to be in exponential growth phase. On the day of transfection, the medium is replaced with fresh medium (15-20 mL / 10 ⁶ cells), then the VSV-G plasmid (5-20 μg / 10 ⁶ cells) and the gene of interest in calcium phosphate buffer pH. 1-2 mL of a solution containing a mixture of plasmids (10-30 μg / 10 ⁶ cells) is added dropwise to the medium (1-2 mL (Promega)).

  The cells are then re-incubated for 16-24 hours at 37 ° C. or preferably at a temperature between 32 and 35 ° C. Again, the medium is replaced with fresh medium. At 32-35 ° C, the cells are incubated for an additional 48 hours. When incubation is complete, the culture supernatant containing the newly formed retroviral vector is collected. A portion of the supernatant from the packaging cell infection is taken as a preparative sample, frozen at -80 ° C, and the other portion is mixed with the medium of mast cells in exponential growth phase. In practice, mast cells in culture are centrifuged and resuspended in a medium containing 50% fresh medium and 50% infection supernatant. The mast cells are incubated at 32-35 ° C. for 24 hours, and then again the medium is replaced with fresh medium.

  48-72 hours after infection of mast cells for analysis by cytofluorimetric analysis in the case of controls with infection with a GFP (green fluorescent protein) fluorescent reporter or using PCR for infection with the gene of interest Samples are taken later. By cell fluorescence analysis, the measurement of transfection efficiency is greater than 20% of total cells.

  The transfection is then performed by adding to the medium a cytotoxic factor (hygromycin, puromycin, G418, zeocin) in which only the mast cells transfected with the retroviral vector carrying the gene of interest and the resistance gene continue to grow. Selected cell populations. By using this method, the target gene is stably integrated and expressed.

  After population selection and cloning, the selection factor can be removed from the medium while simultaneously maintaining expression of the gene of interest. Retroviral vectors produced in this way do not replicate in host mast cells and therefore there is no production of replicating vectors.

  Alternatively, a vector that is subjected to induction of a promoter that controls the expression of a gene of interest can be used using a compound that is added to the medium at a desired time.

(Example 2)
Generation of strains capable of growing in the absence of SCF through stable expression of the mouse c-kit ^G559 gene To obtain porcine mast cells that are growth-independent and SCF-independent for a long time Mast cells were transformed with the c-kit gene. For this purpose, a mouse c-kit gene (gene called c-kit ^G559 ) 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 mast cell line I-2735 was maintained in culture for 929 days in the presence of cytokines.

Mast cells were cultured in MEMα 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). To do. This medium is referred to as complete medium 1. The cells are incubated at 37 ° C. under 5% CO ₂ and seeded at 2 × 10 ⁵ C / mL every 3-4 days.

The mouse plasmid pEF-BOS-ckit ^G559 is obtained by the plasmid pEF-BOS (Mizushima and Nagata, Nucleic Acid Res., 18 (17): 5322, 1990) obtained by circulating the XbaI fragment of the mutated mouse ckit gene with XbaI. As described).

The c-kit gene carries a point mutation that causes modification of valine 559 to glycine (gene called c-kit ^G559 ). This mutation is similar to the c-kit ^G556 mutant in pigs and the c-kit ^G560 mutant in humans.

In order to obtain a mutated mouse ckit ^G559 cDNA, the mouse plasmid pEF-BOS-kit ^G559 was digested with XbaI. The cDNA fragment was cloned into the XbaI site of plasmid pcDNA3.1 (Invitrogen). After complete sequencing of the cDNA, the resulting plasmid was called pBXL2008.

In order to obtain a mutated mouse ckit ^G559 cDNA, plasmid pBXL2008 was digested with XbaI. The cDNA fragment was cloned into the XbaI site of plasmid pcDNA3.1 / Hygro (Invitrogen). The resulting plasmid is called pBXL2015.

  The cDNA fragment comprises the sequence of SEQ ID NO: 1.

  DNA preparations for transfection were made using the “Endofree plasmid maxi” kit (Quiagen).

Transfection selection of hygromycin resistant population After 3 days in culture, 5 × 10 ⁶ mast cells in exponential growth phase were centrifuged at 1000 rpm for 5 minutes and 100 μL of transfection medium (2 mM glutamine, 10 nM PGE2, 80 ng / mL). MEMs supplemented with rpSCF and 2 ng / mL rpIL3). Then 30 μg of pcDNA3.1 / hygro or pBXL2015 is added to the cell suspension. The cells are then transferred into an electroporation cuvette and subjected to 960 μF, 340 V electroshock (gene pulser, Biorad). After electroporation, the cells are transferred into 5 mL of complete medium 1 previously heated to 37 ° C. and then incubated at 37 ° C., 5% CO ₂ . Forty-eight hours after transfection, cells were counted, centrifuged, and at 2 × 10 ⁵ C / mL in selective medium (complete medium 1 supplemented with hygromycin (Invitrogen) at a concentration of 0.2 mg / mL). Sowing.

  Sixteen days after application of hygromycin selection, a hygromycin resistant cell population is obtained. Cells stably transfected with plasmid pcDNA3.1 / hygro are referred to as MCpcDNA3.1 / hygro, and cells stably transfected with plasmid pBXL2015 are referred to as MCpBXL2015.

  Cell cultures are maintained in culture in selective media.

Molecular characterization Detection of mouse ckit ^G559 gene in MCpBXL2015 cells Extracted from MCpcDNA3.1 / hygro and MCpBXL2015 cultures using primers to specifically amplify mouse genes without amplifying endogenous porcine genes The mouse ckit ^G559 gene was detected by PCR on genomic DNA.

Genomic DNA was extracted from 5 × 10 ⁶ cells from MCpcDNA3.1 / hygro and MCpBXL2015 cultures according to the protocol of Qiagen's Dneasy Tissue kit.

  Then, according to the protocol of Advantage 2GC PCR kit (Clontech), the sequence 5′-GCC GAG GCC ACT CGC-3 ′ of SEQ ID NO: 7 and the sequence 5′-AGC CAT GTA CCG TCA CGC TG- of SEQ ID NO: 8 100 ng of these genomic DNAs were amplified by PCR in the presence of 3 ′ antisense primer C19814. After 25 thermal cycles (denaturation at 94 ° C. for 30 seconds, hybridization at 58 ° C. for 45 seconds and extension at 68 ° C. for 45 seconds), 1 × TBE 2% agarose to detect 325 bp amplified fragment PCR reactions were analyzed on the gel.

Under these conditions, an approximately 350 bp PCR product was observed from genomic DNA obtained from MCpBXL2015 culture, but not from genomic DNA obtained from MCpcDNA3.1 / hygro culture. This indicates the presence of the mouse ckit ^G559 gene (FIG. 2A).

Detection of expression of the mouse ckit ^G559 gene in MCpBXL2015 cells Expression of the kitt ^G559 gene was detected by RT-PCR.

According to the protocol Rneasy mini kit Quiagen, RNA was extracted from 10 ⁷ cells from MCpcDNA3.1 / hygro and MCpBXL2015 cultures. 30 μg of RNA was then digested with 20 U of DNase I for 1 hour at 37 ° C. and then purified according to the “cleanup” protocol of Qiagen's Rneasy mini kit.

  Using the oligonucleotide of the sequence 5′-gac cac gcg tat cga tgt cga ctt ttt ttt ttt ttt ttv-3 ′ of SEQ ID NO: 9 as a primer, 2 μL of these RNAs were converted to cDNA by using AMV RT reverse transcriptase (Roche) Reverse transcribed.

  PCR was performed on 1 μL of cDNA under the conditions described in the paragraph above.

Under these conditions, approximately 350 bp of RT-PCR-derived product was observed from RNA derived from MCpBXL2015 culture, but not from RNA derived from MCpcDNA3.1 / hygro culture. This indicates that the mouse ckit ^G559 gene is effectively expressed in the cells (FIG. 2B).

Characterization of growth in the absence of SCF The functionality of ckit ^G559 was assessed by analyzing the ability of cells to grow in media lacking SCF.

To this end, 5-6 weeks after transfection, some of MCpcDNA3.1 / hygro and MCpBXL2015 cells were seeded at 2 × 10 ⁵ C / mL in medium lacking SCF. The medium was replaced with fresh medium twice a week and for 3 weeks. MCpcDNA3.1 / hygro control cells cannot grow in such media. Substantial cell death is observed after growth arrest (FIG. 3A).

In the case of MCpBXL2015 cells, an initial period is observed for about 3 weeks, during which time proliferation is modest (FIG. 3A). This latency period is explained by the fact that not all cells express the functional murine ckit ^G559 receptor, regardless of resistance to hygromycin, which prevents it from growing in the absence of SCF. It is possible. After this incubation period, a population of MCpBXL2015 cells that can grow in the absence of SCF appears. These subpopulations were 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 was 4 × 10 ⁵ and 10 × 10 ⁵ cells / mL with exponential growth doubling times of 24 and 48 hours. Between.

The growth of MCpBXL2015 cells is identical to the growth of MCpcDNA3.1 / hygro control cells in medium supplemented with SCF in the absence of SCF (FIG. 3B). Thus, the expression of the murine ckit ^G559 gene makes it possible to ^dispense with SCF while maintaining the same level of proliferation.

Characterization of proteoglycans contained in cultures by HPLC Studies of the growth of MCpBXL2015 cells in SCF-deficient medium ^indicate that the expression of the ckit ^G559 receptor allows the SCF to be ^{dispensed with} . We also studied the effect of SCF removal on proteoglycan production by mast cells.

  After 5 weeks of culture in the absence of SCF, MCpBXL2015 was analyzed to analyze the proteglycan composition of mast cells according to the protocol described by “Linhardt et al. (Biomethods, 9, 183-197, 1997)”. A sample of the culture was taken.

  Samples are processed as follows.

Proteolysis: Cell samples (2 × 10 ⁶ cells) are centrifuged and rinsed twice in PBS buffer. Each pellet is collected in 100 μL of distilled water to which 10 μL of Alcalase (Novozymes) is added, and then heated at 60 ° C. for 5 hours while stirring. The sample is then diluted with 200 μL of 10 mM tris buffer, pH 7.0 (Prolabo) containing 0.5 M NaCl (Prolabo) prior to centrifugation at 10,000 rpm for 10 minutes. The proteolytic process allows the release of intracellular contents and dissociates protein-polysaccharide bonds.

  Extraction: The supernatant of each sample is purified by ion exchange on SAX quaternary ammonium resin in 96-well plate format, 100 mg / 2 mL (Thermohypersil). After discovery and washing in tris buffer pH 7 containing 0.5 M NaCl, glycosaminoglycan (GAG) is eluted with 500 μL of tris buffer pH 7.0 containing 3 M NaCl.

Desalting / concentration:
The sample is then desalted on a gel permeation column (NAP-5, Pharmacia). After elution with a volume of 1 mL, the sample is concentrated by lyophilization and then taken up in 130 μL of distilled water.

  Depolymerization.

  For HPLC analysis, GAG was depolymerized with a mixture of heparinase I, II and III (Grampian enzymes). Adjust each heparinase solution to 0.5 IU / mL in phosphate buffer. Prepare solutions of heparinase I, II, and III by mixing 1/3 volume for each heparinase solution volume. When analyzing 100 μL of sample, per 30 mL of distilled water, 15 μL of heparinase mixture and 10 μL of acetate buffer containing 0.73 mL of 100% acetic acid (Prolabo), 12.5 mg of bovine albumin (Sigma) and 39.5 mg of calcium acetate (Prolabo) Add.

HPLC analysis Samples are then analyzed by HPLC on a Waters sphere Sorb SAX 5 μm, 250 × 3 mm, thermohypersil column. Inject 50 μL of sample per analysis. The buffer for the mobile phase is composed of 2.5 M sodium dihydrogen phosphate (Na ₂ HPO ₄ , Prolabo) so that its pH is 2.9 with orthophosphoric acid (H ₃ PO ₄ , Prolabo). Adjusted. The elution of the disaccharides constituting GAG extracted from the cell sample is a gradient from 0 to 100% of 2.5 mM Na ₂ HPO ₄ buffer containing 1 M perchloric acid (NaClO ₄ , Prolabo) over 50 minutes. Will be implemented. Disaccharides are detected using their retention times and for standard heparin samples (Aventis) under UV at 234 nm.

  Culture of MCpBXL2015 cells in the absence of SCF improves heparin properties compared to control cells cultured in the presence of SCF.

  Applicants are not bound by any particular theory, but maturation of mast cells through cell selection performed in obtaining cells that grow in the absence of SCF, or in the presence or absence of SCF. It is possible to explain this phenomenon through the difference in crystallization. Indeed, SCF allows cell proliferation but is also involved in cell differentiation.

(Example 3)
Obtaining MCpBXL2015 clones using cloning by limiting dilution MCpBXL2015 cells were cloned by limiting dilution in medium lacking SCF. For this, 10 cells of 96 wells were seeded with 0.3 cells / 100 μL / well. Only clones showing satisfactory growth in 96 well plates were selected and amplified. Once confluence is reached, clones are recovered and seeded with 500 μL of medium in a 48-well plate, then 2 mL of medium is seeded in a 6-well plate, and finally 5 mL of medium is seeded in F25.

At the end of this amplification, 9 clones were obtained and amplified after counting every 3-4 days and seeding at 2 × 10 ⁵ C / mL.

  After analysis of proliferation and mast cell proteoglycan composition, a final selection of clones was performed.

  A comparative growth study was performed using the original MCpBXL2015 culture and the resulting 9 clones. All clones show the same growth as the uncloned MCpBXL2015 culture (FIG. 3).

  After 4 days of culture, samples were taken to analyze the proteoglycan composition of various clones.

According to the HPLC results summarized in Table 2, the polysaccharide synthesized by each clone varies in the relative amount of each disaccharide comprising the heparin chain (eg, the proportion of Is disaccharide is 5-30% according to the clone). A heparin-type property with However, as with the non-cloned MXpBXL2015 population, all clones show a degree of 3-O-sulfation below the detection threshold (IIa IIsglu tetrasaccharide is not detectable).

  MC pBXL2015 clone 6-G4 was selected based on heparin analog productivity and quality criteria for production. This clone was frozen under the name PMC125.

Example 4
Generation of SCF-independent strains with increased 3-O-sulfation activity To increase the biological activity of heparin-type compounds obtained from mast cell cultures, 3-OST-1 (3 O-sulfatase-1 ) Can be stably overexpressed. In this example, porcine genes were used.

  The mast cells used were obtained from the PMC125 culture described in Example 3.

Mast cells are cultured in MEMα medium supplemented with 15% fetal calf serum (Hyclone), 2 mM glutamine (Invitrogen) and 2 ng / mL rpIL-3 (Biotransplant). This medium is referred to as medium 2. Cells were incubated at 37 ° C. under 5% CO ₂ and passaged every 3-4 days.

  HS3ST1-pE-IRES-neo2-, encoding porcine 3-OST1, is available from Aventis Pharma S. A. In application PCT / FR04 / 00902 filed under the name As a residue, plasmid HS3ST1-pE-IRES-neo2 was obtained as follows.

Isolation and Sequencing of the 3 ′ Coding Sequence of the Porcine 3-OST Gene A partial sequence of the porcine gene encoding 3-OST is available in the EST library (GenBank accession number BF075483). Alignment of this sequence with the human sequence shows that it lacks about 650 bp of the 3 ′ coding region.

  The missing portion in the porcine 3-OST gene was identified by combining RT-PCR and 3'-RACE using porcine liver RNA isolated according to the protocol of the Trizol kit (Invitrogen) as the RNA source. .

  Reverse transcription of 2 μg of total RNA into cDNA was performed as follows: sequence 5′-GCA GCA GCC ACG TCG GG-3 ′ of SEQ ID NO: 10 and sequence 5′-TCA GTG YCA GTC RAA TGT TC-3 ′ of SEQ ID NO: 11, This was done by following the protocol of the first strand synthesis system kit (Invitrogen) using a mixture of oligonucleotides BS02 and BS03 as primers.

  Next, the sense primer BS05 of the sequence 5′-CGG NGA CCG CCT NAT CAG-3 ′ of SEQ ID NO: 12 and the sequence 5′-TCA GTG YCA GTC RAA TGT TC- of SEQ ID NO: 12 using KOD hot start polymerase (Novagen) 2 μL of these cDNAs were amplified by PCR in the presence of the antisense primer BS06 of the 3 ′ sequence. 30 thermal cycles (denaturation at 98 ° C. for 15 seconds, hybridization at 60 ° C. for 30 seconds, and extension at 68 ° C. for 30 seconds, the 277 bp amplified fragment was transformed into the vector pCR-Brant II TOPO (Invitrogen, Zero Blunt After cloning into the TOPO PCR cloning kit), the sequence was determined.

  The sequence of this fragment was used to generate two primers BS21 and BS22 to isolate the entire 3 'region by 3'-RACE.

  In 3′-RACE, oligo dT CDSIII of the sequence of SEQ ID NO: 14 (5′-ATT CTA GAG GCC GAG GCG GCC GAC ATG TVN-3 ′) was used as a primer, and according to the protocol of Invitrogen's first strand synthesis system kit 1 μL of porcine liver RNA was reverse transcribed into cDNA.

  The 3 'region of the gene encoding 3-OST was then amplified by two successive PCRs. The first PCR was performed on 2 μL of the cDNA obtained above using the sense primer BS21 and antisense primer CDSIII of the sequence 5′-GCA CCC CCA GAT CGA CCC C-3 ′ of SEQ ID NO: 15. . Thirty thermal cycles (denaturation at 94 ° C. for 10 seconds, hybridization at 60 ° C. for 30 seconds, and extension at 68 ° C. for 120 seconds) were applied. The second PCR then uses the sense primer BS22 and the antisense primer CDSIII of the sequence 5′-CAA ACT CCT CAA TAA ACT GCA CG-3 ′ of SEQ ID NO: 16 and 1 μL of the product obtained from the first PCR. Was performed under the same conditions as in the first PCR.

  Sequencing of the PCR product thus obtained at the end of 3'-RACE made it possible to identify the 3 'sequence of porcine 3-OST and about 250 bp of the non-coding region.

Isolation of the complete coding phase of the porcine 3-OST gene In order to clone the complete coding phase of porcine 3-OST, further RT-PCR experiments were carried out using the information obtained in the first step. The RNA source is the same as in the previous step.

According to the protocol of the first strand synthesis system kit Invitrogen, using oligonucleotide _{dT 24} as a primer, and reverse transcribed to 2μg of RNA to cDNA.

  Subsequently, the gene encoding 3-OST was amplified by two-step PCR. The first PCR makes it possible to amplify a gene that contains part of the 3 ′ non-coding sequence of the gene, and then the second PCR uses primers compatible with the Gateway system (Invitrogen), It became possible to amplify the coding sequence.

  The first PCR consists of the sense primer BS10 of the sequence 5′-AGG CCC GTG ACA CCC ATG AGT-3 ′ that specifically hybridizes in the 5 ′ non-coding region of the porcine 3-OST gene, and 3 ′ in the UTR. The sequence 5′-CAC CTA GTG TAC ACC ACA ATT TAC-3 ′ antisense primer BS30, which hybridizes specifically to the position, was performed on 2 μL of cDNA. Thirty-five thermal cycles (denaturation at 98 ° C. for 10 seconds, hybridization at 64 ° C. for 30 seconds, and extension at 68 ° C. for 150 seconds) were applied.

  A second PCR was then performed on 1 μL of the PCR product to specifically amplify the coding phase. For this, the sequence 5′-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC AGC ATG GCC GCG CTG CTC-3 ′ of the sense primer BS31 and SEQ ID NO: 18 of the sequence 5′-GGG ACC ACT TTG TAC A The antisense primer BS32 of AAA GCT GGG TTT AGT GCC AGT CAA ATG TTC TGC C-3 ′ is used. The PCR program used is the same as that used for the first PCR.

  The 1 kb PCR product was then cloned into the episomal vector pE-IRES-neo2 according to the procedure of Invitrogen's Gateway cloning technology kit. The sequence of the porcine gene was confirmed by sequencing. The resulting nucleotide sequence is the sequence of SEQ ID NO: 5. The deduced protein sequence is SEQ ID NO: 6.

Obtaining plasmid pBXL2033 According to the protocol of Advantage 2GC PCR kit (Clontech), SEQ ID NO: 19 sequence 5'-CGC GGA TCC CAG CAT GGC CGC GCT GCT CC-3 'sense primer C21755 and SEQ ID NO: 20 sequence 5'-CTA The porcine 3-OST1 gene was amplified by PCR from plasmid HS3ST1-pE-IRES-neo2 in the presence of antisense primer C21756 of GTC TAG ATT AGT GCC AGT CAA ATG TTC-3 ′. After 25 thermal cycles (denaturation at 94 ° C. for 30 seconds, hybridization at 58 ° C. for 45 seconds and extension at 68 ° C. for 45 seconds), the PCR reaction is purified according to the protocol of Qiagen's Quiaquick PCR purification kit. All PCR products are then digested with BamHI and XbaI (Biolabs) for 4 hours at 37 ° C. The digestion product is then purified after transfer on a 1 × TBE 1% agarose gel according to the protocol of Qiagen's Quiaquick gel extraction kit. The PCR product digested in this way is cloned into the vector pcDNA3.1 / hygro opened with BamHI-XbaI.

  After complete sequencing of the cDNA, the resulting plasmid was called pBXL2032.

  To obtain porcine 3-OST1 cDNA, plasmid pBXL2032 was digested with XbaI-BamHI.

  The cDNA fragment was cloned into the plasmid pcDNA3.1 (invitrogen) opened with XbaI-BamHI. The plasmid thus obtained was designated pBXL2033.

  The DNA preparation for transfection was performed using the “endotoxin free plasmid” kit (Quiagen).

Transfection-selection of geneticin-resistant population 5 × 10 ⁶ mast cells after 3 days of culture in exponential growth phase are centrifuged at 1000 rpm and collected in 100 μL of nuclear drilling solution 8351 (Amaxa). Then 4 μg of pcDNA3.1 or pBXL2033 is added to the cell suspension. The cells are then transferred into an electroporation cuvette and subjected to electroshock: Program U-28 (Nucleofector, Amaxa).

After electroporation, the cells are transferred into 2 mL of medium 2 that has been pre-heated to 37 ° C. and then incubated in 6-well plates at 37 ° C., 5% CO ₂ . 48 hours after transfection, cells are counted, centrifuged, and seeded at 2 × 10 ⁵ C / mL in selective medium (medium 2 supplemented with geneticin (Invitrogen) at a concentration of 0.8 mg / mL). .

  A geneticin resistant cell population was obtained 20 days after application of the selection. PMC125 cells stably transfected with plasmid pcDNA3.1 are referred to as PMC125 pcDNA3.1, and cells stably transfected with plasmid pBXL2033 are referred to as PMC125pBXL2033.

  Cell cultures are maintained in culture in selective media.

Characterization of proteoglycans contained in cultures by HPLC The functionality of 3-OST1 was assessed by analysis of the degree of 3-O-sulfation of proteoglycans produced by PMC125 pcDNA3.1 control mast cells and PMC125pBXL2033 mast cells . The degree of 3-O-sulfation can be monitored by quantifying the amount of IIa II sglu tetrasaccharide that reveals 3-O-sulfation.

For this, a sample was taken 3 weeks after application of the selection and 4 days after seeding of the culture at 2 × 10 ⁵ C / mL.

Proteoglycans produced by PMC125 pcDNA3.1 control cells do not contain detectable amounts of IIa II sglu tetrasaccharide (Table 3). On the other hand, proteoglycans synthesized by PMC125pBXL2033 cells contain 0.9% of IIa IIsglu , revealing a marked increase in the degree of 3-O-sulfation in these cultures.

  These results indicate that porcine 3-OST1 overexpressed in mast cells is functional, ie induces 3-O-sulfation.

  These samples were then examined for biological activity. For this, anti-Xa and anti-IIa activities were measured.

Characterization of the biological activity of proteoglycans contained in the culture Inactivation of factor Xa and IIa is characteristic of heparin, allowing heparin to distinguish heparan sulfate and dermatan. The method used is that described in the article “European Pharmacopea, 3 ^rd Edition (1997)”.

  The reaction occurs in three stages.

1: ATIII + heparin [ATIII-heparin]
2: [ATIII-heparin] + excess factor remaining [ATIII-heparin-factor] + factor 3: remaining factor + chromophore substrate Colored p-nitroaniline The amount of p-nitroaniline released is the amount of heparin Inversely proportional to the quantity.

  The amount of anti-Xa and anti-IIa is measured with respect to a calibration curve established using SPIM standards (standard international heparin). The sensitivity of this method is 0.006 IU / mL.

  Biological activity is expressed in IU / mg and takes into account the quantification of the disaccharide obtained by HPLC.

  The bioactivity of the same sample as analyzed by HPLC was determined.

  Proteoglycans synthesized by PMC125 pcDNA3.1 control cells do not show detectable biological activity. On the other hand, analysis of the activity of proteoglycans produced by PMC125pBXL2033 cells reveals the presence of anti-Xa and anti-IIa bioactivity between 60 and 80 IU / mg (Table 3).

  It is noted that at this stage of culture (ie, 4 days after sowing), the ratio of the two activities is close to 1, which is characteristic of heparin obtained from extraction from porcine intestinal mucosa.

These results further demonstrate the efficacy of 3-OST1 expression resulting in the appearance of IIa IIsglu and anti-Xa biological activity.

Example 4
Obtaining MCpBXL2033 clone using cloning by limiting dilution PMC125pBXL2033 cells were cloned by limiting dilution technique. For this, 20 96-well plates were seeded with 0.3 cells / 100 μL / well. Only clones showing satisfactory growth in 96 well plates were selected and amplified. Once confluence is reached, clones are recovered and seeded with 0.5 or 1 mL of medium in a 24-well plate, 2 mL of medium in a 12-well plate, and then 4 mL of medium in a 6-well plate. And finally 10 mL of the medium is seeded in F25.

At the end of this amplification, after counting every 3-4 days and seeding at 2 × 10 ⁵ C / mL, 156 clones were obtained and amplified.

  The first analysis of the synthesized proteoglycans was performed by HPLC to identify clones showing 3-O-sulfation activity. At the end of this first study, only 25 clones out of 156 examined were positive by HPLC. These 25 clones were then analyzed to determine the biological activity of the synthesized proteoglycans. At the end of this second analysis, only 19 clones were preserved.

  Nineteen clones selected at the end of the two analyzes produce proteoglycans that show an increased degree of 3-O-sulfation.

  A comparative growth study was performed on the original PMC125pBXL2033 culture and the 12 clones obtained.

  Cell proliferation was observed for all of the clones studied (FIG. 5).

  At the end of the growth curve, samples were taken to analyze the proteoglycans of various clones.

  Analysis of proteoglycans synthesized by the clones by HPLC shows that all clones produce proteoglycans that have 3-O-sulfation in common, but their composition varies (Table 4). Similarly, the anti-Xa biological activity of proteoglycans varies between 17 and 100 IU / mg according to the clone analyzed (Table 5).

  PMC125pBXL2033 clone 19-D4 was selected taking into account the growth criteria and quality of the proteoglycan produced.

The stability of PMC125pBXL2033 clone 19-D4 was monitored for approximately 5 months. This monitoring allows parameters such as productivity (measured by HPLC analysis), degree of 3-O-sulfation (measured by monitoring IIa IIsglu tetrasaccharide) and product biological activity to Similarly, it was shown to be stable during this period (FIG. 6).

  The drawings of the present application are as follows.

Chemical structures of Is, IIs, IIIs and IVs disaccharides corresponding to the N-sulfated disaccharide of heparin, acetylated homologous disaccharides Ia, IIa, IIIa and IVa, and IIa IIs glu and IIa Chemical structure of IVs glu tetrasaccharide. Molecular analysis of MCpcDNA3.1 / hygro and MCpBXL2015 cultures. Figure 2A-Genomic PCR. FIG. 2B—MC pcDNA3.1 / RNA extracted from hygro control cultures (1) or from MCpBXL2015 cultures (2) in the absence (−) or presence (+) of reverser enzyme -PCR was performed (C: control pBXL2015 plasmid DNA). Molecular analysis of MCpcDNA3.1 / hygro and MCpBXL2015 cultures. Figure 2A-Genomic PCR. FIG. 2B—MC pcDNA3.1 / RNA extracted from hygro control cultures (1) or from MCpBXL2015 cultures (2) in the absence (−) or presence (+) of reverser enzyme -PCR was performed (C: control pBXL2015 plasmid DNA). FIG. 3A: Growth of MCpcDNA3.1 / hygro control cells and MCpBXL2015 cells in SCF-free medium. FIG. 3B: Comparison of the growth of MCpcDNA3.1 / hygro control cells in the presence of SCF with the growth of MCpBXL2015 cells in SCF-free medium. FIG. 3A: Growth of MCpcDNA3.1 / hygro control cells and MCpBXL2015 cells in SCF-free medium. FIG. 3B: Comparison of the growth of MCpcDNA3.1 / hygro control cells in the presence of SCF with the growth of MCpBXL2015 cells in SCF-free medium. Growth curves for original MCpBXL2015 cultures and MCpBXL2015 clones. Growth curves for original PM125 culture and PMC125 pBXL2033 clone. Study of the stability of proteoglycan production over time by PMC125 pBXL2033 clone 19-D4. A mucosa extracted and separated under the same conditions as the cells is introduced as a control.

Claims (16)

A porcine mast cell culture or strain characterized by producing a heparin-type molecule containing IIa IIs glu tetrasaccharide.   2. A porcine mast cell culture or strain according to claim 1, characterized in that it produces heparin-type molecules exhibiting anti-Xa activity of at least greater than 50 IU / mg.   2. Porcine mast cell culture or strain according to claim 1, characterized in that it produces heparin-type molecules exhibiting anti-IIa activity of at least more than 50 IU / mg.   The porcine mast cell culture or strain according to any one of claims 1 to 3, wherein an enzyme that acts on sulfation of heparin-type molecules is overexpressed.   The porcine mast cell culture or strain according to any one of claims 1 to 4, comprising a foreign nucleic acid encoding an enzyme that acts on sulfation of heparin-type molecules.   The porcine mast cell culture or strain according to any one of claims 1 to 5, comprising a foreign nucleic acid encoding 3-OST.   The porcine mast cell culture or strain according to any one of claims 1 to 6, comprising a foreign nucleic acid encoding 3-OST1.   The porcine mast cell culture or strain according to any one of claims 1 to 7, comprising a foreign nucleic acid encoding porcine 3-OST.   9. A porcine mast cell culture or strain according to any one of claims 1 to 8, characterized in that it expresses a c-kit protein exhibiting a mutation or deletion. The porcine mast cell culture or strain according to any one of claims 1 to 9, which expresses a c-kit protein having a mutation at a position equivalent to that of mouse c-kit ^G559 . ^11. The porcine mast cell culture or strain according to any one of claims 1 to 10, which expresses mouse c-kit ^G559 , porcine c-kit ^G556 or human c-kit ^G560 protein.   The porcine mast cell culture or strain according to any one of claims 1 to 11, comprising a foreign nucleic acid encoding a c-kit protein exhibiting mutation or deletion. A porcine mast cell culture according to any one of claims 1 to 12, comprising a nucleic acid encoding a c-kit protein that exhibits a mutation at a position equivalent to the position of mouse c-kit ^G559 , or stock.   Shows the mutation or deletion deposited with the number I-2734, I-2735 or I-2735 on October 17, 2001 in the "Collection de Cultures de Microorganisms de l'Institut Paste" at the Pasteur Institute A porcine mast cell line characterized in that it is one of porcine mast cell lines modified to express c-kit protein.   15. The porcine mast cell line according to claim 14, comprising a foreign nucleic acid encoding an enzyme that acts on sulfation of heparin-type molecules.   A method for producing a heparin-type molecule, comprising culturing a porcine mast cell culture or strain according to any one of claims 1 to 15.

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