WO2006040463A1 - Porcine mast cell lines that produce heparin-type molecules - Google Patents

Abstract

The invention relates to porcine mast cell lines, the growth of which is independent of growth factors. The invention also relates to a method of producing bioactive heparin-type molecules, comprising the culturing of said mast cell lines.

Description

 PORCINE MASTOCYTE LINES PRODUCING HEPARIN-LIKE MOLECULES

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 μm 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. After activation, a secondary response is set up during which the synthesis of 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) and chemokines (MCP-1, IL8, RANTES). All of these species participate in the initiation of an inflammatory process and the establishment of a specific immune response dependent on T lymphocytes.

Mast cell cultures have already been obtained in humans, mice and pigs. In pigs, porcine mast cell lines were established by culturing stem cells obtained from fetal liver. They were the subject of the patent application WO 03/035853 filed in the name of the National Institute of Agronomic Research (INRA) and the National Vet School of Maisons Alfort (ENVA). Three lines were deposited with the Collection of Microorganism Cultures of the Institut Pasteur on 17 October 2001 respectively under Nos. I-2734, 1-2735 and I-2736. These cells, immortalized spontaneously, were maintained in culture for more than 170 generations in the presence of growth factors such as rpSCF and rplL3. Under these conditions, these cells are capable of synthesizing heparin-like compounds. These mast cell lines produce significant amounts of heparin-like molecules.

The patent application WO 03/035886 concerning a process for producing heparin from these lines has been filed in the name of Aventis Pharma S.A.

In pigs also Emery et al (Experimental Hematology, 24, 927-935, 1996) maintained, for 7 weeks, cell cultures obtained from bone marrow. Nevertheless it appears that the cultures obtained are mixtures of various cell types and not homogeneous cultures or mast cell lines. In addition, these cultures contain non-differentiated cells in homogeneous cultures of mast cells.

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.

Wang et al (Cire Res., 84, 74-83, 1999) describe the isolation of serous mast cells obtained from rat pleural and peritoneal cavities. Various molecular species are produced but only when the mast cells are co-cultured with rat aortic smooth muscle cells. The application WO99 / 26983 describes close work, and is very imprecise as to the application to other species.

In the mouse, cell lines have been established (Montgomery et al, Proc Natl Acad Sci USA 89, 11327-11331, 1992 and WO90 / 14418) but from mastocytomas. These tumors are extremely rare in pigs. In humans obtaining mast cell cultures has proved difficult. It was first allowed by the use of co-culture system with fibroblasts (Ishizaka et al, Current Opinion in Immunology, 5, 937-943, 1993). Other authors then managed to obtain mast cells from intestinal cells and maintain 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 articles reported only a low production of heparin-like compounds, when however it was measured.

Most mast cell cultures need growth factors such as SCF for optimal growth. 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).

In the case of 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.

Conventionally, the term "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. However, the structure of heparin is in fact very heterogeneous, and there are chains that can contain very different ratios.

Like all GAGs, 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.

Heparin plays an important biological role, especially in hemostasis, and is widely used therapeutically, particularly as an anticoagulant and antithrombotic agent. Most of the heparin used is isolated from the intestinal mucosa of the pig, from which it is extracted by proteolysis, followed by purification on anion exchange resin (for review on the various processes for the preparation of heparin , see DUCLOS, "Heparin: manufacture, structure, properties, analysis", Ed Masson, Paris, 1984). Analysis of the disaccharide composition of heparin pig after depolymerization by heparinase I ₁ II and III and chromatography used to distinguish between heparin. other glycosaminoglycans. In particular, eight major disaccharides are distinguished (Figure 1). HPLC analysis also makes it possible to detect the presence of the tetrasaccharides Ha]] SgIu and llalVsglu (the highlighting symbolizes a sulfation of the disaccharide). The presence of these tetrasaccharides is directly correlated with the presence of affinity site at I ¹ ATHI. In fact, the sulfation of the affinity site with ATIII leads to an incomplete digestion of this site and therefore to the appearance of HaNsglu and IlajVsglu tetrasaccharides. Sulfation of the AIIII affinity site provides biologically active heparin.

The studies mentioned above have allowed the isolation of producing lines. It is nevertheless necessary to improve them in order to allow their industrial use. In particular, it is desirable to make the growth of these lines independent of growth factors and to improve the bioactivity of the molecules produced. The Applicant has surprisingly shown that it is possible to improve these two aspects by the transformation of the mast cell line by mutant c-kit proteins and to obtain, by the overexpression of the 3OST1 protein, the production of heparin type with bioactivity compatible with industrial production.

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. Advantageously these cultures or lines produce detectable amounts of tetrasaccharides lia They glu. Preferably, 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. According to an advantageous embodiment, 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.

According to another advantageous embodiment, 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.

According to yet another advantageous embodiment of such cultures or lines produces heparin-type molecules comprising at least 30% of Is disaccharides (preferably at least 40%, more preferably at least 50%).

According to a preferred embodiment, the lines according to the present invention are modified so as to overexpress an enzyme acting on the sulfation of heparin-like molecules.

Advantageously, 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.

Advantageously, 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.

Alternatively, it is possible to use 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. in 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.

Advantageously, 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. Advantageously, 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] | _Θ g _Ue q _{U a} protein ^G556 porcine c-kit or c-kit ^G560 human. It is preferentially 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.

Because of inter-species conservation of the c-kit gene, murine, human, bovine or other genes having at least 80%, 90%, 95% or 99% identity identity with a 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.

Surprisingly, 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.

It is also possible to use a 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. Finally, it is also possible to use the murine c-kit gene deleted at the amino acid level TQLPYDH 573-579, in pigs this deletion is analogous to amino acids 570-576 in the human 574-580.

More particularly, 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. Preferentially, these lines comprise an exogenous nucleic acid encoding an enzyme acting on the sulfation of heparin-like molecules. The term "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".

The term "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

Developmental Biology, 26, 91-101, 1990).

The term nucleic acid is used to refer to a DNA or an RNA.

Advantageously, it is a complementary or genomic DNA. The "percent identity" between two 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.

By way of illustration, 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). Blast searches for similar / homologous sequences to a reference "query" sequence, using the algorithm of AltschuI et al. The query sequence and the bases of. The data used may be peptide or nucleic, any combination being possible. By "high stringency hybridization conditions" within the meaning of the present invention, the following conditions will be understood:

1- Pre-hybridization of the membranes and:

-Mix: 40μl salmon sperm DNA (10mg / ml) + 40μl human placental DNA (10mg / ml)

- Denature 5 minutes at 96 ⁰ C, then immerse in the ice mixture.

- Remove the 2X SSC and pour 4 ml formamide mix into the hybridization tube containing the membranes.

Add the mixture of the two denatured DNAs. Incubation at 42 ° C for 5 to 6 hours, with rotation.

2- Competition of the marked probe:

Add to the labeled and purified probe 10 to 50 μl Cot I DNA, according to the amount of repetitions.

- Denature 7 to 10 minutes at 95 ^° C. - Incubate at 65 ° C for 2 to 5 hours.

3- Hybridization:

- Remove the mix of pre hybridization.

- Mix 40 μl Salmon sperm DNA + 40 μl human placental DNA; denature 5 minutes at 96 ° C, then dive into the ice. - Add in the hybridization tube 4 ml of formamide mix, the mixture of the two DNAs and the labeled probe / DNA Cot I denatured.

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

4- Washes:

- Washing at room temperature in 2 × SSC, to rinse. - 2 times 5 minutes at room temperature SSC 2X and SDS 0.1% at 65 ° C.

- 2 times 15 minutes at 65 ° C. 1 × SSC and 0.1% SDS at 65 ° C. Wrap the membranes in Saran and expose.

The 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.

It goes without saying that the 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 ^κ 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 (MEMα / DMEM, RPMI, IMDM, etc.).

The media can also be supplemented with bovine serum at a concentration of between 0.5% and 20% (v / v).

The addition of 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. of 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 ^πd 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. In this batch culture mode In addition, 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).

In the context of the present invention, it is particularly possible to use 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.

It may be envisaged to increase quantitatively and qualitatively the content of heparin-like mast cell molecules following treatment with sodium butyrate (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 harvested and separated from the culture medium, generally by centrifugation or filtration.

Various centrifugation systems can be used, for example those described by VOGEL and TODARO (Fermentation and Biochemical Engineering Handbook, 2 ^πd Edition, ^Noys Publication, Westwood, New Jersey, USA).

Alternatively or in combination with the centrifugation, the tangential microfiltration can be carried out using membranes whose porosity is less than the mean diameter of the cells (5 to 20 μm) 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.

Different 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 choose 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 use of membranes of smaller porosity can also be envisaged in the case

10. where 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. In this case, 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

Depending on the size and molecular weight, and possibly the electrical charge or biological properties.

In the context of this embodiment, 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).

However, it will generally be preferred to use methods of producing and harvesting cells to retain heparin in intracellular content.

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.

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. In the particular case of the ionophore A23187, 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 μg / 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. (NaOH, THESIT ™, NP40 ™, TWEEN 20 ™, BRIJ-58 ™, TRITON X ™ -100, ...) or by enzymatic lysis (papain, trypsin, ...) or by combination of two or more of these methods.

To extract and purify the heparin from the cell lysate, separate the polysaccharide chains from the serglycine nucleus, and separate the heparin chains from the other GAGs present in the extraction medium, methods similar to those used in the extraction medium can be used. part of the extraction and purification of heparin from animal tissues, which are known in themselves, and described in general works, such as the manual of DUCLOS, cited above).

As non-limiting examples, to separate heparin from nucleic acids and cellular proteins, and to solubilize it, ie to break the bonds with the serglycine nucleus:

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;

it is also possible to carry out a treatment in an acid medium (for example with cold trichloroacetic acid) in order to destroy the nucleic acids and proteins from cells, supplemented by the use of an ionic solution that dissociates GAG-protein interactions.

It is also possible to carry out an extraction with guanidine, after enzymatic hydrolysis; to purify the solubilized heparin, it may for example be precipitated by potassium acetate, a quaternary ammonium, acetone, etc.

These purification steps may advantageously be completed or replaced by one or more chromatography steps, in particular anion exchange chromatography.

The present invention also relates to heparin preparations that can be obtained from mast cell cultures by implementing a method according to the invention.

The 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.

The figures of the present application are as follows:

• 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

MCpBXL2015 in medium without SCF. Figure 3B: Comparison of the growth of MCpcDNA3.1 / Hygro control cells in the presence of SCF with MCpBXL2015 cells in medium without SCF.

• Figure 4: Growth curve of the MCpBXL2015 culture and MCpBXL2015 clones.

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.

EXAMPLES

EXAMPLE 1 Genetic Modification of Mastocytes

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. introduced. In the case of stable expression, ÂDN can be integrated into the cellular genome or maintained as an episome.

1 Transfection by nucleoporation and electroporation.

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). The resistance to the selection agent is conferred by the integration of the plasmid carrying the gene of interest and the resistance gene.

nucleoporation

This method is used preferentially because it makes it possible to target the DNA directly in the nucleus.

1 to 2.10 ⁶ 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 μl of nucleofection solution (Amaxa, Kit 8351). 2 to 4 μg 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).

The cells are then transferred into 2 ml of complete medium preheated to 37 ^° C. and then incubated at 37 ° C., 5% CO 2.

24 to 48 hours after transfection, 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%.

electroporation

1 to 5.10 ⁶ cells, in the exponential phase, are brought into contact with 1 to 30 μg of DNA. The cells, transferred to a 4 mm electroporation cuvette, are incubated for 5 min in ice before being electroporated at a voltage of between 150 V and 400 V with a capacitance of 500 or 960 μF (Gene Pulser II, Biorad). After electroporation, the cells are again incubated for 5 min in ice to be finally transferred into 5 ml of complete culture medium and incubated at 37 ^° C., 5% CO 2. The process of selecting cells that stably integrate the transgene utilizes the same technique as described above using the resistance conferred by the integration of the plasmid with a selection agent.

2 Transfection by viral vectors, use of pantropic retro-vectors.

Alternatively to transfection methods by electroporation and nucleoporation, recombinant and deleted retroviral vectors can be used for replication. For example, 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.

In this mode of transfection, 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).

In the production of the retroviral vector, 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.

In practice, the GP-293 cells are cultured 48 hours to 72 hours before transfection in order to be in the exponential phase of growth. On the day of transfection, 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 μg per 10 ⁶ cells) and the plasmid carrying the gene of interest (10 to 30 μg 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. At the end of the incubation period, 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. In practice, 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.

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 . By cytofluorimetry, the measurement of the transfection efficiency is greater than 20% of the total cells.

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. By this method the gene of interest is integrated and is expressed in a stable manner.

After selection and cloning of the populations, 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.

Alternatively, one can also use vectors for which the expression is subjected to an induction of the promoter regulating the expression of the gene of interest by a compound added at the desired moment in the culture medium. EXAMPLE 2 Generation of a ^Line Which ^Can Grow in the Absence of SCF by Stable Expression of the C-Kit ^Gene G559 murine

In order to obtain porcine mast cells whose growth would be independent of long-term SCF, the mast cells were transformed with the mutated c-kit gene. For this purpose, the murine c-kit gene carrying a point mutation responsible for the modification of valine 559 to glycine (gene noted c-kit ^G559 ) 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 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). 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).

Res., 18 (17): 5322, 1990) open at Xbal.

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

5.10 ⁶ mast cells, in the exponential phase, after 3 days of culture, are centrifuged at

1000 rpm for 5 minutes and taken up in 100 μl of transfection medium (MEMα supplemented with 2 mM glutamine, 10 nM PGE2, 80 ng / ml rpSCF and 2 ng / ml rplL3). 30 μg 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 μF, 340 V (Gene Pulser, Biorad).

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

(Invitrogen) at a concentration of 0.2 mg / ml).

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.

Molecular characterization

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.

100 ng of these genomic DNAs were then amplified by PCR in the presence of a C19810 sense primer of sequence SEQ ID NO: 7 5'-GCC GAG GCC ACT CGC -3 'and a C19814 antisense primer of sequence SEQ ID No. 8 5'-AGC CAT GTA CCG TCA CGC TG-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 58 ^° C. and 45 sec of elongation at 68 ^° C.), the PCR reaction was analyzed on a 2% agarose gel. TBE1X to detect the amplified fragment of 325 bp. Under these conditions, a PCR product of about 350 bp is observed from the genomic DNA from the MCpBXL2015 culture and not from the genomic DNA from the MCpcDNA3.1 / Hygro culture, which demonstrates the presence of the ckit gene. Murine ^G559 (Figure 2A).

Detection of murin ckit ^G559 gene expression in MCpBXL.2015 cells

The detection of the expression of the ckit ^G559 gene was carried out by RT-PCR.

The RNA was extracted from 10 ⁷ cells from the MCpcDNA3.1 / Hygro and MCpBXL2015 cultures according to the Quiagen Rneasy mini kit protocol. 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 "Cîeanup" protocol of the Quiagen Rneasy mini kit.

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.

Under these conditions, a product resulting from the RT-PCR of approximately 350bp from the RNA resulting from the MCpBXL2015 culture and not with the RNA resulting from the MCpcDNA3.1 / Hygro culture is observed, which demonstrates that the gene murine G559 ^ckit is effectively expressed in cells (Figure 2B).

Characterization of growth in absence of SCF

The functionality of the ckit ^G559 was evaluated by analyzing the ability of cells to grow in a medium lacking SCF.

For this, 5 to 6 weeks after transfection, a portion of the MCpcDNA3.1 / Hygro and MCpBXL2015 cells was seeded at 2.10 ⁵ C / ml in medium without SCF. Twice a week and for three weeks, the medium is renewed with fresh medium.

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). For 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.

The growth of 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.

Characterization, in HPLC, of proteoglycans contained in cultures

The study of the growth of MCpBXL2015 cells, in a SCF deficiency medium, demonstrates that the expression of the ckit ^G559 receptor makes ^it possible to dispense with the SCF. We also investigated the impact of SCF removal on mast cell production of proteoglycans.

After five weeks of culture in the absence of SCF, samples of the MCpBXL2015 culture were taken to analyze the mast cell proteoglycan composition according to the protocol described by Linhardt et al (Biomethodes, 9, 183-197, 1997).

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 μl of distilled water added with 10 μl of alkalase (Novozymes) and then heated for 5 hours at 60 ^° C. with stirring.

Then the samples are diluted with 200 μl of Tris 10mM buffer pH 7.0 (Prolabo) 0.5M

NaCl (Prolabo) before being centrifuged for 10 minutes at 10,000 rpm. The proteolysis step makes it possible to release the intracellular content and to dissociate the protein-polysaccharide bonds.

Extraction: The supernatant of each sample is purified by ion exchange on SAX quaternary ammonium resin in 96 well plate format, 100mg / 2ml

After fixing and washing in Tris pH7 buffer, 0.5M NaCl, GlycoaminoGlycans (GAGs) are eluted with 500 μl of Tris buffer pH7.0, 3M NaCl. Desalting / concentration:

The samples are then desalted on a permeation gel column (NAP-5,

Pharmacia) After elution in a volume of 1 ml, the samples are concentrated by freeze-drying and then taken up in 130 μl of distilled water.

Depolymerization.

For HPLC analysis, 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 μl of sample to be analyzed, add

15 μl of the 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) per 30 ml of distilled water .

HPLC analysis.

The samples are then analyzed by HPLC on Spherisorb SAX 5μm, 250x3mm, Thermohypersil column. 50 μl 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.

When MCpBXL2015 are cultured in the absence of SCF, the heparin profile is improved over control cells grown in the presence of SCF. Without the applicant being bound by a particular theory, it is possible to explain this phenomenon either by the cell selection carried out during the production of the cells growing in the absence of SCF, or by a difference in the maturation of the mast cells in the presence or absence of from SCF. Indeed, SCF allows cells to proliferate but also intervenes in cell differentiation.

EXAMPLE 3: Obtaining an MCpBXL2015 Clone by Limit Dilution Cloning

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 μl / 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 μl 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.

A comparative study of the growth was carried out from the mother culture MCpBXL2015 and the nine clones obtained. All clones show growth comparable to that of the uncloned MCpBXL2015 culture (FIG. 3). After four days of culture, samples were taken to analyze the proteoglycan composition of the different clones.

From the HPLC results summarized in Table 2, the polysaccharides synthesized by each clone exhibit heparin-like characteristics with variations in the relative amounts of each disaccharide constituting the heparin chain (e.g., the proportion of disaccharide Is varies from 5 to 30% depending on the clones). Nevertheless, all clones, such as the MXpBXL2015 uncloned population, show a level of sulfation below the detection threshold (non-detectable tetrasaccharide llallsqlu).

Based on the productivity and quality criteria for the production of heparinoids, the clone MC pBXL2015 6-G4 was retained. This clone was frozen under the name of PMC 125.

EXAMPLE 4: Generation of a SCF Independent Lineage and Increased Sulfation Activity

In order to increase the biological activity of heparin-like compounds derived from mast cell cultures, it is possible to stably overexpress the gene encoding 3-OST-1 (3 O-sulfatase-1). In this example, we used the porcine gene.

The mast cells used are derived from the PMC125 culture described in Example 3.

The mast cells are cultured in MEMά 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)

The reverse transcription of 2 μg of total 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'.

2 .mu.l of these cDNAs were then amplified by PCR in the presence of a BS05 sense primer of sequence SEQ ID NO: 12 5'-CGG NGA CCG CCT NAT CAG-3 'and an antisense primer BS06 of sequence SEQ ID NO: 13 5'-TCA GTG YCA GTC RAA TGT TC-3 'with KOD hot start Polymerase (Novagen). After thermal cycling (15 sec denaturation at 98 ^° C., 30 sec hybridization at 60 ° C. and 30 sec elongation at 68 ° C.), the amplified fragment of 277 bp was cloned into the pCR-vector. Blunt II TOPO (Invitrogen, Zero Blunt TOPO PCR Cloning kit) then sequence.

The sequence of this fragment was used to generate 2 primers BS21 and BS22 to isolate, by 3'-RACE, the entire 3 'region.

In the context of 3'-RACE, 1 .mu.l of porcine liver RNA was reverse transcribed into cDNA according to the protocol of the First Strand Synthesis Sytem kit of Invitrogen, using as primer the oligodT CDSIII of sequence SEQ ID N ° 14 (5'-ATT CTA GAG GCC GAG GCG GCC GAC ATG TVN-3 ').

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 μl 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.

Sequencing of the PCR product thus obtained after 3'-RACE made it possible to identify the 3 'sequence of porcine 3-OST as well as approximately 250 bp of the non-coding region.

Isolation of the complete coding phase of the porcine 3-QST gene.

In order to clone the complete coding phase of the porcine 3-OST, a new RT-PCR experiment was performed using the information obtained in the first step. The source of RNA is the same as in the previous step.

2 μg of RNA 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. For this, we use the BS31 sense primer of sequence SEQ ID No. 17 ^{5 '} GGG GAC AAG TT GTA CAA AAA AGC AGG CTC AGC ATG GCC GCG CTG CTC ^3' and the antisense primer BS32 of sequence SEQ ID N ° 18 ^{5 '} GGG ACC ACT TTG TAC AAG AAA GCT GGG TT AGT GCC AGT CAA ATG TTC TGC C ^3' . 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.

Obtaining plasmid pBXL2033 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. and 45 sec of elongation at 68 ° 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.

The plasmid obtained, after complete sequencing of the cDNA, was named pBXL2032. 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.

DNA preparations for transfections were performed with the kit "Endotoxin free plasmid" (Quiagen).

Transfection-Selection of a Population Resistant to Geneticin 5.10 ⁶ mast cells, in the exponential phase, after 3 days of culture, are centrifuged at 1000 rpm for 5 minutes and taken up in 100 μl of nucleoporation solution 8351 (Amaxa). 4 μg of pcDNA3.1 or pBXL2033 are then added to the cell suspension. The cells are then transferred to the electroporation cuvette and subjected to an electric shock: program U-28 (Nucleofector, Amaxa). After electroporation, the cells are transferred into 2 ml of culture medium 2 preheated to 37 ^° C. and then incubated in a 6-well plate 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 (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

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.

For this, three weeks after the application of the selection, samples were taken 4 days after seeding the cultures at 2.10 ⁵ C / ml. The proteoglycans produced by the PMC125pcDNA3.1 control cells do not contain detectable amounts of llallsglu tetrasaccharide (Table 3). In contrast, proteoglycans synthesized by PMC125pBXL2033 cells contain 0.9% llallsglu indicative of a significant increase in the level of sulfation in these cultures.

These results demonstrate that porcine 3OST1 overexpressed in mast cells is functional, ie it induces a 3-0 sulfation. The biological activity of these samples was then examined. For this, a measurement of the anti-Xa and anti-lla activity was performed.

Characterization of the biological activity of the proteoglycans contained in the culture

Inactivation of factors Xa and IIa is characteristic of heparin and makes it possible to differentiate it from heparan sulfate and dermatan. The method used is that described in the monograph of the European Pharmacopoeia 3rd edition (1997).

The reaction takes place in three stages:

1: ATIII + heparin [ATIII - Heparin]

2: [ATIIII - Heparin] + Excess factor [ATIII-Heparin-Factor] + Residual factor

3: Residual Factor + Chromophoric Substrate Colored Para-nitroaniline

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). 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.

The proteoglycans synthesized by the PMC125pcDNA3.1 control cells do not exhibit any detectable biological activity. On the other hand, 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.

These results further demonstrate the expression efficiency of 3OST1 resulting in the appearance of llallsglu and antiXa biological activity.

EXAMPLE 4: Obtaining an MCpBXL2033 Clone by Limit Dilution Cloning

PMC125 pBXL2033 cells were cloned by the limiting dilution technique. For this, 20 96-well plates were inoculated with 0.3 cells / 100 μl / 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.

A comparative study of the growth was carried out starting from the mother culture PMC125pBXL2033 and twelve clones obtained.

For all the studied clones cell proliferation is observed (FIG. 5).

At the end of the growth curve, samples were taken to analyze the proteoglycans of the different clones. Analysis of the proteoglycans synthesized by the HPLC clones shows that all the clones produce proteoglycans having in common a sulfation but varying in their composition (Table 4). Similarly, the biological anti-Xa activity of the proteoglycans varies, according to the analyzed clones, between 17 and 100 IU / mg (Table 5).

Considering the growth criteria and the quality of the proteoglycans produced, clone PMC125 pBXL2033 19-D4 was retained.

The stability of the clone PMC125pBXL2033 19-D4 was followed for nearly five months. This monitoring showed that parameters such as productivity (determined by HPLC analysis), level of sulfation (determined by tetrasaccharide monitoring IlaHsgJu) and the biological activity of the product were stable during this period as well as cell growth. (Figure 6).

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.

HPLC Biological Activity

Ilallsqlu Anti Xa (Ul / mg) Anti lia (Ul / mg) Xa / lla

PMC125pcDNA3.1 <0.1 Not detectable Not detectable -

PMC125pBXL2033 0.9 60 80 0.8

Table 3: Analysis of the structure and biological activity of proteoglycans synthesized by cultures PMC125pcDNA3.1 and PMC125pBXL2033.

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Table 4: Analysis, by HPLC, of the proteoglycans synthesized by clones PMC125pBXL2033.

Table 5: Analysis of the biological activity of the proteoglycans synthesized by clones PMC125pBXL2033.

Claims

1. Culture or line of porcine mast cells characterized in that it produces heparin-type molecules comprising tetrasaccharides lia They glu.

2. Culture or line of porcine mast cells according to claim 1 characterized in that it produces heparin-type molecules having anϋ-Xa activity greater than at least 50 IU / mg.

3. Culture or line of porcine mast cells according to claim 1 characterized in that it produces heparin-type molecules having an anti-lia activity greater than at least 50 IU / mg.

4. Culture or line of porcine mast cells according to one of claims 1 to 3 characterized in that it overexpresses an enzyme acting on the sulfation of heparin-type molecules.

5. Culture or line of porcine mast cells according to one of claims 1 to 4 characterized in that it comprises an exogenous nucleic acid encoding an enzyme acting on the sulfation of heparin-type molecules.

6. culture or line of porcine mast cells according to one of claims 1 to 5 characterized in that it comprises an exogenous nucleic acid encoding a 3-OST.

7. Culture or line of porcine mast cells according to one of claims 1 to 6, characterized in that it comprises an exogenous nucleic acid encoding a 3-

OST1.

8. culture or porcine mast cell line according to one of claims 1 to 7 characterized in that it comprises an exogenous nucleic acid encoding a porcine 3-OST1.

9. Culture or line of porcine mast cells according to one of claims 1 to 8 characterized in that it expresses a c-kit protein having a mutation or a deletion.

10. Culture or line of porcine mast cells according to one of claims 1 to 9 characterized in that it expresses a c-kit protein having a mutation at a position equivalent to that of the c-kit murine ^G559 .

11. culture or line of porcine mast cells according to one of claims 1 to 10 characterized in that it expresses a murine ^G559 c-kit ^protein , c-kit ^G556 porcine or c-kit ^G560 human.

12. Culture or line of porcine mast cells according to one of claims 1 to 11 characterized in that it comprises an exogenous nucleic acid encoding a c-kit protein having a mutation or a deletion.

13. Culture or porcine mast cell line according to one of claims 1 to 12 characterized in that it comprises a nucleic acid encoding a c-kit protein having a mutation at a position equivalent to that of the c-kit murine ^G559 .

14. Porcine mast cell line characterized in that it is one of the porcine mast cell lines deposited with the Collection of Microorganism Cultures of the Institut Pasteur on 17 October 2001 under n ° I-2734, I-2735 or I-2736 modified to express a c-kit protein having a mutation or deletion.

15. Line of porcine mast cells according to claim 14 characterized in that it comprises an exogenous nucleic acid encoding an enzyme acting on the sulfation of heparin-type molecules.

A method for producing heparin-like molecules comprising culturing a porcine mast cell porcine mast cell culture or line according to one of claims 1 to 15.