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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/069—Vascular Endothelial cells
  • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
• • A—HUMAN NECESSITIES
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  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• Cell marker / ligand system where the marker is of the Eph type, cellular material comprising this system, method of preparation and proangiogenic use
• the present invention relates to a new technical solution implementing a system comprising a cell marker and a ligand specific for said marker, wherein said marker is selected from the group consisting of Eph, in particular the EphB4 marker. It also relates to the cellular material comprising this system as well as its method of preparation and its therapeutic use as a proangiogenic agent.
• Eph receptors members of the family of ephrins and their tyrosine kinase Eph receptors, which were first identified in the nervous system for the guidance of neurons, are factors involved in angiogenesis. Many isoforms for Eph receptors and their ephrin ligands have been described with tissue expression specificities.
• Eph receptors In vertebrates, at least 16 Eph receptors are known, namely: 10 EphA receptors (EphA1 to EphAlO) and 6 EphB receptors (EphB1 to EphB6), on the one hand, and at least 9 Ephrin ligands, namely: ephrin- Al to Ephrin-A6 and Ephrin-B to Ephrin-B3, on the other hand.
• EphA and EphB were based on the homology of the sequence of their extracellular domain, but this subdivision also corresponds to the preferential binding of their ligands, the ephrin-A ligands being in general linked to the membrane by a glycosylphosphatidilinositol (GPI) and the Ephrin-B ligands being transmembrane with an intracytoplasmic domain possessing a PDZ binding domain.
• GPI glycosylphosphatidilinositol
• the Eph receptor has in the extracellular domain, repeated units of fibronectin type III ("fibronectin type III repeats"), a region rich in cysteine.
• cyst rich region and a binding domain for a ligand specific (“ligand-binding domain”); it further has an intracellular domain extension comprising a tyrosine kinase domain, an ⁇ -sterile motif (“SAM”) and a PDZ binding motif (PDZ), and the ephrin-A ligand is attached to the membrane by a GPI
• the ephrin-B transmembrane ligand has an intracellular prolongation comprising a cytoplasmic tail (in English: "cytoplasmic tail”) and a PDZ binding motif.
• cytoplasmic tail in English: "cytoplasmic tail”
• a transmembrane protein there is a "bi-directional signaling" which includes a signaling induced by the Eph receptor in the cell expressing Eph on its surface ("forward signaling"), and a “reverse” signaling induced by the ephrin ligand in the cell expressing ephrin on its surface (“reverse signaling").
• This bidirectional signaling plays a role in cell / cell contacts, migration, and cell adhesion.
• EphB4 regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes and blood vessels," Blood, 2004; 103: 100-109, EphB4 appears to be involved in the differentiation of mouse embryonic (ES) cells into endothelial cells.
• Eph receptors notably EphB4
• EphB4 EphB4
• Eph / ephrine relate to methods relating in particular to:
• ephrin-B2 as a marker of tumor vasculature, see in particular WO 02/058538 A cited above.
• the Fc protein ie the Fc fragment of an antibody
• US 6610534 B an adenovirus having the coding sequence of sphingosine kinase and a coding sequence of an angiogenic protein.
• the purpose of this patent is the expression of sphingosine kinase and angiogenic protein, which may be ephrin-B2, after local injection of said adenovirus.
• US 2005/0049194 A discloses a method for decreasing the abnormal proliferation of hematopoietic stem cells, which comprises administration by injection of a water soluble inhibitor of ephrin-B2. Purpose of the invention
• the invention it is proposed to provide a new solution, not described or suggested by the aforementioned prior art, to the technical problem of stimulating angiogenesis. More specifically, it is proposed here to provide a new technical solution implementing a specific cell marker / ligand system of said marker, which is applied to particular cells, namely endothelial cell precursors, to solve the problem of angiogenic stimulation. .
• the aim is to improve previously known cell therapies by activating the cells before their injection, to have a higher efficiency revascularization processes, including post-ischemic revascularization.
• the new solution according to the invention implements a cell marker / ligand system, wherein said marker is associated with an endothelial cell precursor cell.
• the marker is an Eph receptor
• the Eph-specific ligand is an ephrin ligand.
• a novel proangiogenic system of the cell marker / specific ligand type said system, wherein the cell marker is present on the outer membrane of the cell, characterized in that it comprises:
• an endothelial cell precursor comprising a cell marker selected from the group consisting of Eph, in particular EphB4 or EphB1, and
• EPCs having the Eph marker by the specific L ligand belonging to the family of ephrines may also consist of a peptide fragment of an ephrin, for example ephrin-B2, which would then have the same biological activity.
• a cellular material capable of stimulating angiogenesis characterized in that it has the following structure:
• said ligand L is associated with or fused with a binding protein K.
• This cellular material is capable of (i) being in the form of a substantially purified cell culture or in the form of a cell culture in association with other precursor cells, especially mononuclear cells, and (ii) to be; if necessary, frozen.
• a process for the preparation of said cellular material comprising the steps of:
• LK protein material where L is a ligand specific for said marker and K is a binding protein associated or fused with L.
• a drug that can be used to reconstitute the damaged vessels, characterized in that it comprises, in association with a physiologically acceptable excipient, a therapeutically acceptable amount of the cellular material according to the invention. , especially as a proangiogenic active ingredient.
• a new use of said cellular material is provided, said use being characterized in that said cellular material, as a proangiogenic active ingredient, is used in association with an excipient. physiologically acceptable for the preparation of a composition for therapeutic use in the treatment of vascular insufficiency, particularly in the revascularization of ischemic, cardiac, cerebral or peripheral tissues.
• FIG. 1 illustrates the previous state of knowledge and schematically represents the tyrosine kinase receptors, Eph, and their ephrin-A or ephrin-B type ligands; and FIGS. 2 to 4 show graphically the proangiogenic properties, with regard to the tests which have been undertaken, of the cellular material according to the invention.
• the two components of the pair may each be in the form of an amino acid sequence or, where appropriate, in the form of a sequence of amino acids. nucleic acids.
• an Eph receiver expressed on the EPC membrane as an amino acid sequence.
• the ephrin ligand will be very advantageously used in the form of an amino acid sequence or a peptide fragment ephrin. Indeed, it is in the form of amino acids that Eph and ephrine bind, the protein / protein interaction being necessary to observe the proangiogenic activity. Accordingly, in the following, Eph and ephrin each occur exclusively in the protein form of an amino acid sequence.
• EphA or EphB can be used here.
• the Eph marker is an EphB, since the EphB markers are involved predominantly in angiogenesis, whereas (in the current state of knowledge) the EphA markers seem to intervene mainly. at the level of the nervous system.
• the EphB marker will be EphB4 or EphB1; among the EphB markers, EphB4 will be preferred to EphBl.
• the ephrin-B ligands in particular ephrin-B2 or ephrin-B1, are preferred.
• an ephrin-B2 variant for example, which corresponds to a peptide fragment of ephrin-B2 having the same biological activity may also be appropriate.
• the EphB4 or EphBl marker will advantageously be used, on the one hand, and the ephrin-B2 or ephrin-B1 ligand, on the other hand, the Ephrin / Ephrin couple, more particularly preferred according to the invention.
• invention being EphB4 / Ephrin-B2.
• ligand L For the ligand L to activate EPC-Eph, it is important, in the majority of cases, that it is associated with or fused with a binding protein K in the form of a proteinaceous material of structure:
• the recombinant ephrin-B2 protein being, to the knowledge of the Applicant, a substance capable of activating EPC-Eph alone without the contribution of said binding protein K.
• the binding proteins K which are suitable according to the invention,
• the numerous antibody Fc fragments (for example those obtained by cleavage of antibodies by pepsin, papain or any other appropriate substance) can be cited.
• an Fc fragment readily available on the market as a byproduct of the antibody preparation such as Fab 'and F (ab) 2 , is recommended here.
• said L-K protein material is likely to be replaced by the recombinant ephrin-B2 protein, preferably human.
• the EPCs which are suitable according to the invention, are cells which comprise an Eph cell marker (in particular an EphB, preferably EphB1 or better EphB4) expressed at their outer membrane.
• Eph cell marker in particular an EphB, preferably EphB1 or better EphB4
• Such EPCs are obtained from mononuclear cells or from cells expressing CD34 or CD133, which come from bone marrow, peripheral blood or better umbilical cord blood.
• Mononuclear cells are produced in the bone marrow, where they are at a high concentration, pass into the bloodstream and end up in the cord blood and peripheral blood. They constitute a material of choice, in that, as a whole, a large number of cells possess the genetic material required to (i) express the Eph marker (and more particularly the EphB4 marker), or (ii) have on their membrane said marker already expressed.
• Preferred mononuclear cells according to the invention are those which are CD34 + or CD133 4 " , since they provide after differentiation a relatively large amount of EPCs that express or substantially comprise the EphB4 marker.
• the concentration of differentially produced EPCs which are contained in the mononuclear cell population, varies according to the origin of the cells. This concentration is approximately equivalent in bone marrow and umbilical cord blood. On the other hand, it is lower in the peripheral blood.
• the cellular material according to the invention which is represented by structure II above, in which the LK protein material is capable of being replaced by the recombinant ephrin-B2 protein, consists of one or more cells, each having on its outer membrane an Eph marker, in particular EphB4 or EphB1, bound to its specific ligand.
• said cellular material may consist of a culture containing several cells of structure II in combination, if appropriate, with other cells not activated by said protein material; such a culture can therefore be a cell mixture of activated EPCs, unactivated EPCs and mononuclear cells.
• said cellular material is obtained by incubation
• an endothelial cell precursor comprising a cell marker selected from the group consisting of Eph, in particular EphB4 or EphB 1, with
• the incubation is carried out in vitro for a period of 10 to 60 minutes, especially for 30 minutes, this incubation time being just before the administration of the cellular material.
• said cellular material may be in the form of a cell culture which is purified or which is a mixture of cells containing (i) LK-activated EPCs cells or recombinant ephrin-B2 protein, and (ii) non-activated precursors, for example mononuclear cells and / or EPCs, which are not activated by LK or said recombinant ephrin-B2 protein.
• the cellular material may be stored frozen.
• the preferred cellular material according to the invention is of structure II where said marker is EphB4 or EphB 1, and wherein said ligand is ephrin-B2 or ephrin-B 1.
• the method of the invention for preparing said cellular material comprises the following steps:
• step (2 °) differentiate said cells from the previous step to obtain EPCs provided with the Eph marker, and (3) activate in vitro the EPCs, thus obtained in step (2 °), by fixing ephrin on Eph .
• Said method comprises, if necessary, an additional step between steps (1 °) and (2 °), namely:
• Step (Ia 0 ) implies a ternary process (1 °) + (Ia 0 ) + (2 °).
• step (°) increases production costs compared to the binary process (1 °) + (2 °).
• unactivated EPCs cells serve to dilute the activated EPCs cells in the cellular medium containing them, without hindering their action, the binary process is at present clearly more cost-effective than the ternary process.
• said ligand L is associated with or fused with a binding protein K to provide a cellular material of structure:
• Eph marker is EphB4 or EphB1
• said ligand L is ephrin-B2 or ephrin-B1.
• the cellular material according to the invention can be used to treat, in particular, vascular insufficiencies, in particular in the revascularization of cardiac, cerebral or peripheral ischemic tissues.
• the cellular material according to the invention can be packaged in unit dose form, each dose containing the material of structure II.
• a drug composition which is characterized in that it comprises a therapeutically acceptable amount of the two components of the proangiogenic system according to the invention, which are packaged separately, each in a physiologically acceptable excipient, both said components being incubated before administration to give a cellular material of structure IL
• the cellular material according to the invention is useful for regenerating the vascular tissues that have been damaged in particular at the cardiac, cerebral or peripheral level. It is particularly suitable for the preparation of a composition for the treatment of arteritis, coronary vascular or cardiac insufficiency, and cerebrovascular insufficiency. On an animal model, it has provided good results in the treatment of critical ischemia of the lower limbs, on the one hand, and the assurance of healing to prevent amputation.
• the cellular material according to the invention can be administered in the mammal and in particular in humans in a manner known per se.
• said cellular material is capable of being (i) injected at or near the vascular lesion, (ii) injected into the blood by the IV route, or (iii) brought to the site of the lesion by means of an appropriate vector.
• the EPC-Eph cells, on the one hand, and the protein material bound to a vector known in the field of gene therapy, on the other hand can be administered separately by injection (especially by the IV route).
• structure II cells contained, if appropriate, in a cellular mixture of nonactivated EPCs can be administered by IV injection.
• a cell mixture may comprise a total of about 10 5 to 10 9 cells per injection.
• Other advantages and characteristics of the invention will be better understood on reading the following examples of preparation and pharmacological tests. Of course, all of these elements is not limiting but is given by way of illustration, the cellular material used being: EPC-EphB4-ephrin-B2-Fc.
• Example 2 The cell mixture, obtained at the end of Example 1 (A), is placed in the wells of a 6-well plate coated with type I collagen.
• Example 1 From the cell mixture obtained at the end of Example 1 (A), the CD34 + cells of the non-adherent cells are isolated and purified by a standard immunomagnetic separation technique, in particular using the "CD34 isolation” material. Kit “(marketed by the company called MILTENYI BIOTECH, Paris France), which comprises an anti-CD34 monoclonal antibody.
• Kit “(marketed by the company called MILTENYI BIOTECH, Paris France) which comprises an anti-CD34 monoclonal antibody.
• the resulting cell mixture which contains 1.5 x 10 6 to 3.5 x 10 6 CD34 + cells, can be placed in the wells of a 6-well plate coated with a fibronectin-containing matrix, laminin, heparan sodium sulfate, and type I and IV collagen (products sold by the aforementioned company SIGMA-ALDRICH) and in a culture medium containing hVEGF, bFGF and IGF1 (products marketed by the company called R & D Systems Inc., Oxford, United Kingdom). After 15 days of culture, a cell mixture enriched in EPC-EphB4 is collected.
• Example 3 Example 3
• Example 1 (B) which contains EPCs cells provided with the EphB4 marker, it is treated with 3 ⁇ g / ml of Ephrin-B2-Fc fusion protein, EphB4-Fc or CD6-Fc (CD6 Fc acting as a negative control for the demonstration of the effect observed by the activation of EPCs by ephrin-B2) for a 30 minute incubation time at 37 ° C. Rinse each non-melting fusion protein linked (at least two rinses are performed here).
• Three cellular mixtures are obtained, one of which contains the cellular material according to the invention of EPC-EphB4-ephrin-B2-Fc structure, the second contains the cellular material resulting from the activation of EPC by the EphB4-Fc fusion protein. and the third contains the cellular material resulting from the activation of EPC by the CD6-Fc fusion protein.
• Example 4 Obtaining EPC-EphB4-ephrin-B2-Fc
• Example 3 The procedure is as indicated in Example 3 for obtaining EPC-EphB4-ephrin-B2-Fc structure cells, with the difference that the starting cell mixture is that obtained after Example 2 (B ). The announced activated cells are obtained.
• Test protocol
• Angiographic score (i.e. vessel density) was determined by micro-angiography. More specifically, the density of the vessels in the ischemia member relative to the non-ischemic limb is measured by high-definition micro-angiography (see procedures in the article by Sivestre J. S et al, Cir Res., 2001; 89: 259-264). The results obtained, presented as a ratio (ischemic paw / non-ischemic paw), are recorded graphically in Figure 2.
• the capillary density of the ischemia muscle was studied by labeling sections of the gastrocnemius muscle by means of an antibody directed against the CD31 marker, which is specific for endothelial cells, compared to sections of the same muscle of the non-ischemic limb.
• the results obtained, presented in the form of a ratio (ischemic paw / non-ischemic paw), are graphically recorded in FIG.
• RNA interference or "siRNA”, in English, capable in particular to degrade specifically
• RNA coding for a given gene and in particular here for the gene expressing the EphB4 marker.
• the specific action of these RNA interference is called transfection.
• progenitor endothelial cells, or EPCs are grown up to 80% confluency.
• Solutions of EphB4 siRNA, that is to say containing the interference RNA, capable of inhibiting the protein synthesis of the EphB4 marker are diluted in an M199 medium containing no antibiotics or serum and incubated for five minutes at room temperature. ambient temperature.
• a "siRNA control" solution corresponding to no particular gene is diluted under the same conditions. This latter diluted solution, which therefore has no biological effect on the expression of the EphB4 marker, will simply make it possible to verify that the interference RNAs do not have any specific activity, regardless of their inhibitory role.
• a transfection agent Dharmafect2 (Dharmacon, Perbio) is prepared under the same conditions as the aforementioned solutions, and then mixed with these solutions, which are then incubated.
• the interference RNAs do not have their own activity independently of their inhibitory role and that the EPCs endogenous progenitor cells expressing the EphB4 marker do not have more activity than the EPCs endogenial progenitor cells. not expressing the marker and which are associated with the ephrin-B2-Fc protein material.

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• 2017
  • 2017-06-21 JP JP2017121385A patent/JP2017169590A/ja active Pending

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