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WO2003101201A1 - Injection intramyocardique de moelle osseuse autologue - Google Patents

Injection intramyocardique de moelle osseuse autologue Download PDF

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Publication number
WO2003101201A1
WO2003101201A1 PCT/US2003/015529 US0315529W WO03101201A1 WO 2003101201 A1 WO2003101201 A1 WO 2003101201A1 US 0315529 W US0315529 W US 0315529W WO 03101201 A1 WO03101201 A1 WO 03101201A1
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bone marrow
cells
vector
days
patient
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Stephen Epstein
Shmuel Fuchs
Ran Kornowski
Martine B. Leon
Kenneth W. Carpenter
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Myocardial Therapeutics Inc
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Myocardial Therapeutics Inc
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Priority to CA2487410A priority Critical patent/CA2487410C/fr
Priority to AU2003232152A priority patent/AU2003232152A1/en
Priority to JP2004508572A priority patent/JP4111950B2/ja
Priority to EP03756182A priority patent/EP1513404A4/fr
Publication of WO2003101201A1 publication Critical patent/WO2003101201A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • This application is directed to methods for injecting autologous bone marrow and bone marrow cells. More specifically, this invention is directed to intramyocardial injection of autologous bone marrow and transfected bone marrow cells to enhance collateral blood vessel formation and tissue perfusion.
  • the bone marrow (BM) is a natural source of a broad spectrum of cytokines (e.g., growth factors) and cells that are involved in the control of angiogenic processes. It is therefore believed that the intramyocardial injection of autologous (A) BM or bone marrow cells derived therefrom, by taking advantage of the natural ability of these cells to secrete many angiogenic factors in a time-appropriate manner, provides an optimal intervention for achieving therapeutic collateral development in ischemic myocardium.
  • cytokines e.g., growth factors
  • autologous bone marrow, or cells derived therefrom is injected, either as a "stand alone” therapeutic agent or combined with any pharmacologic drug, protein or gene or any other compound or intervention that may enhance bone marrow production of angiogenic growth factors and/or promote endothelial cell proliferation, migration, and blood vessel tube formation.
  • the "combined" angiogenic agents can be administered directly into the patient or target tissue, or incubated ex- vivo with bone marrow prior to injection of bone marrow into the patient.
  • Non-limiting examples of these "combined" angiogenic agents are Granulocyte- Monocyte Colony Stimulator-y Factor (GM-CSF), Monocyte Chemoattractant Protein 1 (MCP-1), and Hypoxia Inducible Factor-1 (HIF-1).
  • GM-CSF Granulocyte- Monocyte Colony Stimulator-y Factor
  • MCP-1 Monocyte Chemoattractant Protein 1
  • HIF-1 Hypoxia Inducible Factor-1
  • filtered bone marrow and/or bone marrow cells (either transfected with a gene encoding an angiogenic factor, or not) is directly administered into the ischemic target tissue accompanied by an angiogenic agent known as RGTA (ReGeneraTing Agent).
  • RGTA is a family of agents that has properties mimicking those of heparan sulfates toward heparin-binding growth factors (or HBGF) and which stimulate tissue repair and protection.
  • RGTAl 1 one member of the RGTA family, RGTAl 1, has previously been shown to prevent most of the damage resulting from acute skeletal muscle ischemia (FASEB J. (1999) 13:761-766).
  • RGTAl 1 a chemically defined member of the RGTA family, is a Dextran derivative obtained by controlled and sequential substitutions on the glucose residues of Dextran T as previously described (M. Mauzac and J. Josefonvicz (1984) Biomaterials 5:301-304).
  • RGTAl 1 the percentages of substitutions are 69, 2.5 and 36.5% for the carboxymethyl, carboxymethylbenzylamme, and carboxymethyl-benzylaminde sulfonate and sulfate groups, respectively.
  • the anticoagulant activity of RGTAl 1 is 3.5 IU/mg (compared with heparin, usually at about 170-180IU/mg).
  • simultaneous injection is not required in practice of the invention methods, RGTAl 1 can conveniently be injected simultaneously with autologous bone marrow cells, either fresh or treated as described herein, into a site of muscle ischemia to promote growth of blood vessels therein.
  • RGTAl 1 can be added to a carrier solution containing filtered bone marrow cells prior to injection of the cells. If the RGTAl 1 is added to the carrier containing the bone marrow, since it has activity as a blood anticoagulant, the amount of heparin administered with the bone marrow cells, if present, can be reduced accordingly.
  • Another example of an intervention that enhances bone marrow production of angiogenic factors is ex-vivo exposure of bone marrow cells to hypoxia.
  • the autologous bone marrow, alone or with "combined" angiogenic agents can be delivered to the patient directly via either trans-endocardial or trans-epicardial approaches into either ischemic and/or non-ischemic myocardium, or directly into any other ischemic organ (including a peripheral limb) to enhance and/or promote the development of collateral blood vessel formation and therefore collateral flow to ischemic myocardium or ischemic limbs.
  • This approach can also be used to promote the development of newly implanted dedifferentiated and/or differentiated myocardial cells by the process of cardiac myogenesis.
  • the invention provides methods for enhancing transfection efficiency in bone marrow cells.
  • bone marrow cells are cultured under suitable culture conditions in a container for a period of time sufficient to promote production by the bone marrow of early attaching cells.
  • the early attaching cells are transfected with a vector comprising a polynucleotide that encodes one or more angiogenic cytokines, growth factors or mammalian angiogenesis- promoting factors.
  • the efficiency with which the early attaching cells are transfected is enhanced as compared with that obtained by transfecting fresh bone marrow or non-adherent bone marrow cells with the vector.
  • the invention provides methods for enhancing collateral blood vessel formation in a subject in need thereof by obtaining autologous bone marrow from the patient, growing the autologous bone marrow under suitable culture conditions in a container for a period of time sufficient to promote production by the bone marrow of early attaching cells, and transfecting the early attaching cells with a vector comprising a polynucleotide that encodes one or more angiogenic cytokines, growth factors or mammalian angiogenesis-promoting factors.
  • An effective amount of the transfected cells is directly administered to a desired site in the patient to cause expression of the one or more agents leading to enhanced collateral blood vessel formation at the site in the patient.
  • the mvention provides compositions comprising early attaching cells derived from autologous bone marrow that have been transfected with a vector comprising a polynucleotide that encodes one or more cytokines, growth factors or mammalian angiogenesis-promoting factors as described herein.
  • Fig. 1 is a graph of the proliferation of PAEC's vs. the quantities of conditioned medium
  • Fig. 2 is a graph of the proliferation of endothelial cells vs. the quantities of conditioned medium
  • Fig. 3 is a graph of the concentration of VEGF in conditioned medium over a four- week period of time.
  • Fig. 4 is a graph of the concentration of MCP-1 in conditioned medium over a four- week period of time.
  • Bone marrow is a natural source of a broad spectrum of cytokines, growth factors and angiogenesis-promoting factors that are involved in the control of angiogenic and inflammatory processes.
  • the angiogenic cytokines, growth factors and angiogenesis- promoting factors expressed comprise mediators known to be involved in the maintenance of early and late hematopoiesis (IL-lalpha and IL-lbeta, IL-6, IL-7, IL-8, IL- 11 and IL-13; colony-stimulating factors, thrombopoietin, erythropoietin, stem cell factor, fit 3-ligand, hepatocyte cell growth factor, tumor necrosis factor alpha, leukemia inhibitory factor, transforming growth factors beta 1 and beta 3; and macrophage inflammatory protein 1 alpha), angiogenic factors (fibroblast growth factors 1 and 2, vascular endothelial growth factor) and mediators whose usual target (and source) is the connective tissue-forming cells (platelet-
  • NEGF polypeptides are present in platelets and rnegacaryocytes, and are released from activated platelets together with the release of beta-thromboglobulin. Wartiovaara, U., et al., Thromb Haemost 1998; 80:171- 5; Mohle, R, Proc NatlAcad Sci USA 1997; 94:663-8.
  • angiogenesis is needed to support bone marrow function and development of hematopoietic cells, including stem cells and progenitor cells, that may enter the circulation and target to sites of wound healing and/or ischemia, ultimately contributing to new blood vessel formation.
  • Monoclonal antibodies that specifically recognize undifferentiated mesenchymal progenitor cells isolated from adult human bone marrow have been shown to recognize cell surface markers of developing microvasculature, and evidence suggests such cells may play a role in embryonal angiogenesis. Fleming, J.E., Jr., Dev Dyn 1998; 212: 119- 32.
  • Bone marrow angiogenesis may become exaggerated in pathologic states where the bone marrow is being activated by malignant cells (such as in multiple myeloma) where bone marrow angiogenesis has been shown to increase simultaneously with progression of human multiple myeloma cells.
  • malignant cells such as in multiple myeloma
  • bone marrow angiogenesis has been shown to increase simultaneously with progression of human multiple myeloma cells.
  • Ribatti, D., et al., Er J Cancer 1999; 79:451-5 vascular endothelial growth factor (N ⁇ GF) has been shown to play a role in the growth of hematopoietic neoplasms such as multiple myeloma, through either a paracrine or an autocrine mechanism.
  • N ⁇ GF vascular endothelial growth factor
  • HIF-1 a potent transcription factor that binds to and stimulates the promoter of several genes involved in responses to hypoxia.
  • Induction and activation of HIF-I is tightly controlled by tissue pO 2 ; HIF-1 expression increases exponentially as pO 2 decreases, thereby providing a positive feedback loop by which a decrease in pO causes an increase in the expression of gene products that serve as an adaptive response to a low oxygen environment.
  • Activation of HIF-1 leads, for example, to the induction of erythropoietin, genes involved in glycolysis, and to the expression of N ⁇ GF.
  • HIF-1 is a heterodimer with a basic helix-loop-helix motif, consisting of the subunits HIF-1 ⁇ and HIF-1 ⁇ . Its levels are regulated by pO2 both transcriptionally and posttranscriptionally — HIF-1 induction is increased by hypoxia, and its half-life is markedly reduced as pO 2 levels increase.
  • HIF-1 as determined in HeLa cells
  • pO 2 the inflection point of the curve occurs at an oxygen saturation of 5%, with maximal activity at 0.5% and Vi maximal activity at 1.5- 2.0%.
  • hypoxia relatively low levels
  • myocardial or lower limb ischemia i.e., levels present in the absence of tissue necrosis (myocardial infarction, and leg ulcerations, respectively).
  • tissue necrosis myocardial infarction, and leg ulcerations, respectively.
  • bone marrow cells could have the capacity to secrete angiogenic factors and thereby enhance collateral development.
  • HIF-1 will provide optimal expression of many of the hypoxia-inducible angiogenic genes present in the bone marrow implant.
  • the HIF-1 can be injected either as the protein, or as the gene. If as the latter, it can be injected either in a plasmid or viral vector, or any other manner that leads to functionally relevant protein levels. It is emphasized, however, that HIF-1 is used as an example of an intervention that could enhance production of angiogenic substances by bone marrow.
  • This invention also covers use of other angiogenic agents, which by enhancing HIF-1 activity (i.e., prolonging its half-life), or by producing effects analogous to HIF-1, stimulate the bone marrow to increase expression of angiogenic factors.
  • HIF-1 activity i.e., prolonging its half-life
  • HIF-2 endothelial PAS domain protein 1
  • bone marrow cells can be exposed ex- vivo in culture to hypoxia or other forms of energy, such as, for example, ultrasound, RF, or electromagnetic energy. This intervention increases NEGF and other gene expression. By this effect it may augment the capacity of bone marrow to stimulate angiogenesis.
  • the invention involves the ex-vivo stimulation of aspirated autologous bone marrow by HIF-1 (or products that augment the effects of HIF-1 or produce similar effects to HIF-1 on bone marrow) or direct exposure of bone marrow to hypoxic environment followed by the delivery of activated bone marrow cells to the ischemic myocardium or peripheral organ (e.g., ischemic limb) to enhance collateral-dependent perfusion in cardiac and/or peripheral ischemic tissue.
  • HIF-1 or products that augment the effects of HIF-1 or produce similar effects to HIF-1 on bone marrow
  • ischemic myocardium or peripheral organ e.g., ischemic limb
  • Current data indicate the importance of monocyte-derived cytokines for enhancing collateral function. Monocytes are activated during collateral growth in vivo, and monocyte chemotactic protein-1 (MCP-1) is upregulated by shear stress in vitro.
  • monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis).
  • MCP-1 was also shown to enhance collateral growth after femoral artery occlusion in the rabbit chronic hindlimb ischemia model (Ito et al., Circ Res 1997; 80:829-3).
  • Activation of monocytes seems to play an important role in collateral growth as well as in capillary sprouting.
  • Increased monocyte recruitment by LPS is associated with increased capillary density as well as enhanced collateral and peripheral conductance at 7 days after experimental arterial occlusion (Arms M. et al., JClin Invest 1998; 101:40-50.).
  • a further aspect of the invention involves the ex-vivo stimulation of aspirated autologous bone marrow by MCP-1, followed by the direct delivery of activated bone marrow cells to the ischemic myocardium or peripheral organ (e.g., ischemic limb) to enhance collateral-dependent perfusion and muscular function in cardiac and/or peripheral ischemic tissue.
  • the stimulation of the bone marrow could be by the direct exposure of the bone narrow to MCP-1 in the form of the protein, or the bone marrow cells can be transfected with a vector carrying the MCP-1 gene.
  • bone marrow, or early attaching cells derived from bone marrow can be transfected with a plasmid vector, or with an adenoviral vector, carrying the MCP-1 trans gene.
  • Granulocyte-macrophage colony-stimulating factor GM-CSF
  • Granulocyte-Colony Stimulatory Factor G-CSF
  • stimulatory cytokines for monocyte maturation and are multipotent hematopoietic growth factors, which are utilized in clinical practice for various hematological pathologies such as depressed white blood cell count (i.e., leukopenia or granulocytopenia or monocytopenia) which occurs usually in response to immunosuppressive or chemotherapy treatment in cancer patients.
  • GM-CSF has also been described as a multilineage growth factor that induces in vitro colony formation from erythroid burst-forming units, eosinophil colony-forming units (CSF), and multipotential (CSF), as well as from granulocyte-macrophage CSF and granulocyte CFU.
  • CSF erythroid burst-forming units
  • CSF eosinophil colony-forming units
  • CSF multipotential
  • GM-CSF carries multiple stimulatory effects on macrophage/monocyte proliferation, differentiation, motility and survival (reduced apoptotic rate). Consistent with the combined known effects on bone marrow derived endothelial progenitor cells and monocytes, it is another aspect of the invention to use GM-CSF as an adjunctive treatment to autologous bone marrow injections aimed to induce new blood vessel formation and differentiation in ischemic cardiovascular organs.
  • GM-CSF may further enhance therapeutic myocardial angiogenesis caused by bone marrow, by augmenting the effect of bone marrow, or by further stimulating, administered either in vivo or in vitro, bone marrow that is also being stimulated by agents such as HIF-1, EPAS 1, hypoxia, or MCP-1.
  • ABM cells collected from a subject can be transfected, ex vivo, with a plasmid vector, or with an adenoviral vector, carrying an angiogenic cytokine growth factor or mammalian angiogenesis promoting factor transgene, such as the HIF-1 or EPSA1 transgene, for expression thereof in the cells and/or in the subject when the transfected cells are injected into a treatment site as described herein.
  • a plasmid vector or with an adenoviral vector, carrying an angiogenic cytokine growth factor or mammalian angiogenesis promoting factor transgene, such as the HIF-1 or EPSA1 transgene, for expression thereof in the cells and/or in the subject when the transfected cells are injected into a treatment site as described herein.
  • an angiogenic cytokine growth factor or mammalian angiogenesis promoting factor transgene such as the HIF-1 or EPSA1 transgene
  • Early attaching cells as the term is used herein means the cells from the culture medium containing bone marrow, or from bone marrow cells seeded into the container, that do not wash away after growth at suitable culture conditions for about 8 hours (e.g., overnight) to about 24 hours.
  • the early attaching cells are mostly monocytes, endothelial precursor cells, or other hematopoietic lineage cells. Inoculation takes place after culture of the cells for a period of from about 3 to about 28 days, for example after about 3 to about 14 days, or after about 3 to about 7 days.
  • the early attaching cells can be inoculated with a vector encoding one or more angiogenic cytokines, growth factors and/or factors that promote angiogenesis in mammalian cell by any method known in the art, for example by in vitro contact for a period of about 2 hours to about 3 days after the inoculation.
  • the vector used can be selected from any of those known in the art and include, but without limitation thereto, those described herein.
  • the vector e.g. a virus
  • the vector is generally washed out about 2 hours to about 3 days after the inoculation before the cells are prepared for administration to the patient.
  • the ABM can be filtered prior to placement in the container to remove particles larger than about 300 ⁇ to about 200 ⁇ .
  • Bone marrow cells can also be separated from the filtered ABM for growth in the container leading to production of early attaching cells. Suitable culture conditions are well known in the art and include, but are not limited to, those described in the Examples herein.
  • Suitable transgenes for transfecting bone marrow-derived early attaching cells include, but without limitation thereto, those encoding such angiogenesis-promoting agents as HJF-l, EPASl (also known as HJF-2), MCP-1, CM-CSF, and the like.
  • An effective amount of the transfected early attaching cells derived from bone marrow prepared as described herein can be directly administered to (i.e. injected into) a desired site in a patient to enhance collateral blood vessel formation at the site in the patient.
  • Particularly effective sites for administration of cells transfected with an angiogenesis-promoting agent include heart muscle or skeletal muscle, such as in the leg, to enhance collateral-dependent perfusion in cardiac and/or peripheral ischemic tissue.
  • the polynucleotide encoding the therapeutic protein may be "functionally appended to”, or “operatively associated with”, a signal sequence that can "transport” the encoded product across the cell membrane.
  • signal sequences are known and can be used by those skilled in the art without undue experimentation.
  • Gene transfer vectors contemplated for such purposes are recombinant nucleic acid molecules that are used to transport nucleic acid into host cells for expression and/or replication thereof.
  • Expression vectors may be either circular or linear, and are capable of incorporating a variety of nucleic acid constructs therein.
  • Expression vectors typically come in the form of a plasmid that, upon introduction into an appropriate host cell, results in expression of the inserted nucleic acid.
  • Suitable viral vectors for use in gene therapy have been developed for use in particular host systems, particularly mammalian systems and include, for example, retroviral vectors, other lentivirus vectors such as those based on the human immunodeficiency virus (HIN), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, and the like (see Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Nerma and Somia. Nature 389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which is incorporated herein by reference).
  • retroviral vectors such as those based on the human immunodeficiency virus (HIN)
  • adenovirus vectors such as those based on the human immunodeficiency virus (HIN)
  • adeno-associated virus vectors such as those
  • Preferred gene transfer vectors are replication-deficient adenovirus carrying the cD ⁇ A to effect development of collateral arteries in a subject suffering progressive coronary occlusion (Barr et al., "PCGT Catheter-Based Gene Transfer Into the Heart Using Replication-Deficient Recombinant Adenoviruses," Journal of Cellular Biochemistry, Supplement 17D, p. 195, Abstract PlOl (Mar. 1993); Barr et al., "Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus," Gene Therapy, vol. 1:51-58 (1994)).
  • the gene of interest maybe transferred to the heart (or skeletal muscle), including cardiac myocytes (and skeletal myocytes), in vivo and direct constitutive production of the encoded protein.
  • adenoviral vectors based on the human adenovirus 5 are missing essential early genes from the adenoviral genome (usually El A/EIB), and are therefore unable to replicate unless grown in permissive cell lines that provide the missing gene products in trans.
  • a transgene of interest can be cloned and expressed in tissue/cells infected with the replication deficient adenovirus.
  • adenovirus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1% adenovirus-mediated transfections result in transgene incorporation into host DNA), and therefore is not stable, adenoviral vectors can be propagated in high titer and transfect non-replicating cells well.
  • the amount of exogenous nucleic acid introduced into a host organism, cell or cellular system can be varied by those of skill in the art according to the needs of the individual being treated.
  • the amount of nucleic acid introduced to the cells to be transfected can be varied by varying the amount of plaque forming units (PFU) of the viral vector.
  • PFU plaque forming units
  • a subject in need thereof by obtaining ABM from the patient; growing the ABM under suitable culture conditions in a container for a period of time sufficient to promote production by the bone marrow of early attaching cells, which early attaching cells adhere to the container.
  • the early attaching cells are transfected in culture as described above (i.e.
  • angiogenesis-promoting agent can be transiently expressed in the subject into which the transfected cells are injected, thus delivering the therapeutic angiogenesis-promoting agents, or a combination thereof, to the ischemic site and leading to enhanced collateral blood vessel formation at the site of administration in the patient.
  • transcription regulatory region refers to that portion of a nucleic acid or gene construct that controls the initiation of mRNA transcription.
  • a minimal promoter when combined with a regulatory element, functions to initiate mRNA transcription in response to a ligand/functional dimer complex. However, transcription will not occur unless the required inducer (ligand therefor) is present.
  • certain of the invention chimeric protein heterodimers activate or repress mRNA transcription even in the absence of ligand for the DNA binding domain.
  • operatively associated with refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
  • operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • the transcription regulatory region further comprises a binding site for ubiquitous transcription factor(s).
  • binding sites are preferably positioned between the promoter and the regulatory element.
  • Suitable ubiquitous transcription factors for use herein are well known in the art and include, for example, Spl .
  • Exemplary eukaryotic expression vectors include eukaryotic constructs, such as the pSV-2 gpt system (Mulligan et al, (1979) Nature, 277:108-114); PBLUESKRIPT® vector (Stratagene, La Jolla, CA), the expression cloning vector described by Genetics Institute (Science, (1985) 228:810-815), and the like. Each of these plasmid vectors is capable of promoting expression of the protein of interest.
  • a gene transfer vector contemplated for use herein is a viral vector, such as Adenovirus, adeno-associated virus, a herpes-simplex virus based vector, a synthetic vector for gene therapy, and the like (see, e.g., Suhr et al, Arch, of Neurol. 50: 1252-1268, 1993).
  • a gene transfer vector employed herein can be a retroviral vector.
  • Retroviral vectors contemplated for use herein are gene transfer plasmids that have an expression construct containing an exogenous nucleic acid residing between two retroviral LTRs.
  • Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Patent 4,650,764).
  • Suitable retroviral vectors for use herein are described, for example, in U.S. Patents 5,399,346 and 5,252,479; and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, each of which is hereby incorporated herein by reference, in its entirety. These documents provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors.
  • retroviral vectors include, for example, mouse mammary tumor virus vectors (e.g., Shackleford et al, (1988) PNAS, USA, 85:9655-9659), human immunodeficiency virus (e.g., Naldini et al. (1996) Science 272:165-320), and the like.
  • helper cells that produce retroviral vector particles that are essentially free of replicating virus. See, for example, U.S. Patent 4,650,764; Miller, Human Gene Therapy, 1:5-14, 1990; Markowitz, et al.
  • Patent 4,650,764 Miller, supra 1990; Markowitz, et al, supra 1988; Watanabe, et al, supra 1983; Danos, et al, PNAS, 85:6460-6464, 1988; and Bosselman, et al, Molecular and Cellular Biology, 7(5): 1797-1806, 1987.
  • Bone Marrow Cultured Media-on Endothelial Cell Proliferation Studies were conducted to determine whether aspirated pig autologous bone marrow cells obtained secreted NEGF, a potent angiogenic factor, and MCP-1, which recently has been identified as an important angiogenic co-factor. Bone marrow was cultured in vitro for four weeks. The conditioned medium was added to cultured pig aortic endothelial cells (PAECs), and after four days proliferation was assessed. NEGF and MCP-1 levels in the conditioned medium were assayed using ELISA. During the four weeks in culture, BM cells secreted NEGF and MCP-1, such that their concentrations increased in a time-related manner.
  • PAECs cultured pig aortic endothelial cells
  • BM cells are capable of secreting potent angiogenic cytokines such as NEGF and MCP-1 and of inducing proliferation of vascular endothelial cells.
  • Bone marrow (BM) cells were harvested under sterile conditions from pigs with chronic myocardial ischemia in preservative free heparin (20 units/ml BM cells) and filtered sequentially using 300 ⁇ and 200 ⁇ stainless steel mesh filters. BM cells were then isolated by Ficoll-Hypaque gradient centrifugation and cultured in long-term culture medium (LTCM) (Stem Cell Tech, Nancouver, British Columbia, Canada) at 330° C with 5% CO 2 in T-25 culture flask. The seeding density of the BMCs in each culture was 7 x 106/ml. Weekly, one half of the medium was removed and replaced with fresh LTCM. The removed medium was filtered (0.2 ⁇ filter) and stored at -200° C for subsequent Enzyme-linked Immunosorbent Assay (ELISA) and cell proliferation assays.
  • LTCM long-term culture medium
  • Fresh pig aortic endothelial cells were isolated using conventional methods. Endothelial cell growth medium (EGM-2 medium, Clonetics, San Diego, CA), containing 2% FBS, hydrocortisone, human FGF, NEGF, human EGF, IGF, heparin and antibiotics, at 37° C with 5% carbon dioxide. When the cells became confluent at about 7 days, they were split by 2.5% trypsin and cultured thereafter in medium 199 with 10% FBS. Their identity was confirmed by typical endothelial cell morphology and by immunohistochemistry staining for factor NIII. Passage 3-10 was used for the proliferation study.
  • Cell proliferation assay PAECs (Passage 3-10) were removed from culture flasks by trypsinization. The detached cells were transferred to 96-well culture plates and plated at a seeding density of 5,000 cells/well. Cells were cultured for 2-3 days before being used in proliferation and D ⁇ A synthesis experiments. The conditioned medium of BM cells cultures were collected at 4 weeks, medium from 7 culture flasks were pooled and used in the bioassay. Aliquotes (10 ⁇ L, 30 ⁇ L, 100 ⁇ L or 200 ⁇ L) of pooled conditioned medium, or LTCM (200 ⁇ L, as control), were added to confluent PAECs in 96-well plates in triplicate. Four days following culture with conditioned medium or control medium, the PAECs were trypsinized and counted using a cell counter (Coulter Counter Beckman Corporation, Miami FL).
  • NEGF and MCP-1 in conditioned medium by ELISA NEGF The concentration of NEGF in conditioned medium was measured using a sandwich ELISA kit (Chemicon International Inc., Temecula, CA). Briefly, a plate pre- coated with anti-human NEGF antibody was used to bind NEGF in the conditioned medium or to a known concentration of recombinant VEGF. The complex was detected by the biotinylated anti-NEGF antibody, which binds to the captured NEGF. The biotinylated NEGF antibody in turn was detected by streptavidin-alkaline phosphatase and color generating solution. The anti-human NEGF antibody cross-reacts with porcine NEGF.
  • the concentration of MCP-1 in conditioned medium was assayed by sandwich enzyme immunoassay kit (R &D Systems, Minneapolis, M ⁇ ): a plate pre-coated with anti human MCP-1 antibody was used to bind MCP-1 in the conditioned medium or to a known concentration of recombinant protein.
  • the complex was detected by the biotinylated anti-MCP-1 antibody, which binds to the captured MCP-1.
  • the biotinylated MCP-1 antibody in turn was detected by streptavidin-alkaline phosphatase and color generating solution.
  • the anti-human MCP-1 antibody cross-reacts with porcine MCP-1.
  • NEGF and MCP-1 in the BM conditioned medium increased gradually to 10 and 3 times the 1st week level, respectively (P ⁇ 0.001 for both comparisons) (Fig. 3).
  • NEGF and MCP-1 levels in a control culture medium, not exposed to BM were 0 and 11 ⁇ 2 pg/ml, respectively, as shown in Fig. 4.
  • hypoxia markedly increases the expression of NEGF by cultured bone marrow endothelial cells, results indicating that ex-vivo exposure to hypoxia, by increasing expression of hypoxia-inducible angiogenic factors, can further increase the collateral enhancing effect of bone marrow cells and its conditioned media to be injected in ischemic muscular tissue.
  • Pig bone marrow was harvested and filtered sequentially using 300 ⁇ and 200 ⁇ stainless steel mesh filters. BMCs were then isolated by Ficoll-Hypaque gradient centrifugation and cultured at 33° C with 5% CO 2 in T-75 culture flasks.
  • Chronic myocardial ischemia was created in 14 pigs by the implantation of ameroid constrictors around the left circumflex coronary artery.
  • 7 animals underwent transendocardial injections of freshly aspirated ABM into the ischemic zone using a transendocardial injection catheter (2.4 ml per animal injected at 12 sites) and 7 control animals were injected with heparinized saline.
  • Bone Marrow Aspiration and Preparation and Intramyocardial Injection [0064] Immediately after completion of the baseline assessment, all animals underwent BM aspiration from the left femoral shaft using standard techniques. BM was from aspirated 2 sites (3 ml per site) using preservative free heparinized glass syringes (20 unit heparin 1 ml fresh BM). The aspirated bone marrow was immediately macro- filtered using 300 ⁇ and 200 ⁇ stainless steel filters, sequentially. Then, the bone marrow was injected using a transendocardial injection catheter into the myocardium in 12 sites (0.2 ml per injection site for total of 2.4 ml) directed to the ischemic myocardial territory and its borderline region.
  • Transthoracic echocardiography images of short and long axis views at the mid-papillary muscle level were recorded in animals at baseline and during pacing, at baseline and during follow-up evaluation at four weeks after ABM implantation.
  • Fractional shortening measurements were obtained by measuring the % wall thickening (end-systolic thickness minus end-diastolic thickness/end-diastolic thickness) x 100. Those measurements were taken from the ischemic territory (lateral area) and remote territory (anterior-septal area).
  • a temporary pacemaker electrode was inserted via a right femoral venous sheath and positioned in the right atrium. Animals were paced at 180/minute for 2 minutes and echocardiographic images were simultaneously recorded.
  • Histopathology assessment was performed on sampled heart tissue.
  • 7-mm thick short-axis slices were examined under UN light to identify fluorescent-tagged areas. Each identified area was cut into 3 full thickness adjacent blocks (central, right and left) that were immersion-fixed in 10% buffered formaldehyde. Subsequently, each such block was cut into 3 levels, of which 2 were stained with Hematoxylin and Eosin (H&E) and one with PAS.
  • H&E Hematoxylin and Eosin
  • one fresh fluorescent- labeled tissue block was obtained from the ischemic region of each animal and was embedded in OCT compound (Sakura Finetek USA Inc., Torrance, CA) and frozen in liquid nitrogen.
  • the density of the endothelial population was determined by Sigma- Scan Pro morphometry software using the intensity threshold method.
  • the total endothelial area for each sample as well as for each specimen were obtained along with the relative percent endothelial area (endothelial area /area of the myocardium studied).
  • the total endothelial area was also calculated as the relative percent of the non-infarcted (viable) area of the myocardium studied.
  • the trichrome stained sections were digitized and the area occupied by the blue staining collagen as well as the total area of the section excluding the area occupied by the epicardium (which normally contained collagen) were measured using Sigma-Scan Pro.
  • the infarcted area was then calculated as the area occupied by the blue staining.
  • ABM indicates autologous bone marrow.
  • ABM indicates autologous bone marrow. Histopathology and Nascularity Assessment
  • Chronic myocardial ischemia was created in 16 pigs by the implantation of ameroid constrictors around the left circumflex coronary artery.
  • 8 animals underwent subcutaneous injection of GM-CSF for 3 consecutive days (dose 10 ⁇ g per day) followed (on the fourth day and exactly 4 weeks after ameroid implantation) by transendocardial injections of freshly aspirated ABM into the ischemic zone using a transendocardial injection catheter (2.4 ml per animal injected at 12 sites) and 8 control animals without GM-CSF stimulation were injected with heparinized saline.
  • Bone marrow ( ⁇ 5 ml) will be aspirated from the iliac crest at approximately 60 minutes prior to initiation of the cardiac procedure using preservative-free heparinized glass syringes (20 unit heparin/1 ml fresh BM). The aspirated bone marrow will be immediately macro-filtered using 300 ⁇ and 200 ⁇ stainless steel filters, sequentially. An experienced hematologist will perform the procedure under sterile conditions. The bone marrow smear will be evaluated to confirm a normal histomorphology of the bone marrow preparation.
  • any of several procedures for delivery of an agent to the myocardium can be used. These include direct transepicardial delivery, as could be achieved by a surgical approach (for example, but not limited to, a transthoracic incision or transthoracic insertion of a needle or other delivery device, or via thoracoscopy), or by any of several percutaneous procedures. Following is one example of percutaneous delivery. It should be emphasized that the following example is not meant to limit the options of delivery to the specific catheter-based platform system described in the example — any catheter-based platform system can be used.
  • an introducer sheath of at least SF is inserted in the right or left femoral artery.
  • heparin is administered and supplemented as needed to maintain an ACT for 200-250 seconds throughout the LN mapping and ABM transplantation portion of the procedure.
  • ACT will be checked during the procedure at intervals of no longer than 30 minutes, as well as at the end of the procedure to verify conformity with this requirement.
  • FIG. 1 Left ventriculography is performed in standard RAO and/or LAO views to assist with guidance of ⁇ OGA-STARTM and injection catheters, and an LN electromechanical map is obtained using the ⁇ OGA-STARTM catheter.
  • the 8F I ⁇ JECTIO ⁇ - STAR catheter is placed in a retrograde fashion via the femoral sheath to the aortic valve. After full tip deflection, the rounded distal tip is gently prolapsed across the aortic valve and straightened appropriately once within the LN cavity.
  • the catheter (incorporating an electromagnetic tip sensor) is oriented to one of the treatment zones (e.g., anterior, lateral, inferior-posterior or other).
  • the treatment zones e.g., anterior, lateral, inferior-posterior or other.
  • needle insertion and injection is allowed only when stability signals will demonstrate an LS value of ⁇ 3.
  • a single injection of 0.2 cc of freshly aspirated ABM will be delivered via trans-endocardial approach to the confines of up to two treatment zones with no closer than 5 mm between each injection site.
  • the density of injection sites will depend upon the individual subject's LN endomyocardial anatomy and the ability to achieve a stable position on the endocardial surface without catheter displacement or premature ventricular contractions (PNCs).
  • ABM may be an optimal source for cellular (an example would be endothelial progenitor cells, but the invention is not limited to such cells as many other cells in the bone marrow may contribute importantly to the angiogenic effect) and secreted, e.g., angiogenic growth factors, elements necessary to promote new blood vessel growth and restore function when transferred to another tissue, such as ischemic heart or peripheral limbs.
  • a patient's own bone marrow can be used as the key therapeutic source to induce therapeutic angiogenesis and/or myogenesis in ischemic tissues, e.g., heart muscle and/or ischemic limb, with compromised blood perfusion due to arterial obstructions.
  • the patient's own bone marrow is aspirated, i.e., ABM donation, processed as described herein, and injected directly into ischemia and/or adjacent non-ischemic tissue, e.g., heart muscle and/or ischemic limb, to promote blood vessel growth.
  • ABM donation i.e., ABM donation
  • non-ischemic tissue e.g., heart muscle and/or ischemic limb
  • the ABM and/or bone marrow products are injected into the heart muscle, e.g., the myocardium, by use of either a catheter-based trans-endocardial injection approach or a surgical (open chest or via thoracoscopy) trans-epicardial thoracotomy approach.
  • Those two delivery strategies can be used to achieve the same therapeutic goal by promoting the incorporation and integration of angiogenic bone marrow elements in the target organ tissue, e.g., heart muscle and/or ischemic limb.
  • ABM effective amounts of ABM, bone marrow cells or bone marrow cells transfected with an angiogenesis-promoting agent are administered for treatment.
  • the amount administered will depend upon many factors, including, but not limited to, the intended treatment, the severity of a condition being treated, the size and extent of an area to be treated, etc.
  • a representative protocol would be to administer quantities of from about 0.2 to about 0.5 ml of ABM in each of from about 12 to about 25 injections, for a total of from about 2.4 to about 6 ml of ABM being administered.
  • Each dose administered could preferably comprise from about 1 to about 2 percent by volume of heparin or another blood anticoagulant, such as coumadin.
  • the quantity of ABM present should be approximately the same in each dose and/or the total of the ABM administered should be about the same as described above. It is believed that the total number of cells of ABM administered in each treatment should be on the order of from about 10 7 to 5X10 8 .
  • optimization of angiogenic gene expression may be enhanced by co-administration of various angiogenic stimulants with the ABM.
  • ABM transplantation is injected either as a "stand alone” therapeutic agent or combined with any pharmacologic drug, protein or gene or any other compound or intervention that may enhance bone marrow production of angiogenic growth factors and/or promote endothelial cell proliferation, migration, and blood vessel tube formation.
  • the "combined" agent(s) can be administered directly into the patient or target tissue, or incubated ex-vivo with bone marrow prior to injection of bone marrow into the patient.
  • GM- CSF Granulocyte-Monocyte Colony Stimulatory Factor
  • MCP 1 Monocyte Chemoattractant Protein 1
  • EPASl Monocyte Chemoattractant Protein 1
  • HIF-1 Hypoxia Inducible Factor-1
  • the stimulation of the bone marrow could be by the direct exposure of the bone marrow to the factors in the form of proteins, or the bone marrow cells can be transfected with vectors carrying the relevant genes.
  • bone marrow can be transfected with a plasmid vector, or with an adenoviral vector, carrying the HIF-1 or EPASl transgenes.
  • an intervention that may enhance bone production of angiogenic factors is ex-vivo exposure of bone marrow cells to hypoxia.
  • This intervention can be used alone with bone marrow, or in combination with any of the factors outlined above.
  • These optimization strategies are designed to increase the production of vascular endothelial growth factor (NEGF) expression and/or other cytokines with angiogenic activity prior to the direct injection of the bone marrow into the heart or any peripheral ischemic tissue, hi a broad sense, the invention comprises intramyocardial injection of ABM with any agent that would become available to cause stimulation of bone marrow and/or ex-vivo or in vivo stimulation of any angiogenic growth factor production by the bone marrow or its stromal microenvironment.
  • NEGF vascular endothelial growth factor
  • patients with refractory coronary artery disease or ischemic peripheral vasculopathy will be candidates for a bone marrow aspiration procedure followed by ABM myocardial or limb transplantation directed into the ischemic tissue or its borderline zone and/or normal tissue that may serve as the source for collateral or cellular supply to the diseased tissue for the purposes of therapeutic angiogenesis and/or myogenesis.
  • patients with refractory coronary artery disease or ischemic peripheral vasculopathy will be candidates for a bone marrow aspiration procedure followed by ABM myocardial or limb transplantation directed into the ischemic tissue or its borderline zone and/or normal tissue that may serve as the source for collateral or cellular supply to the diseased tissue for the purposes of therapeutic angiogenesis and/or myogenesis.
  • This procedure will involve the use of a bone marrow aspiration procedure, bone marrow harvesting and processing, followed by the use of the ABM or its elements (growth factors and/or cellular elements being isolated from the patient's own bone marrow), with or without any ex-vivo stimulation of its delivery forms, to be injected into the ischemic or non ischemic myocardium and/or peripheral ischemic tissue (such as limb ischemia).
  • the bone marrow will be kept in standard anticoagulation/anti-aggregation solution (containing sodium citrate and EDTA) and kept in 4° C in sterile medium until the time of its use. [0089]
  • the bone marrow filtered to avoid injecting remaining blood clots or macroaggregates into the target tissue.
  • the bone marrow with or without a stimulatory agent in any of its delivery forms, or with or without having been transfected with a vector carrying a transgene that is designed to enhance the angiogenesis effect of the bone marrow, will be injected into the heart muscle, i.e., in therapeutic myocardial angiogenesis or therapeutic myogenesis, using either any catheter-based trans-endocardial injection device or via a surgical (open chest) trans-epicardial thoracotomy approach, or any other approach that allows for transepicardial delivery.
  • the bone marrow will be transferred by a direct injection of the bone marrow or it elements, with or without ex- vivo or in vivo stimulation in any of its delivery forms, into the muscles of the leg.
  • the volume of inj ection per treatment site will probably range between 0.1-5.0 cc per injection site, dependent upon the specific bone marrow product and severity of the ischemic condition and the site of injection.
  • the total number of injections will probably range between 1-50 injection sites per treatment session.
  • Bone marrow cells are harvested under sterile conditions from pigs in preservative free heparin (20 units/ml BM cells) and filtered sequentially using 300 ⁇ and 200 ⁇ stainless steel mesh filters. BMCs are then isolated by Ficoil-Hypaque gradient .centrifugation, seeded in T-75 flasks, and cultured overnight in long-term culture medium (LTCM) (Stem Cell Tech, Nancouver, British Columbia, Canada) at 33°C with 5% CO2 in T-75 culture flasks. The medium is then changed and the non-attaching cells washed out.
  • LTCM long-term culture medium
  • the attached cells are mostly monocytes, endothelial precursor cells, or other hemopoietic lineage cells. By lac-Z staining testing, these cells have been shown to be permissive for adenovirus by expression of the marker protein.
  • the seeding density of the BMCs in each culture dish is 7 x 106 /ml. When the cells become confluent at about 7 days, they are split 1 to 3 by 0.25% trypsin. Passages 3-8 were used for this study.
  • BMCs are first cultured in 6-cm Petri dishes for 3 to 14 days to allow for production of a lining of early attaching cells that adhere to the Petri dish.
  • the non- adherent cells are washed away the day after initial seeding.
  • the early attaching cells are inoculated with a vector encoding one or more cytokines, growth factors, or other mammalian angiogenesis promoting factors, such as, but not limited to, the transcription factors HJJ or HIF-2.
  • This inoculation can occur from 3 to 28 days after seeding. This virus is washed out from the transfected cells about 2 hours to 3 days after inoculation.
  • the transfected cells can then be injected into the patient's target tissue, such as the muscle of heart or leg.
  • target tissue such as the muscle of heart or leg.

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Abstract

L'invention concerne des procédés permettant de renforcer le rendement de transfection de cellules de moelle osseuse par transfection de cellules de fixation précoce dérivées de moelle osseuse en culture. L'invention concerne également l'utilisation de ces cellules dérivées de moelle osseuse autologue en vue d'administrer au patient des transgènes favorisant l'angiogenèse. Les cellules de fixation précoce tranfectées sont introduites dans un tissu ischémique, par exemple le coeur, afin de renforcer la formation de vaisseaux sanguins collatéraux. L'invention concerne en outre des procédés de traitement de muscle ischémique par co-administration de cellules de moelle osseuse et de RGTA, par exemple RGTA11.
PCT/US2003/015529 2002-05-30 2003-05-16 Injection intramyocardique de moelle osseuse autologue Ceased WO2003101201A1 (fr)

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EP3097922A1 (fr) 2015-05-28 2016-11-30 Denis Barritault Composition pour le traitement des lesions tissulaires
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007525214A (ja) * 2004-01-30 2007-09-06 ケムジェネックス・ファーマシューティカルズ・インコーポレイテッド Ｎ−アセチルトランスフェラーゼ遺伝子型解析によって投与するナフタルイミド
WO2011121036A2 (fr) 2010-03-30 2011-10-06 Vib Vzw Induction de l'artériogenèse à l'aide de facteurs spécifiques ou par thérapie cellulaire avec des cellules myéloïdes polarisées
CN107809999A (zh) * 2015-05-28 2018-03-16 生物组织再生和修复公司 用于治疗组织病变的组合物
EP3097922A1 (fr) 2015-05-28 2016-11-30 Denis Barritault Composition pour le traitement des lesions tissulaires
WO2016189087A1 (fr) * 2015-05-28 2016-12-01 Organes Tissus Regeneration Reparation Remplacement - Otr3 Composition pour le traitement des lesions cerebrales
WO2016189088A1 (fr) 2015-05-28 2016-12-01 Organes Tissus Regeneration Reparation Remplacement - Otr3 Composition pour le traitement des lesions tissulaires
EP3097928A1 (fr) * 2015-05-28 2016-11-30 Organes Tissus Régénération Réparation Remplacement Composition pour le traitement des lesions cerebrales
CN107864626A (zh) * 2015-05-28 2018-03-30 生物组织再生和修复公司 用于治疗脑损伤的组合物
EP3626256A1 (fr) 2015-05-28 2020-03-25 Denis Barritault Composition pour le traitement des lésions tissulaires
US11351190B2 (en) 2015-05-28 2022-06-07 Organes Tissus Regeneration Reparation Remplacement—Otr3 Composition for treating tissue lesions
US11701392B2 (en) 2018-04-05 2023-07-18 Medeor Therapeutics, Inc. Compositions for establishing mixed chimerism and methods of manufacture thereof
US11819521B2 (en) 2018-04-05 2023-11-21 Medeor Therapeutics, Inc. Cellular compositions derived from prior organ donors and methods of manufacture and use thereof
US11813376B2 (en) 2018-09-18 2023-11-14 Medeor Therapeutics, Inc. Cellular compositions derived from deceased donors to promote graft tolerance and manufacture and uses thereof

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