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WO2005104766A2 - Injection d'un milieu conditionne derive de la moelle osseuse pour favoriser l'angiogenese - Google Patents

Injection d'un milieu conditionne derive de la moelle osseuse pour favoriser l'angiogenese Download PDF

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WO2005104766A2
WO2005104766A2 PCT/US2005/014513 US2005014513W WO2005104766A2 WO 2005104766 A2 WO2005104766 A2 WO 2005104766A2 US 2005014513 W US2005014513 W US 2005014513W WO 2005104766 A2 WO2005104766 A2 WO 2005104766A2
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bone marrow
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WO2005104766A3 (fr
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Ran Kornowski
Shmuel Fuchs
Stephen Epstein
Martin B. Leon
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Myocardial Therapeutics Inc
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Myocardial Therapeutics Inc
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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    • C12N2510/00—Genetically modified cells

Definitions

  • This application is directed to methods for treating a site of damaged blood supply by injecting bone marrow and bone marrow cell products. More specifically, this invention is directed to intramyocardial injection of bone marrow derived conditioned medium produced by culturing bone marrow cells, to enhance collateral blood vessel formation (angiogenesis) and tissue perfusion.
  • the present invention is based on the premise that multiple complex processes, involving the differential expression of dozens if not hundreds of genes, are necessary for optimal collateral development. Based on this concept, it follows that optimal development of collateral blood vessels and tissue perfusion cannot be achieved by the administration of single genes whose encoded products are known to be related to angiogenesis nor, because of the complexity of the angiogenesis processes, by the administration of a combination ofangiogenesis-related genes.
  • This invention relies on the capacity of bone marrow cells to secrete the growth factors and cytokines involved in angiogenesis in a time and concentration-dependent coordinated and appropriate sequence.
  • VEGF vascular endothelial growth factor
  • FGF vascular endoietin-1
  • angiopoietin-1 vascular endothelial growth factor 1
  • VEGF vascular endothelial growth factor 1
  • FGF vascular endoietin 1
  • angiopoietin-1 vascular endothelial growth factor 1
  • complex interactions among several growth factor systems are probably necessary for the initiation and maintenance of new blood vessel formation. More specifically, it is believed important to induce a specific localized angiogenic milieu with various angiogenic cytokines interacting in concert and in a time-appropriate manner to initiate and maintain the formation and function of new blood vessels.
  • the invention provides methods for promoting collateral blood vessel formation and tissue perfusion in tissue by injecting conditioned medium of bone marrow early attaching cells into the tissue to promote formation of collateral blood vessels in the tissue.
  • the invention provides methods for enhancing collateral blood vessel formation in a patient in need thereof by growing bone marrow under suitable culture conditions for a period of time sufficient to promote production by the bone marrow of early attaching cells; transfecting at least a portion of the early attaching cells with a vector comprising a polynucleotide that encodes one or more agents selected from angiogenic cytokines, growth factors and mammalian angiogenesis- promoting factors for expression by the early attaching cells, and culturing the transfected early attaching cells in a culture medium and for a time suitable to allow expression by the cells of the one or more agents, thereby producing conditioned medium.
  • An effective amount of the transfected early attaching cells and/or the conditioned medium is then directly administered to a desired site in the patient, thereby enhancing collateral blood vessel formation at the site in the patient.
  • the invention provides a therapeutic composition
  • a therapeutic composition comprising early attaching cells derived from bone marrow, which cells have been transfected with a vector comprising a polynucleotide that encodes one or more agents selected from angiogenic cytokines, growth factors, and angiogenesis-promoting factors.
  • the therapeutic composition can further comprise a conditioned medium in which the transfected cells have been grown in culture for a time sufficient to allow expression of one or more of the transgenic agents as well as other agents normally produced by such cells in culture.
  • 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.
  • Fig. 5A is a graph showing in- vitro production of NEGF, MCP-1 and bFGF by CD34+ cells and bone marrow-derived stromal cells from mice.
  • Fig. 5B is a graph showing % increase of human umbilical vein endothelial cells caused by MSC conditioned media as a function of conditioned media (CM) concentration.
  • CM conditioned media
  • Fig. 6 is a graph showing the effect of bone marrow-derived stromal cells on development of collateral flow when injected into adductor muscles of ischemic hind limb of mice as determined by Laser/Doppler perfusion imaging. Flow is expressed as the ratio of flow in the ischemic limb to flow in the normal hind limb.
  • MSC marrow- derived stromal cells
  • Media non-conditioned media
  • MAEC mouse aortic endothelial cells.
  • Fig. 7 is a graph showing the effect on release of NEGF and bFGF in vitro from mouse marrow-derived stromal cells (MSCs) transfected with an adenovirus encoding HIF-1I-VP16.
  • Fig. 9 is a graph showing VEGF values for human coronary artery patients ( ⁇ SD) under baseline conditions, following hypoxia exposure, and following Ad.HIF- . l ⁇ NP16 transduction. * p ⁇ 0.05 vs normoxia;
  • 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 delivery of autologous bone marrow(ABM) or bone marrow cells derived therefrom, or media derived from these cells while the cells are grown in culture, 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, or media derived from these cells while the cells are grown in culture 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 or bone marrow cells into the patient.
  • bone marrow cells means any cells that are produced by culturing of aspirated bone marrow under cell growth conditions.
  • Non-limiting examples of these "combined" angiogenic agents are Granulocyte-Monocyte Colony Stimulatory Factor (GM-CSF), Monocyte Chemoattractant Protein 1 (MCP-1), and Hypoxia Inducible Factor-1 (HIF-1).
  • GM-CSF Granulocyte-Monocyte Colony Stimulatory Factor
  • MCP-1 Monocyte Chemoattractant Protein 1
  • HIF-1 Hypoxia Inducible Factor-1
  • Bone marrow is also 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-1 alpha and IL-1 beta, 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-derived growth factor A
  • 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 Natl Acad 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., DevDyn 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
  • NEGF vascular endothelial growth factor
  • the media in which such bone marrow cells are cultured contains such a mixture of interactive growth factor proteins that produce therapeutic angiogenesis and/or myogenesis.
  • the therapeutic effects can be produced by culturing non-autologous bone marrow cells for a time suitable to allow production by the bone marrow cells of the interactive growth factor proteins and delivering the "conditioned" media to a region of ischemic tissue to produce the therapeutic angiogenesis and/or myogenesis.
  • transfected early attaching bone marrow cells such bone marrow cells and conditioned medium obtained by growing the transfected bone marrow cells in culture, or the conditioned medium alone is injected into tissue associated with ischemia or delivered by injection into the blood stream, such as an artery supplying an ischemic tissue, or any other artery or vein
  • the resulting angiogenesis is greater than is achieved by injection of transfected bone marrow stem cells alone.
  • 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 2 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 VEGF.
  • HIF-1 is a heterodimer with a basic helix-loop-helix motif, consisting of the subunits HIF-l ⁇ 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 the presence of functionally relevant protein levels.
  • HIF-1 is a transcription factor that plays a critical role in the transcriptional activation of hypoxia inducible genes. It functions as a heterodimer composed of HIF-1 ⁇ and HIF-l ⁇ subunits. HIF-1 activity is controlled by the stability of the HIF-l ⁇ subunit. Thus, HIF-1 ⁇ is ubiquitinated under normoxic conditions, which targets the molecule for proteasomal degradation. Hypoxia leads to decreased ubiquitination, and therefore greater protein stability. This enhances heterodimer formation and therefore increases HIF-1 activity.
  • the fact that the functional activity of HIF-1 is tightly and inversely coupled to oxygen levels indicates its critical role as a molecular sensor of oxygen, and thereby in modulating the adaptive responses of cells to hypoxia.
  • HIF-1 the transcriptional activity of HIF-1 derives from the capacity of the heterodimer, which as noted forms only under hypoxic conditions, to bind to a specific DNA hypoxia- responsive recognition element (HRE) present in the promoter of many genes involved in the response of the cell to hypoxia, including VEGF, VEGFR1, VEGFR2, Ang-2, Tie-1, and nitric oxide synthase.
  • HRE DNA hypoxia- responsive recognition element
  • HIF-1 ⁇ Because of the lability of HIF-1 ⁇ in the absence of hypoxia, to assure its constitutive activity even under normoxic conditions, a chimeric construct of the HIF-l ⁇ gene has been constructed, consisting of the DNA-binding and dimerization domains from HIF-l ⁇ and the transactivation domain from herpes simplex virus VP16 protein as described in Example 8 below.
  • the VP 16 domain abolishes the ubiquitination site in HIF- l ⁇ , and therefore eliminates the proteasomal-mediated degradation of the protein.
  • the resulting stable levels of HIF-l ⁇ lead to constitutive transactivation of the genes targeted by HIF-1.
  • 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.
  • a similar approach involves the exposure of autologous bone marrow to endothelial PAS domain protein 1 (EPAS1).
  • EPAS1 shares high structural and functional homology with HIF-1 and is also known as HIF-2.
  • 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 VEGF 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 or media derived from these cells while the cells grow in culture, 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
  • monocyte-derived cytokines are activated during collateral growth in vivo, and monocyte chemotactic protein- 1 (MCP-1) is upregulated by shear stress in vitro. It has been shown that 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 hind limb 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., J Clin 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 or media derived from these cells while the cells grow in culture, 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 adeno viral vector, carrying the MCP-1 transgene.
  • 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.
  • agents such as HIF-1 , EPAS 1 , hypoxia, or MCP-1.
  • bone marrow cells that are injected into regions in which collateral blood vessel development is desired in order to enhance the delivery of blood to ischemic regions may not produce optimal angiogenic effects when certain "at risk" conditions prevail. For example, there is evidence demonstrating that angiogenesis is impaired in the presence of hypercholesterolemia, and it is also compromised with aging.
  • Hypercholesterolemia is a dominantly inherited genetic condition that results in markedly elevated low-density lipoprotein cholesterol levels beginning at birth, and resulting in myocardial infarctions at an early age. "Aging" as the term is used herein is not necessarily measured in years, but is measured in terms of deterioration of the body's ability to maintain the vascular system in a healthy condition. Nevertheless, the ability of the body to maintain vasular health tends to deteriorate withtime (i.e., with age) as well.
  • the present invention recognizes the confounding effects of these and other "risk factors", and describes throughout this application methods that are designed to enhance the angiogenic potential of such functionally compromised bone marrow cells by transducing these cells with polynucleotides encoding proteins that will enhance the capacity of such impaired bone-marrow cells to foster development of collateral blood vessels.
  • Example 9 and Fig. 9 describe the enhanced production in vitro of recombinant VEGF by MSCs transfected with an adenoviral vector encoding HIF-l ⁇ NP16 obtained from coronary artery disease patients.
  • the invention provides methods for transforming bone marrow cells with a gene encoding one or species of nitric oxide synthase ( ⁇ OS).
  • ⁇ OS nitric oxide synthase
  • ⁇ OS gene or a "polynucleotide encoding ⁇ OS” as the term is used herein means any of the known isoforms of ⁇ OS, including inducing ⁇ OS (i ⁇ OS) and endothelial ⁇ OS (e ⁇ OS), as well as ⁇ OS genes that have been mutated such that the magnitude of their expression is altered, or that they encode an altered protein, either of which results in a more potent angiogenic effect.
  • i ⁇ OS inducing ⁇ OS
  • e ⁇ OS endothelial ⁇ OS
  • VEGF vascular endothelial growth factor
  • NO nitric oxide
  • transfecting bone marrow cells with NOS augments the intrinsic capacity of bone marrow cells to secrete multiple angiogenic cytokines and growth factors and also stimulates expression of multiple angiogenesis-related genes.
  • the invention also provides such NOS-transfected bone marrow cells, especially ABM cells, or media derived from these cells while the cells grow in culture.
  • FGF fibroblast growth factor
  • the invention provides a method for using bone marrow cells transfected with a polynucleotide encoding one of the FGF family of peptides to enhance the capacity of bone marrow cells to increase development of collateral blood vessel development, such as bone marrow cells that may have an impaired capacity to enhance angiogenesis because of diverse risk factors, including but not limited to hypercholesterolemia and aging.
  • the invention also provides such FGF- transfected bone marrow cells, especially ABM cells, or media derived from these cells while the cells grow in culture.
  • PR39 another gene expressed by monocytes/macrophages, is another gene that this invention describes as being able to enhance the angiogenic potential of bone marrow cells to improve collateral formation.
  • the rationale for transducing bone marrow cells with the gene encoding this protein derives from the fact that PR39 inhibits the proteasomal degradation of HIF-l ⁇ , resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice.
  • HIF-1 ⁇ By increasing the steady state levels of HIF-1 ⁇ , the heterodimer— HIF-1 ⁇ /HIF-1 ⁇ — forms, which is a transcription factor that induces the expression of HIF-1 -related genes.
  • the protein products of many of these genes promote the development of angiogenesis.
  • the invention also provides such PR39-transfected bone marrow cells, especially ABM cells, or media derived from these cells while the cells are grown in culture.
  • 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 EPAS1 transgene, or a transgene encoding PR39, or a member of the NOS or FGF families, for expression thereof in the cells and/or in the subject when the transfected cells are injected into a treatment site as described herein, or media derived from these cells while the cells are grown in culture is injected into a treatment site.
  • 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 EPAS1 transgene, or a transgene encoding PR39, or a member of the NOS or FGF families, for expression thereof in the cells and/or
  • 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 several hours, and the inoculated cells begin to produce the transgene products after about 12 hours to 3 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 cells 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 or plasmid
  • 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 culture 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 early attaching cells include, but without limitation thereto, those encoding such angiogenesis-promoting agents as HIF-1, EPAS1 (also known as HIF-2), MCP-1, CM-CSF, NOS, FGF, 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 cells or media derived from such cells can also be injected into the vascular system so that they are delivered to the desired site by the blood.
  • 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 (HIV), 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; Verma 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 (HIV)
  • adenovirus vectors such as those based on the human immunodeficiency virus (HIV)
  • adeno-associated virus vectors such as those based
  • 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 Adeno viruses, " Journal of Cellular Biochemistry, Supplement 17D, p. 195, Abstract PI 01 (Mar. 1993); Barr et al., "Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus," Ge/7e Therapy, vol. 1 :51-58 (1994)).
  • the gene of interest may be 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/El B), 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 D ⁇ A), 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.
  • marrow-derived stromal cells means CD34 minus/ CD45 minus early attaching cells that can be obtained from a sample of bone marrow.
  • 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 an RNA polymerase that specifically recognizes, binds to and transcribes the DNA initiates transcription of such DNA from the promoter.
  • 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, 277:108-1 14); 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 naked plasmid, 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. ofNeurol. 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.
  • 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
  • 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, Journal of Virology, 61 (4): 1120-1124, 1988; Watanabe, et al, Molecular and Cellular Biology, 3(12 :2241 -2249, 1983; Danos, et al, PNAS, 85:6460- 6464, 1988; and Bosselman, et al, Molecular and Cellular Biology, 7(5): 1797- 1806, 1987, which disclose procedures for producing viral vectors and helper cells that minimize the chances for producing a viral vector that includes a replicating virus.
  • retroviral virions suitable for prepackaging with polynucleotides that encode therapeutic proteins, such as angiogenic growth factors, are produced employing well-known methods for producing retroviral virions. See, for example, U.S. 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 VEGF, 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. VEGF and MCP-1 levels in the conditioned medium were assayed using ELISA. During the four weeks in culture, BM cells secreted VEGF 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 VEGF 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, Vancouver, 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 10 6 /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, VEGF, 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 VIII. 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 DNA 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).
  • the concentration of VEGF in conditioned medium was measured using a sandwich ELISA kit (Chemicon International Inc., Temecula, CA). Briefly, a plate pre- coated with anti-human VEGF antibody was used to bind VEGF in the conditioned medium or to a known concentration of recombinant VEGF. The complex was detected by the biotinylated anti-VEGF antibody, which binds to the captured VEGF. The biotinylated VEGF antibody in turn was detected by streptavidin-alkaline phosphatase and color generating solution. The anti-human VEGF antibody cross-reacts with porcine VEGF.
  • the concentration of MCP-1 in conditioned medium was assayed by sandwich enzyme immunoassay kit (R &D Systems, Minneapolis, MN): 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.
  • VEGF 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).
  • VEGF and MCP-1 levels in a control culture medium, not exposed to BM were 0 and 1 1 ⁇ 2 pg/ml, respectively, as shown in Fig. 4.
  • hypoxia markedly increases the expression of VEGF 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. When cells became confluent at about 7 days, they were split 1 :3 by trypsinization.
  • the BMCs were either exposed to hypoxic conditions (placed in a chamber containing 1% oxygen) for 24 to 120 hrs, or maintained under normal conditions.
  • the resulting conditioned medium was collected and VEGF, MCP-1 were analyzed by ELISA.
  • 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.
  • animals Under baseline and 4 weeks later, animals underwent rest and pacing echocardiogram to assess regional contractility (% myocardial thickening), and microsphere study to assess collateral-dependent perfusion at rest and during adenosine infusion.
  • 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.
  • MSCs injected into the region of developing collaterals express collateral-enhancing- related cytokines in vivo and incorporate into developing collaterals.
  • 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 UV 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.
  • other parameters of vascularity including % area occupied by any blood vessel and number of blood vessels > 50 ⁇ m were similar in the ischemic and non- ischemic territories in both groups.
  • 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-STAR3 and injection catheters, and an LV electromechanical map is obtained using the NOGA-STAR3 catheter.
  • the 8F INJECTION- 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 LV 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 LV endomyocardial anatomy and the ability to achieve a stable position on the endocardial surface without catheter displacement or premature ventricular contractions (PVCs).
  • ABM may be an optimal source for cellular (an example would be mononuclear stromal 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 muscle, to promote blood vessel growth.
  • 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.
  • angiogenesis-promoting agent is 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 maybe 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
  • EPAS1 Hypoxia Inducible Factor-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 EPAS1 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 (VEGF) 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.
  • 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.
  • patients with refractory coronary artery disease or ischemic peripheral vasculopathy are 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 are 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 involves 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 muscle tissue (such as limb muscle 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.
  • the bone marrow is filtered to avoid injecting remaining blood clots or macro aggregates 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, is 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 cells i.e., early attaching cells
  • the bone marrow cells are 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 injection per treatment site can range between 0.1 -5.0 cc per injection site, dependent upon the specific bone marrow product, severity of the ischemic condition and the site of injection.
  • the total number of injections can range between 1-50 injection sites per treatment session.
  • Bone marrow cells were 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 were then isolated by Ficoll-Hypaque gradient centrifugation, seeded in T-75 flasks, and cultured overnight in long-term culture medium (LTCM) (Stem Cell Tech, Vancouver, British Columbia, Canada) at 33°C with 5% CO2 in T-75 culture flasks. The medium was then changed and the non-attaching cells washed out.
  • LTCM long-term culture medium
  • the attached cells were mostly mononuclear cells, endothelial precursor cells, or other hemopoietic lineage cells.
  • mononuclear cells in early attaching cells are marrow-derived stromal 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.
  • 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 were 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 were 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 HIP-1 or HIF-2. This inoculation can occur from 3 to 28 days after seeding, for example 3 to 12 days or 3 to 8 days.
  • the virus was washed out from the transfected cells about 2 hours to 3 days after inoculation.
  • the transfected cells were then injected into the patient's target tissue, such as the muscle of heart or leg.
  • MSCs have the capacity to secrete biologically active collateral-enhancing factors in vitro.
  • This isolation procedure involves negatively selecting cells not expressing cell markers CD34 and CD45 by using magnetic beads labeled with commercially available antibodies to these markers MSCs were purified from the heterogeneous cultured cells.
  • the CD34 minus-/CD45 minus- fraction was isolated by labeling with FITC-labeled anti-CD34 antibody (Pharmingen, San Diego, CA) followed by simultaneous incubation with anti-FITC and anti-CD45 magnetic beads (Miltenyi Biotech, Sunnyvale, CA). Cells were passed through a magnetic column and the double-negative fraction collected. Subsequently, the bead-negative and bead- positive populations were separately cultured.
  • the bead-negative population demonstrated typical fibroblastic morphology of the MSCs, while the bead-positive population appeared to mainly consist of small, spherical cells consistent with lymphohematopoietic cells (Figs. 5A and 5B).
  • FACS analysis was performed and demonstrated that cells did not express the surface makers CD31, CD34, CD45, and CD117 typical of lymphohematopoietic cells, but did express high levels of CD44 (95 ⁇ 0.6%), CD90 (99J ⁇ 0J%), and CD105 (89 ⁇ 2J%) typical of marrow derived-stromal cells.
  • CD34 minus/CD45 minus cells are also referred to herein as "marrow- derived stromal cells", or "MSCs").
  • the isolated MSCs were replated, and the conditioned media subsequently collected for 24 hours.
  • Conditioned media prepared as above was analyzed for the presence of angiogenic cytokines by ELISA. Cytokine levels were corrected for total cell culture protein. The data reflect at least 3 different cell populations, with each population containing cells pooled from 2 mice. The results show (Fig. 5) that MSCs express such known collateral-enhancing factors as VEGF, MCP-1, and bFGF (also, angiopoietin-1 and P1DGF (not shown)). In contrast, CD34+ cells (progenitor endothelial cells) do not express these factors.
  • MSC-conditioned media prepared as above was collected and found to indeed increase the proliferation of cultured human umbilical vein endothelial cells.
  • MAECs or SMC's (1 x 10 4 /well) were plated in 24- well plates in MEM with 0.1% fetal calf serum for 24- hours. The media was then replaced with varying dilutions of MSC CM or control wells of DM- 10 only. Cultures were continued for 72-hours, after which the cells were recovered and counted using a Coulter counter. Data is reported as the mean % change in proliferation when compared with control.
  • VEGF endothelial cells were cultured in non-conditioned medium containing recombinant VEGF protein in concentrations found in conditioned media (using the above ELISA data).
  • 10 ⁇ g of VEGF blocking antibody was added to block the effect of VEGF in the conditioned media.
  • the results shown in Fig. 5B show that bone marrow-derived progenitor cells, such as MSCs secrete factors that increase endothelial cell proliferation.
  • VEGF is one of the factors that contribute to this effect as is shown by blocking antibody to VEGF reducing cell proliferation compared to conditioned medium alone.
  • VEGF endothelial growth factor
  • MSCs increase collateral flow in the mouse ischemic hind limb.
  • mice Twelve week-old Balb/C male mice underwent right distal femoral artery ligation using a method known in the art. Twenty-four hours later, mice were randomized to 3 groups— one group received lxlO 6 MSCs prepared as above described from syngenic mice, one group received lxlO 6 mature endothelial cells isolated from syngenic mice, and one group received non-conditioned media injected into the adductor muscles of the ischemic hind limb. Laser Doppler perfusion imaging (LDP I) was utilized to follow ischemic hind limb flow recovery over the ensuing 28 days (Fig. 6).
  • LDP I Laser Doppler perfusion imaging
  • MSCs injected into the region of developing collaterals express collateral-enhancing- related cytokines in vivo and incorporate into developing collaterals.
  • MSCs were incubated ex-vivo with carboxyfluorescein diacetate succinimidyl esters (CFSD) to stain cytoplasm green and injected into adductor tissue of the ischemic hind limb of mice as described. Mice were dilled a 1 7 days and adductor muscles snap frozen. Eight-micron sections were taken and stained for VEGF using HRP-conjugated anti-VEGF antibodies. Micrographs of the stained sections showed CFSE positive MSCs surrounded by VEGF staining, reflecting secreted VEGF. Thus, the injected MSCs express VEGF in vivo after injection.
  • CFSD carboxyfluorescein diacetate succinimidyl esters
  • MSCs As an initial step to determine whether MSCs provide an appropriate target for genetic alteration, the viability of MSCs in-situ following ex-vivo transduction with an adenoviral vector was examined. To this purpose, two separate experiments were performed, one utilizing an adenovirus comprising a gene encoding for Green Fluorescent Protein (GFP) and one comprising a gene encoding ⁇ -galactosidase. MSCs prepared as above were transduced ex-vivo. Preliminary studies determined that over 90% of MSCs were successfully transduced with an adenovirus containing a reporter transgene at an MOI of 150 (data not shown). To track protein expression, cells were incubated with Ad.GFP or Ad.
  • Ad Ad.GFP or Ad.
  • ⁇ -galactosidase at an MOI of 150 for 2-hours, rinsed three times, recovered and immediately injected into the adductor muscle (24-hours post-surgery).
  • GFP+/MSCs To follow the fate of injected GFP+/MSCs, multiple sections of adductor and calf muscle were examined using a Nikon inverted fluorescent microscope.
  • ⁇ - gal+/MSCs sections were developed with a commercially available X-gal kit (Invitrogen) and immediately injected into the adductor muscle of mice that had undergone femoral artery ligation 24-hours previously. Mice were sacrificed at day-3, day-7 and day- 14. Adductor muscle sections were subsequently either examined under a fluorescent microscope or stained with X-gal depending on the appropriate protocol as known in the art.
  • HIF-l ⁇ NP16 transfection of MSCs in vitro leads to an increase in collateral- enhancing-related factors greater than those induced by hypoxia.
  • Murine MSCs were isolated and plated as described above. Three groups of MSCs were compared. Group l ⁇ MSCs cultured under normoxic conditions; Group 2— MSCs cultured in 1% O 2 ; Group 3 ⁇ MSCs transfected with an adenovirus encoding HIF- l ⁇ /NP16 prepared as described above. MSCs were incubated with the virus at a multiplicity, of infection of 200 for 2 hr, followed by 48 hr of culture to allow time for gene expression.
  • the culture-conditioned media was subsequently collected for 24 hr from all 3 groups of cells. Using commercially available ELISA kits, media was analyzed for the presence of angiogenic cytokines VEGF and b-FGF. Cytokine levels were corrected for total cell culture protein. The results shown in Fig. 7 demonstrate that HIF-l ⁇ NP16 transfection increases expression and secretion by MSCs of both VEGF and b-FGF to levels substantially greater than those achieved by hypoxia.
  • MSC conditioned medium (MSC conditioned medium, or MSC CM ) was also added to cultures of endothelial cells (EC) and smooth muscle cells (SMC) to assess the effect of MSC CM on cell proliferation.
  • MSC conditioned medium or MSC CM
  • EC endothelial cells
  • SMC smooth muscle cells
  • Smooth muscle cells were isolated using a modification of a previously described protocol. 8 Briefly, after collecting MAECs as above, collagenase in Hanks Balanced Salt Solution (lmg/ml) was added and incubated in 37°C for up to 3 hours with gentle agitation every 15-30 min. Floating cells were again harvested, washed and re-suspended in Medium 199 supplemented with 10% FBS. Cells stained uniformly for smooth-muscle actin. Passages 3-8 for both cells were used for the purposes of the study.
  • MSC CM from HIF-1 ⁇ NP16-transduced MSCs increased EC proliferation (290% vs. 31% vs. 79% compared to proliferation in control media, p ⁇ 0.001) and SMC proliferation (220% vs. 26% vs. 58%, ⁇ .001).
  • Conditioned media from non-transfected MSCs and MSCs transfected with HIF-l ⁇ /VP16 vector were collected as described above. Human umbilical vein endothelial cells were cultured in varying dilutions of the conditioned media for 72 hr. Cells were then trypsinized and counted. Conditioned medium derived from the MSCs transfected with HIP-l ⁇ /VP16 vector markedly increased HUVEC proliferation. The increase was even greater than that observed following the addition to endothelial cells of conditioned medium from non-transfected cells subjected to 24 hr of hypoxia.
  • HIF-l ⁇ /NP16 transfection of MSCs leads to an increase in collateral flow.
  • this experiment shows that transfection with HIF-l ⁇ /VP16 in an adenoviral vector significantly and markedly enhances the in-vitro angiogenic effects of MSCs. More importantly, in vivo studies indicate that this strategy results in an increase in the collateral-improving effects over that achieved by injection of MSCs alone. These studies indicate that transduction of MSCs with HIF-l ⁇ , (and also with genes encoding other angiogenic-related cytokines, such as the FGF family of proteins, and NOS) will optimize the collateral-enhancing effects of a cell-based strategy for increasing collateral flow in ischemic tissue.
  • VEGF secretion observed in the MSCs of coronary artery disease patient are substantially increased by Ad.HIF-l ⁇ NP16 transduction.
  • This example demonstrates that production of angiogenic cytokines by bone marrow-derived progenitor cells can be substantially increased by transduction with a viral vector encoding recombinant HIF-1 ⁇ /VP 16.
  • a viral vector encoding recombinant HIF-1 ⁇ /VP 16 Freshly aspirated and filtered BM cells of human coronary artery disease patients were used to obtain hMSCs as described above. The hMSCs were transfected with vector HIF-l ⁇ /VP16 and cultured to obtain conditioned medium as above. Levels of VEGF in the conditioned medium of transfected hMSCs were measured by ELISA and compared with those of untransfected hMSCs under normoxic and hypoxic conditions (Fig. 9).

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Abstract

La présente invention se rapporte à des procédés permettant de favoriser la formation d'un apport de sang collatéral au niveau d'un site ischémique dans un tissu, par culture de cellules à fixation précoce dérivées de la croissance de moelle osseuse obtenue par aspiration in vitro, collecte des cellules à fixation précoce produites par la culture de moelle osseuse et injection du milieu conditionné produit par la culture de ces cellules à fixation précoce dans un site ischémique du coeur ou d'un membre. Les cellules à fixation précoce préférées pouvant être utilisées dans les procédés de cette invention sont des cellules stromales dérivées de la moelle. Toute moelle osseuse d'un donneur peut être utilisée pour la préparation du milieu conditionné. Eventuellement, les cellules à fixation précoce peuvent être transfectées au moyen d'un transgène favorisant l'angiogenèse et codant le facteur 1 alpha induisant l'hypoxie, un facteur de croissance des fibroblastes et/ou une monoxyde d'azote-synthase. L'invention se rapporte également à des milieux conditionnés contenant des cytokines angiogéniques produites à partir de ces cellules et destinées à être injectées dans un tissu, par exemple dans le coeur ou un muscle de membre périphérique, nécessitant la formation d'un apport de sang collatéral.
PCT/US2005/014513 2004-04-28 2005-04-27 Injection d'un milieu conditionne derive de la moelle osseuse pour favoriser l'angiogenese Ceased WO2005104766A2 (fr)

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US9512403B2 (en) 2008-01-30 2016-12-06 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions for methods of treating tissue injury
US8257947B2 (en) 2008-01-30 2012-09-04 Naughton Gail K Extracellular matrix compositions produced under hypoxic culture conditions
US11274276B2 (en) 2008-01-30 2022-03-15 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions
US10538736B2 (en) 2008-01-30 2020-01-21 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions
US8524494B2 (en) 2008-01-30 2013-09-03 Histogen, Inc. Low oxygen tension and BFGF generates a multipotent stem cell from a fibroblast in vitro
US8530415B2 (en) 2008-01-30 2013-09-10 Histogen, Inc. Repair and/or regeneration of cells with a composition produced by culturing fibroblast cells under hypoxic conditions
US8535913B2 (en) 2008-01-30 2013-09-17 Histogen, Inc. Soluble composition for promoting hair growth produced by hypoxic culture of fibroblasts cells
AU2009209022B2 (en) * 2008-01-30 2014-06-12 Histogen, Inc. Extracellular matrix compositions
EP2262891A4 (fr) * 2008-01-30 2011-04-20 Histogen Inc Compositions de matrice extracellulaires
CN102497871A (zh) * 2008-11-14 2012-06-13 希斯托金公司 用于癌症治疗的胞外基质组合物
AU2010271212B2 (en) * 2009-07-10 2015-07-16 Histogen, Inc. Conditioned medium and extracellular matrix compositions from cells cultured under hypoxic conditions
CN103194426B (zh) * 2012-01-05 2016-03-16 台北荣民总医院 细胞移植物的制备
EP3219793A1 (fr) * 2012-01-05 2017-09-20 Taipei Veterans General Hospital Préparation de transplant cellulaire
CN103194426A (zh) * 2012-01-05 2013-07-10 台北荣民总医院 细胞移植物的制备
EP2612907A1 (fr) * 2012-01-05 2013-07-10 Taipei Veterans General Hospital Préparation de transplant cellulaire

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