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  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Definitions

• the present invention relates to methods of ex-vivo expansion (self-renewal) of hematopoietic stem cells present in the hematopoietic mononuclear cells fraction of a blood sample and to expanded (self-renewed) populations of hematopoietic stem cells obtained thereby.
• the present invention further relates to therapeutic applications in which these methods and/or the expanded hematopoietic stem cell populations obtained thereby are utilized.
• Differentiation in the hematopoietic system involves, among other changes, altered expression of surface antigens (Sieff C, Bicknell D, Caine G, Robinson J, Lam G, Greaves MF (1982) Changes in cell surface antigen expression during hematopoietic differentiation. Blood 60:703).
• most of the hematopoietic pluripotent stem cells and the lineage committed progenitor cells are CD34+.
• the majority of cells are CD34+CD38+, with a minority of cells ( ⁇ 10 %) being CD34+CD38-.
• the CD34+CD38- phenotype appears to identify the most immature hematopoietic cells, which are capable of self-renewal and multilineage differentiation.
• the CD34+CD38- cell fraction contains more long-term culture initiating cells (LTC-IC) pre-CFU and exhibits longer maintenance of their phenotype and delayed proliferative response to cytokines as compared with CD34+CD38+ cells.
• CD34+CD38- cells can give rise to lymphoid and myeloid cells in vitro and have an enhanced capacity to repopulate SCID mice (Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE (1997) Purification of primitive human hematopoietic cells capable of repopulating immune- deficient mice. Proc Natl Acad Sci USA 94:5320).
• CD34+CD38- cells infused correlates positively with the speed of hematopoietic recovery.
• CD34+CD38- cells have been shown to have detectable levels of telomerase.
• such agents preferably include transition metal chelators that are capable of binding copper, such as, for example, linear polyamines (e.g., tetraethylenepentamine, TEPA).
• TEPA tetraethylenepentamine
• stem cells cannot be expanded unless first substantially enriched or isolated to homogeneity and therefore the presently known methods of ex-vivo expanding stem cell populations are limited by the laborious and costly process of stem cells enrichment prior to initiation of cultures.
• the present invention discloses the use of various agents in expanding hematopoietic stem cells present in the hematopoietic mononuclear cells fraction of a blood sample, without the use of a prior stem cells enrichment procedure, to expanded
• the method comprises providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation ofthe hematopoietic stem cells ex-vivo.
• the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation ofthe stem cells ex-vivo.
• the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, retinoid-X receptor and/or Vitamin D receptor, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation ofthe hematopoietic stem cells ex-vivo.
• the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation ofthe hematopoietic stem cells ex-vivo.
• the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, with a PI 3-kinase inhibitor, thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• the method comprises providing the hematopoietic mononuclear cells with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, with one or more copper chelator(s) or copper chelate(s), thereby expanding the population of the hematopoietic stem cells while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• ex-vivo expanded populations of hematopoietic stem cells obtained by the methods described hereinabove.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex- vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to responding to signaling pathways involving the retinoic acid receptor, the retinoid X receptor and/or the Vitamin D receptor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to responding to signaling pathways involving PI-3 kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a PI 3-kinase inhibitor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing the expression and/or activity of PI 3- kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex- vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more copper chelator(s) or chelate(s), thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) transplanting or implanting the hematopoietic stem cells to a recipient.
• the donor and the recipient in the methods above can be a single individual or different individuals, for example, allogeneic or xenogeneic individuals.
• transplantable hematopoietic cell preparations there are provided transplantable hematopoietic cell preparations.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent for reducing an expression and/or activity of CD38, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent for reducing an expression and/or activity of PI 3-kinase, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent, the agent reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent, the agent reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid receptor, retinoid X receptor and or Vitamin D receptor signaling, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent, the agent reducing a capacity of the hematopoietic mononuclear cells in responding to PI 3-kinase signaling, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative and a nicotinamide or a nicotinamide analog metabolite, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of a PI 3-kinase inhibitor, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a transplantable hematopoietic cell preparation of the present invention comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of one or more copper chelator(s) or copper chelate(s), while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• a method of adoptive immunotherapy is provided.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding a population of the stem cells, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor and/or the retinoid X receptor and/or the Vitamin D receptor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a PI 3-kinase inhibitor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more copper chelator(s) or chelate(s), thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells; and (c) transplanting said hematopoietic stem cells to the recipient.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of PI 3-kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor and/or the retinoid X receptor and/or the Vitamin D receptor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a PI 3-kinase inhibitor, thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• the method comprises (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more copper chelator(s) or chelate(s), thereby expanding a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo; and (c) genetically modifying said hematopoietic stem cells with the exogene.
• genetically modifying the cells is effected by a vector which comprises the exogene, which vector is, for example, a viral vector or a nucleic acid vector.
• a hematopoietic stem cells collection/culturing bag supplemented with an effective amount of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and/or a Vitamin D receptor antagonist, with an effective amount of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative and a nicotinamide or a nicotinamide analog metabolite, with an effective amount of a PI 3- kinase inhibitor, or with an effective amount of a copper chelator or chelate, each of which substantially inhibits cell differentiation of a hematopoietic stem cells fraction of hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells.
• an assay of determining whether an agent/molecule is an effective hematopoietic stem cell expansion agent comprises culturing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells in the presence of tested agent/molecule and monitoring expansion of the hematopoietic stem cells, wherein if increased expansion and decreased differentiation of the hematopoietic stem cells occurs, as compared to non-treated hematopoietic mononuclear cells, the tested agent/molecule is an effective hematopoietic stem cell expansion agent.
• the agent/molecule can be a retinoic acid receptor antagonist, a retinoid X receptor antagonist, a Vitamin D receptor antagonist, nicotinamide and an analog, a derivative and a metabolite thereof, a PI 3-kinase inhibitor, a copper chelator and a copper chelate.
• reducing the expression and/or activity of CD38 is effected by an agent that downregulates CD38 expression.
• the agent that downregulates CD38 expression is selected from the group consisting of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and a Vitamin D receptor antagonist.
• this agent is an antagonist for reducing a capacity of the stem cells in responding to retinoic acid, retinoid and/or Vitamin D.
• the agent that downregulates CD38 expression is a PI 3-kinase inhibitor.
• the agent that downregulates CD38 expression is a polynucleotide.
• the polynucleotide encodes an anti CD38, an anti retinoic acid receptor, an anti retinoid X receptor, an anti Vitamin D receptor or an anti PI 3-kinase antibody or intracellular antibody.
• the polynucleotide is a small interfering polynucleotide molecule directed to cause intracellular CD38, retinoic acid receptor, retinoid X receptor, Vitamin D receptor or
• the small interfering polynucleotide molecule is selected from the group consisting of an RNAi molecule, an anti-sense molecule, a rybozyme molecule and a DNAzyme molecule.
• reducing the expression and/or activity of CD38 is effected by an agent that inhibits CD38 activity.
• the agent can be, for example, nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite.
• the nicotinamide analog is preferably selected from the group consisting of benzamide, nicotinethioamide, nicotinic acid and ⁇ -amino-3-indolepropionic acid.
• reducing the expression and/or activity of CD38 is effected by an agent that inhibits PI 3-kinase activity.
• providing the stem cells with the conditions for ex-vivo cell proliferation comprises providing the cells with nutrients and with cytokines.
• the cytokines are early acting cytokines, such as, but not limited to, stem cell factor, FLT3 ligand, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-10, interleukin-12, tumor necrosis factor- ⁇ and thrombopoietin.
• the cytokines are late acting cytokines, such as, but not limited to, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, erythropoietin,
• FGF FGF, EGF, NGF, NEGF, LIF, Hepatocyte growth factor and macrophage colony stimulating factor.
• the hematopoietic mononuclear cells are derived from a source selected from the group consisting of bone marrow, peripheral blood and neonatal umbilical cord blood. According to still further features in the described preferred embodiments reducing the capacity of the hematopoietic mononuclear cells in responding to signaling pathways is reversible, e.g., inherently reversible.
• reducing the capacity of the hematopoietic mononuclear cells in responding to the above antagonists and/or signaling pathways of the above receptors is by ex-vivo culturing the hematopoietic mononuclear cells in a presence of an effective amount of at least one retinoic acid receptor antagonist, at least one retinoid X receptor antagonist and/or at least one Vitamin D receptor antagonist, preferably, for a time period of 0.1-50 %, preferably, 0.1-25 %, more preferably, 0.1-15 %, of an entire ex- vivo culturing period ofthe hematopoietic mononuclear cells.
• the retinoic acid receptor antagonist is selected from the group consisting of: AGN 194310; AGN 193109; 3-(4-Methoxy-phenylsulfanyl)-3-methyl-butyric acid; 6-Methoxy-2,2-dimethvl-thiochroman-4-one,2,2-Dimethyl-4-oxo-thiochroman-6- yltrifluoromethane-sulfonate; Ethyl 4-((2,2 dimethyl-4-oxo-thiochroman-6- yl)ethynyl)-benzoate; Ethyl 4-((2,2-dimethy 1-4-trifIouromethanensulfonyloxy -(2H)- thiochromen-6-yl)ethynyl)-benzoate(41); Thiochromen-6-yl]-ethynyl]-benzoate(yl); (p-[(E)-2-[
• the retinoid X receptor antagonist is selected from the group consisting of: LGN100572, LGN100574, l-(3-hydroxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)ethanone, 1 -(3-propoxy-5, 6,7, 8-tetrahydro-5, 5,8,8- tetramethylnaphthalene-2-yl)ethanone, 3-(3-propoxy-5,6,7,8-tetrahydro-5, 5,8,8- tetramethylnaphthalene-2-yl)but-2-enenitrile, 3-(3-propoxy-5,6,7,8-tetrahydro-
• the Vitamin D receptor antagonist is selected from the group consisting of: 1 alpha, 25- (OH)-D3-26,23 lactone; 1 alpha, 25-dihydroxyvitamin D (3); the 25-carboxylic ester ZK159222; (23S)- 25-dehydro-l alpha-OH-D (3); (23R)-25-dehydro-l alpha-OH-D (3); 1 beta, 25 (OH) 2 D 3 ; 1 beta, 25(OH) 2 -3-epi-D 3 ; (23S) 25-dehydro-l alpha(OH) D3-26,23-lactone; (23R) 25-dehydro-l alpha(OH)D3-26,23-lactone and Butyl- (5Z,7E,22E-( 1 S,7E,22E-( 1 S,3R,24R)- 1 ,3,24-trihydroxy-26,27-cyclo-9, 10- secocholesta-5 ,7, 10( 19),
• PI 3-kinase inhibitor is selected from the group consisting of wortmannin and
• the copper chelate(s) or chelator(s) used in the various aspects of the present invention described hereinabove preferably comprise a polyamine chelator.
• the polyamine chelator is capable of forming an organometallic complex with a transition metal other than copper.
• the transition metal can be, for example, zinc, cobalt, nickel, iron, palladium, platinum, rhodium and ruthenium.
• the polyamine chelator is a linear polyamine.
• the linear polyamine has a general formula I:
• A is an alkylene chain having a general formula II: R, R 2 R g
• Ri, R 2 and Rg is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroalicyclic, heteroaryl, halo, amino, alkylamino, arylamino, cycloalkylamino, heteroalicyclic amino, heteroarylamino, hydroxy, alkoxy, aryloxy, azo, C-amido, N-amido, ammonium, thiohydroxy, thioalkoxy, thioaryloxy, sulfonyl, sulfinyl, N-sulfonamide, S-sulfonamide, phosphonyl, phosphinyl, phosphonium, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, C-thiocarboxy, O-thiocarboxy, N- carb
• each of Bl and Bn is independently an alkylene chain having a general formula III: Rp R(P+1) Rq i i r
• At least one of C], C 2 and Cg and/or at least one of Cp, Cp+1 and Cq is a chiral carbon atom.
• a preferred linear polyamine according to the present invention is tetraethylenepentamine.
• the polyamine chelator is a cyclic polyamine, such as cyclam.
• Formula IV wherein m is an integer from 1 to 10; n is an integer from 0 to 20; X and Z are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; Yi and Yn are each independently selected from the group consisting of an oxygen atom, a sulfur atom and a -NH group; A is an alkylene chain having between 1 and 10 substituted and/or non-substituted carbon atoms; Bi and Bn are each independently an alkylene chain having between 1 and 20 substituted and/or non-substituted carbon atoms; and D is a bridging group having a general formula V:
• Formula V whereas U and V are each independently selected from the group consisting of substituted hydrocarbon chain and non-substituted hydrocarbon chain; and W is selected from the group consisting of amide, ether, ester, disulfide, thioether, thioester, imine and alkene, provided that at least one ofthe X, Z, Yi and Yn is a -NH group and/or at least one ofthe carbon atoms in the alkylene chains is substituted by an amine group.
• a and each of Bl and Bn in Formula IV are alkylene chains having the general formulas II and III, as is described hereinabove.
• the polyamine chelator includes at least one linear polyamine and at least one cyclic polyamine.
• Such a polyamine chelator preferably has a general formula XI:
• n is an integer greater than 1; each of f, g, h, i, j, k, 1, o and t is independently an integer from 0 to 10; each of Ei, E 2 and En is independently a linear polyamine as is described hereinabove; each of Gi, G 2 and Gn is independently a cyclic polyamine as is described hereinabove; and each of Qi, Q 2 and Qn is independently a linker linking between two ofthe polyamines, provided that at least one of the Qi, Q 2 and Qn is an amine group and/or at least one of the linear polyamine and the cyclic polyamine is having at least one free amine group.
• each of Qi, Q 2 and Qn is independently selected from the group consisting alkylene, alkenylene, alkynylene, arylene, cycloalkylene, hetroarylene, amine, azo, amide, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphonium, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether, carbamate, thiocarbamate, urea, thiourea, borate, borane, boroaza, silyl, siloxy and silaza.
• the polyamine chelator is selected from the group consisting of ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, tetraethylenepentamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, captopril, penicilamine, N,N'-bis(3-aminopropyl)-l,3-propanediamine, N,N'-Bis-(2- animoethyl)-l,3-propanediamine, l,7-dioxa-4,10-diazacyclododecane, 1,4,8,11- tetraaza cyclotetradecane-5,7-dione, 1 ,4,7-triazacyclononane, l-oxa-4,7,10- triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and
• FIGs. la-b illustrates the effect of TEPA chelator on the expansion of CD34+ hematopoietic stem cells in a culture of hematopoietic mononuclear cells.
• Cord-blood mononuclear cells MNCs
• MNC-TEPA TEPA chelator
• MNC control TEPA chelator
• purified CD34+ cells were similarly seeded in culture-bags in the presence of cytokines with no supplementation of TEPA chelator (CD34+ culture). All cultures were incubated for
• the CD34+ cells were purified from cultures using miniMacs columns and enumerated;
• FIG. 2 illustrates the FACS-analysis of the density of CD34+CD38" cells in the untreated NMCs, TEPA-treated MNCs and CD34+ cell cultures described above; and
• FIG. 3 presents the comparative numbers of colony-forming cells (CFUs) measured from the untreated MNCs, TEPA-treated MNCs and CD34+ cell cultures described above, at weekly intervals.
• CFUs colony-forming cells
• the present invention is of methods of ex-vivo expanding a population of hematopoietic stem cells present, as a minor fraction, in hematopoietic mononuclear cells, without first enriching the stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells.
• the present invention can be used to efficiently provide ex-vivo expanded populations of hematopoietic stem cells, using hematopoietic mononuclear cells that comprise a major fraction of hematopoietic committed cells and a minor fraction of the hematopoietic stem and progenitor cells as a source of stem cells, without prior enrichment of the hematopoietic mononuclear cells for stem cells.
• the expanded populations of hematopoietic stem cells of the present invention can be used in, for example, hematopoietic cell transplantation, in generation of stem cells suitable for genetic manipulations for cellular gene therapy, as well as in additional application such as, but not limited to, adoptive immunotherapy, implantation of stem cells in an in vivo cis-differentiation and trans-differentiation settings, as well as, ex-vivo tissue engineering in cis-differentiation and trans-differentiation settings.
• the methods of the present invention utilize various molecules (also referred to herein as agents), that interfere with CD38 expression and/or activity and/or with intracellular copper content, for inducing the ex-vivo expansion of hematopoietic stem cell populations described above, thereby providing an efficient, simplified and yet versatile technology for ex-vivo expansion of hematopoietic stem cells.
• PCT/IL03/00062 discloses that copper chelates, namely, copper chelators that are complexed with a copper ion, also promote proliferation and inhibit differentiation of stem and progenitor cells when added to the culture media of such cells. According to the teachings of PCT/IL03/00062, these finding suggest that this effect of copper chelates on proliferation and differentiation of stem and progenitor cells is not associated solely with the content of cellular copper but rather with additional regulatory pathways.
• 60/452,545 which are incorporated by reference as if fully set forth herein, disclose that a series of molecules that are capable of interfering with CD38 expression and/or activity, repress the process of differentiation of stem cells and stimulates and prolongs, for up to 16-18 weeks, the phase of active cell proliferation and expansion (renewal) ex-vivo, in a reversible manner.
• these references teach methods of expanding stem cell populations ex-vivo, which involve the addition of agents that either downregulate CD38 expression or inhibit the activity of CD38 to the culture media of stem cells.
• 60/452,545 therefore utilize molecules such as retinoic acid receptor antagonists of the RAR and RXR superfamilies, Vitamin D receptor antagonists, polynucleotides encoding antibodies such as anti CD38, anti retinoic acid receptor, anti retinoid X receptor, anti Vitamin D receptor, polynucleotides that are directed to cause degradation of endogenous polynucleotides encoding for these receptors, molecules that are capable of interfering with expression and/or activity of PI 3-kinase and CD38 inhibitors such as nicotinamide and its related compounds.
• retinoic acid receptor antagonists of the RAR and RXR superfamilies Vitamin D receptor antagonists
• polynucleotides encoding antibodies such as anti CD38, anti retinoic acid receptor, anti retinoid X receptor, anti Vitamin D receptor
• polynucleotides that are directed to cause degradation of endogenous polynucleotides encoding for these receptors molecules that are capable of
• WO 99/40783, WO 00/18885, PCT/IL03/00064 and U.S. Provisional Patent Application No. 60/452,545 all teach the use of various molecules that modulate, via diverse pathways and/or mechanisms, the balance between self-renewal and differentiation of stem cells, hematopoietic stem cells in particular, in methods for ex-vivo expanding of stem cell populations.
• the regulation of self-renewal and differentiation of stem cells by these molecules is obtained, according to the teachings of these references, when the cultured cells are first enriched for stem and/or progenitor cells and hence, in line with other present day technologies in this field, require preliminary stem cells enrichment.
• Hematopoietic cell transplantation Transplantation of hematopoietic cells has become the treatment of choice for a variety of inherited or malignant diseases. While early transplantation procedures utilized the entire bone marrow (BM) population, recently, more defined populations, enriched for stem cells (CD34 + cells) have been used (Van Epps DE, et al. Harvesting, characterization, and culture of CD34+ cells from human bone marrow, peripheral blood, and cord blood. Blood Cells 20:411, 1994). In addition to the marrow, such cells could be derived from other sources such as peripheral blood (PB) and neonatal umbilical cord blood (CB) (Emerson SG.
• PB peripheral blood
• CB neonatal umbilical cord blood
• An additional advantage of using PB for transplantation is its accessibility.
• the limiting factor for PB transplantation is the low number of circulating pluripotent stem/progenitor cells.
• PB-derived stem cells are "harvested" by repeated leukophoresis following their mobilization from the marrow into the circulation by treatment with chemotherapy and cytokines (Brugger W, et al. Reconstitution of hematopoiesis after high-dose chematotherapy by autologous progenitor cells generated in-vivo. N Engl J Med 333:283, 1995; Williams SF, et al. Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer. Blood 87: 1687, 1996). Such treatment is obviously not suitable for normal donors.
• ex-vivo expanded stem cells for transplantation has the following advantages (Koller MR, Emerson SG, Palsson BO. Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood 82:378, 1993; Lebkowski JS, et al. Rapid isolation and serum-free expansion of human CD34+ cells. Blood Cells 20:404, 1994):
• the cultures provide a significant depletion of T lymphocytes, which may be useful in the allogeneic transplant setting for reducing graft-versus-host disease.
• ex- vivo expanded cells include, in addition to stem cells, more differentiated progenitor cells in order to optimize short-term recovery and long-term restoration of hematopoiesis.
• Such cultures may be useful in restoring hematopoiesis in recipients with completely ablated bone marrow, as well as in providing a supportive measure for shortening recipient bone marrow recovery following conventional radio- or chemotherapies.
• Prenatal diagnosis of genetic defects in scarce cells involves the collection of embryonic cells from a pregnant woman, in utero, and analysis thereof for genetic defects.
• a preferred, non-invasive, means of collecting embryonic cells involves separation of embryonic nucleated red blood cell precursors that have infiltrated into peripheral maternal circulation.
• a further application of the present invention would be the expansion of such cells according to methods described herein, prior to analysis. The present invention, therefore, offers a means to expand embryonic cells for applications in prenatal diagnosis.
• Gene therapy For successful long-term gene therapy, a high frequency of genetically modified stem cells with transgenes stably integrated within their genome, is an obligatory requirement.
• stem cells In BM tissue, while the majority of cells are cycling progenitors and precursors, stem cells constitute only a small fraction of the cell population and most of them are in a quiescent, non-cycling state.
• Viral-based (e.g., retroviral) vectors require active cell division for integration of the transgene into the host genome. Therefore, gene transfer into fresh BM stem cells is highly inefficient.
• the ability to expand a purified population of stem cells and to regulate their cell division ex-vivo would provide for an increased probability of their genetic modification (Palmiter RD. Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc Natl Acad Sci USA 91(4): 1219-1223, 1994).
• Adoptive immunotherapy Ex-v/v ⁇ -expanded, defined lymphoid subpopulations have been studied and used for adoptive immunotherapy of various malignancies, immunodeficiencies, viral and genetic diseases (Freedman AR, et al. Generation of T lymphocytes from bone marrow CD34+ cells in-vitro. Nature Medicine 2: 46, 1996; Heslop HE, et al. Long term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nature Medicine 2: 551, 1996; Protti MP, et al. Particulate naturally processed peptides prime a cytotoxic response against human melanoma in-vitro. Cancer Res 56: 1210, 1996).
• antigen-presenting cells could be grown from a starting population of CD34 + PB cells in cytokine-supported cultures, as well. These cells can present soluble protein antigens to autologous T cells in-vitro and, thus, offer new prospects for the immunotherapy of minimal residual disease after high dose chemotherapy. Ex-vivo expansion of antigen-presenting dendritic cells has been studied as well, and is an additional promising application of the currently proposed technology (Bernhard H, et al. Generation of immunostimulatory dendritic cells from human CD34+ hematopoietic progenitor cells of the bone marrow and peripheral blood. Cancer Res 10: 99, 1995; Fisch P, et al.
• These molecules therefore represent a wide variety of molecules that are capable of inducing the effect of expanding a hematopoietic stem cells population that is present in a mixed hematopoietic cells population.
• a method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, thereby expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation ofthe hematopoietic stem cells ex-vivo.
• the phrase "hematopoietic mononuclear cells” refers to the entire repetoir of white blood cells present in a blood sample.
• the white blood cells comprise a mixture of hematopoietic lineages committed and differentiated cells (typically over 99 % of the mononuclear cells are lineages committed cells) including, for example: Lineage committed progenitor cells CD34 + CD33 + (myeloid committed cells), CD34 + CD3 + (lymphoid committed cells) CD34 + CD41 + (megakaryocytic committed cells) and differentiated cells - CD34 " CD33 + (myeloids, such as granulocytes and monocytes), CD34 " CD3 + , CD34 " CD19 + (T and B cells, respectively), CD34 " CD41 + (megakaryocytes), and hematopoietic stem and early progenitor cells such as CD34 + Lineage negative (Lin ' ), CD34-Lineage negative CD34 +
• hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells
• hematopoietic stem and progenitor cells any portion of the white blood cells fraction, in which the majority of the cells are hematopoietic committed cells, while the minority of the cells are hematopoietic stem and progenitor cells, as these terms are further defined hereinunder.
• Hematopoietic mononuclear cells are typically obtained from a blood sample by applying the blood sample onto a Ficoll-Hypaque layer and collecting, following density-cussion centrifugation, the interface layer present between the Ficoll-Hypaque and the blood serum, which interface layer essentially entirely consists of the white blood cells present in the blood sample.
• hematopoietic committed cells refers to differentiated hematopoietic cells that are committed to a certain hematopoietic cell lineage and hence can develop under physiological conditions substantially only to this specific hematopoietic lineage.
• hematopoietic stem cells refers to pluripotent hematopoietic cells that, given the right growth conditions, may develop to any cell lineage present blood.
• This phrase refers both to the earliest renewable hematopoietic cell populations responsible for generating cell mass in the blood (e.g., CD347AC133 + , CD347AC1337Lineage " , CD34 + /AC133 + cells) and the very early hematopoietic progenitor cells, which are somewhat more differentiated, yet are not committed and can readily revert to become a part of the earliest renewable hematopoietic cell population (e.g., CD34 + cells, especially CD34 + CD38 " cells).
• CD34 + In normal human, most of the hematopoietic pluripotent stem cells and the lineage committed progenitor cells are CD34 + . The majority of cells are CD34 + CD38 + , with a minority of cells ( ⁇ 10 %) being CD34 + CD38 " .
• the CD34 + CD38 " stem cells fraction identifies the most immature hematopoietic cells, which are capable of self-renewal and multilineage differentiation. This fraction contains more long-term culture initiating cells (LTC- IC) pre-CFU and exhibits longer maintenance of the stem cells and delayed proliferative response to cytokines as compared with the CD34 + CD38 + cell fraction.
• LTC- IC long-term culture initiating cells
• hematopoietic stem cells are obtained by further enrichment of the hematopoietic mononuclear cells obtained by differential density centrifugation as described above.
• This further enrichment process is typically performed by immuno- separation such as immunomagnetic-separation or FACS and results in a cell fraction that is enriched for hematopoietic stem cells.
• hematopoietic mononuclear cells as a direct source for obtaining expanded population of hematopoietic stem cells circumvents the need for stem cell enrichment prior to expansion, thereby substantially simplifying the process in terms of both efficiency and cost.
• the term “inhibiting” refers to slowing, decreasing, delaying, preventing or abolishing.
• the term “differentiation” refers to relatively generalized or specialized changes during development. Cell differentiation of various lineages is a well-documented process and requires no further description herein. As used herein the term differentiation is distinct from maturation which is a process, although some times associated with cell division, in which a specific cell type mature to function and then dies, e.g., via programmed cell death.
• cell expansion is used herein to describe a process of cell proliferation substantially devoid of cell differentiation.
• Cells that undergo expansion hence maintain their cell renewal properties and are oftentimes referred to herein as renewable cells, e.g., renewable stem cells.
• Expansion of hematopoietic stem cells using hematopoietic mononuclear cells as a source for the hematopoietic stem cells therefore result in converting the minor fraction (of less than 1 %) of hematopoietic stem and progenitor cells present in the mononuclear cells into at least the major, if not the sole hematopoietic cells population post expansion, whereby in the course of stem cells expansion the committed cells are either substantially diluted and/or die.
• ex-vivo refers to a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube).
• ex-vivo does not refer to a process by which cells known to propagate only in-vitro, such as various cell lines (e.g., HL-60, MEL, HeLa, etc.) are cultured.
• cells expanded ex-vivo according to the present invention do not transform into cell lines in that they eventually undergo differentiation.
• Providing the ex-vivo grown cells with conditions for ex-vivo cell proliferation include providing the cells with nutrients and preferably with one or more cytokines, as is further detailed hereinunder.
• the cells are short-term treated or long-term treated to reduce the expression and/or activity of CD38.
• reducing the activity of CD38 is effected by providing the cells with an agent that inhibits CD38 activity (i.e., a CD38 inhibitor).
• a CD38 inhibitor refers to an agent which is capable of downregulating or suppressing CD38 activity in stem cells.
• a CD38 inhibitor according to this aspect of the present invention can be a
• direct inhibitor which inhibits CD38 intrinsic activity
• an indirect inhibitor which inhibits the activity or expression of CD38 signaling components (e.g., the cADPR and ryanodine signaling pathways) or other signaling pathways which are effected by CD38 activity.
• nicotinamide is a possible CD38 inhibitor.
• the method according to this aspect of the present invention is effected by providing the hematopoietic mononuclear cells either with nicotinamide itself, or with a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite.
• nicotinamide analog refers to any molecule that is known to act similarly to nicotinamide.
• Representative examples of nicotinamide analogs include, without limitation, benzamide, nicotinethioamide (the thiol analog of nicotinamide), nicotinic acid and ⁇ -amino-3-indolepropionic acid.
• a nicotinamide or a nicotinamide analog derivative refers to any structural derivative of nicotinamide itself or of an analog of nicotinamide. Examples of such derivatives include, without limitation, substituted benzamides, substituted nicotinamides and nicotinethioamides and N-substituted nicotinamides and nicotinthioamides.
• a nicotinamide or a nicotinamide analog metabolite refers to products that are derived from nicotinamide or from analogs thereof such as, for example, NAD, NADH and NADPH.
• a CD38 inhibitor according to this aspect of the present invention can be an activity-neutralizing antibody that binds, for example, to the CD38 catalytic domain, thereby inhibiting CD38 catalytic activity.
• a fragmented antibody such as a Fab fragment (described hereinunder) is preferably used.
• antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab') 2 , and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows:
• Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
• Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab' fragments are obtained per antibody molecule; (Fab') 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
• Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains;
• Single chain antibody a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
• Antibody fragments according to the present invention can be prepared by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659-
• variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
• the Fv fragments comprise V H and V L chains connected by a peptide linker.
• These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins recipient antibody in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe
• FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
• human can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
• the method according to this aspect of the present invention can be effected by providing the ex-vivo cultured hematopoietic mononuclear cells with an agent that downregulates CD38 expression.
• An agent that downregulates CD38 expression refers to any agent which affects CD38 synthesis (decelerates) or degradation (accelerates) either at the level of the mRNA or at the level of the protein.
• a small interfering polynucleotide molecule which is designed to down regulate the expression of CD38 can be used according to this aspect ofthe present invention.
• RNA or siRNA such as, for example, the morpholino antisense oligonucleotides described by in Munshi et al. (Munshi CB, Graeff R, Lee HC, J Biol Chem 2002 Dec 20;277(51):49453-8), which includes duplex oligonucleotides which direct sequence specific degradation of mRNA through the previously described mechanism of RNA interference (RNAi) (Hutvagner and Zamore (2002) Curr. Opin. Genetics and Development 12:225-232).
• RNAi RNA interference
• duplex oligonucleotide refers to an oligonucleotide structure or mimetics thereof, which is formed by either a single self- complementary nucleic acid strand or by at least two complementary nucleic acid strands.
• the "duplex oligonucleotide” of the present invention can be composed of double-stranded RNA (dsRNA), a DNA-RNA hybrid, single-stranded RNA (ssRNA), isolated RNA (i.e., partially purified RNA, essentially pure RNA), synthetic RNA and recombinantly produced RNA.
• dsRNA double-stranded RNA
• ssRNA single-stranded RNA
• isolated RNA i.e., partially purified RNA, essentially pure RNA
• synthetic RNA recombinantly produced RNA.
• the specific small interfering duplex oligonucleotide of the present invention is an oligoribonucleotide composed mainly of ribonucle
• the small interfering polynucleotide molecule according to the present invention can be an RNAi molecule (RNA interference molecule).
• a small interfering polynucleotide molecule can be an oligonucleotide such as a CD38-specific antisense molecule or a rybozyme molecule, further described hereinunder.
• Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art.
• Oligonucleotides used according to this embodiment of the present invention are those having a length selected from a range of 10 to about 200 bases preferably 15-150 bases, more preferably 20-100 bases, most preferably 20-50 bases.
• the oligonucleotides of the present invention may comprise heterocyclic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
• oligonucleotides are those modified in either backbone, internucleoside linkages or bases, as is broadly described hereinunder. Such modifications can oftentimes facilitate oligonucleotide uptake and resistivity to intracellular conditions.
• oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S.
• Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
• Various salts, mixed salts and free acid forms can also be used.
• modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
• the base units are maintained for complementation with the appropriate polynucleotide target.
• An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• a PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
• the bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
• United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference.
• Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.
• Oligonucleotides of the present invention may also include base modifications or substitutions.
• "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted
• 5-substituted pyrimidines include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
• 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
• oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
• moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmity
• the oligonucleotides of the present invention are preferably antisense molecules, which are chimeric molecules.
• "Chimeric antisense molecules” are oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target polynucleotide. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
• RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.
• Activation of RNase H therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
• Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
• Chimeric antisense molecules of the present invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, as described above.
• Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711 ; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein fully incorporated by reference.
• the oligonucleotides of the present invention can further comprise a ribozyme sequence.
• Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs.
• Several rybozyme sequences can be fused to the oligonucleotides of the present invention. These sequences include but are not limited ANGIOZYME specifically inhibiting formation of the VEGF-R (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway, and HEPTAZYME, a rybozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, (Rybozyme Pharmaceuticals, Incorporated - WEB home page).
• VEGF-R Vascular Endothelial Growth Factor receptor
• HEPTAZYME a rybozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA
• a small interfering polynucleotide molecule can be a DNAzyme.
• DNAzymes are single-stranded catalytic nucleic acid molecules.
• a general model (the "10-23” model) for the DNAzyme has been proposed.
• "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
• This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM Curr Opin Mol Ther 2002;4:119-21). Examples of construction and amplification of synthetic, engineered
• DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al., 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
• retinoid receptor superfamily inhibitors e.g., antagonists, siRNA molecules, antisense molecules, antibodies, etc.
• retinoid receptor superfamily inhibitors which downregulate or suppress retinoid receptor activity and/or expression can be used to downregulate CD38 expression.
• retinoid receptors such as retinoic acid receptor (RAR), retinoid X receptor (RXR) and vitamin D receptor (VDR) have been reported to be involved in the regulation of gene expression pathways associated with cell proliferation and differentiation and in particular in the regulation of CD38 expression [Kapil M., Maria M., Taghi M., Michael A., Steven C, Maher A.. Involvement of retinoic acid receptor mediated signaling pathway in induction of CD38 cell surface antigen, Blood. 1997; 89:3607-3614; Ueno H, Kizaki M, Matsushita H, Muto A, Yamato K, Nishihara T, Hida T, Yoshimura H, Koeffler HP, Ikeda Y.
• RAR retinoic acid receptor
• RXR retinoid X receptor
• VDR vitamin D receptor
• a novel retinoic acid receptor (RAR)- selective antagonist inhibits differentiation and apoptosis of HL-60 cells: implications of RAR alpha-mediated signals in myeloid leukemic cells. Leuk Res. 1998; 22:517- 25].
• preferred agents that downregulate CD38 expression according to the present invention include RAR antagonists, RXR antagonists and VDR antagonists or, alternatively, antagonists for reducing the capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoid and/or Vitamin D.
• antagonist refers to an agent that counteracts or abrogates the effects of an agonist or a natural ligand of a receptor. Further features relating to such antagonists are detailed hereinunder. Further alternatively, as is described in detail in U.S. Provisional Patent
• down regulation of CD38 expression can be obtained by downregulating the expression and or activity of phosphatidyl inositol 3-kinase, which is also referred to herein throughout as PI 3-kinase.
• PI 3-kinase plays a critical function in the activation of nuclear receptors such as the retinoid receptor superfamily and the vitamin D receptor, as an obligatory factor for proper receptor signaling pathways and is hence involved in cell differentiation.
• agents that interfere with PI 3-kinase expression and/or activity are also preferred agents for downregulating CD38, according to the present invention.
• agents that inhibit PI 3-kinase activity include, but are not limited to, the known PI 3-kinase inhibitors wortmannin and LY294002, and analogs, derivatives, and metabolites thereof. Additional examples of PI 3-kinase inhibitors are described in U.S. Patent No. 5,378,725, which is incorporated by reference as if fully set forth herein.
• agents that downregulate PI 3- kinase expression according to the present invention include, but are not limited to, polynucleotides, such as small interfering RNA molecules, antisense ribozymes and DNAzymes, as well as intracellular antibodies, using the methodologies described hereinabove with respect to downregulating CD38 expression.
• each of the agents described hereinabove may reduce the expression or activity of CD38 individually.
• the present invention aims to also encompass the use of any subcombination of these agents.
• protein agents e.g., antibodies
• protein agents of the present invention can be expressed from a polynucleotide encoding same and provided to ex- vivo cultured hematopoietic mononuclear cells employing an appropriate gene delivery vehicle/method and a nucleic acid construct as is further described hereinunder.
• suitable constructs include, but are not limited to pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
• retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, including Retro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter.
• Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
• the method of ex-vivo expanding a population of hematopoietic stem cells is effected by modulating CD38 expression and/or activity, either at the protein level, using RAR, RXR or VDR antagonists, a PI-3 kinase inhibitor or a CD38 inhibitor such as nicotinamide and analogs thereof, or at the at the expression level via genetic engineering techniques, as is detailed hereinabove, there are further provided, according to the present invention, several preferred methods of ex-vivo expanding a population of hematopoietic stem cells of hematopoietic mononuclear cells.
• a method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, so as to expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• Reducing the capacity of the cells in responding to retinoic acid, retinoids and/or Vitamin D, or to retinoic acid, retinoid X and/or Vitamin D receptor signaling may be effected, for example, by the administration of chemical inhibitors, including receptor antagonists.
• the method of ex-vivo expanding a population of stem cells, while at the same time, substantially inhibiting differentiation of the stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, retinoid-X receptor and/or Vitamin D receptor, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation ofthe hematopoietic stem cells ex-vivo.
• Reducing the capacity of the cells to respond to retinoic acid, retinoid X and/or Vitamin D receptor signaling events includes treating the cells with antagonists supplied continuously or for a short-pulse period, and is effected by a diminution or abrogation of cellular signaling pathways through their respective, cognate receptors.
• antagonists supplied continuously or for a short-pulse period As is described and exemplified in PCT/IL01/00064, reducing the capacity of hematopoietic cells in responding to the disclosed signaling pathways is reversible, e.g., inherently reversible. In other words, cells expanded using the protocols of the present invention do not transform into cell lines.
• cis-differentiation refers to differentiation of adult stem cells into a tissue from which they were derived. For example, the differentiation of CD34+ hematopoietic cells to different committed/mature blood cells constitutes cis- differentiation.
• trans-differentiation refers to differentiation of adult stem cells into a tissue from which they were not derived. For example, the differentiation of CD34+ hematopoietic cells to cells of different tissue origin, e.g., myocites constitutes trans-differentiation.
• Reducing the capacity of the hematopoietic mononuclear cells in responding to the above antagonists and/or signaling pathways of the above receptors and kinase is effected by ex-vivo culturing hematopoietic mononuclear cells in a presence of an effective amount of at least one retinoic acid receptor antagonist, at least one retinoid X receptor antagonist and/or at least one Vitamin D receptor antagonist, preferably, for a time period of 0.1-50 %, preferably, 0.1-25 %, more preferably, 0.1-15 %, of an entire ex-vivo culturing period of the hematopoietic mononuclear cells or for the entire period.
• an initial pulse exposure to an antagonist is sufficient to exert cell expansion long after the antagonist was removed from the culturing set up.
• Final concentrations of the antagonists may be, depending on the specific application, in the micromolar or millimolar ranges. For example, within about 0.1 ⁇ M to about 100 mM, preferably within about 4 ⁇ M to about 50 mM, more preferably within about 5 ⁇ M to about 40 mM.
• retinoic acid receptor antagonist include, without limitation, AGN 194310; AGN 109; 3-(4-Methoxy-phenylsulfanyl)-3- methyl-butyric acid; 6-Methoxy-2,2-dimethvl-thiochroman-4-one,2,2-Dimethyl-4- oxo-thiochroman-6-yltrifluoromethane-sulfonate; Ethyl 4-((2,2 dimethyl-4-oxo- thiochroman-6-yl)ethynyl)-benzoate; Ethyl 4-((2,2-dimethy 1-4- trifTouromethanensulfonyloxy -(2H)- thiochromen-6-yl)ethynyl)-benzoate(41);
• retinoid X receptor antagonist include, without limitation, LGN100572, l-(3-hydroxy-5,6,7,8-tetrahydro-5,5,8,8- tetramethylnaphthalene-2-yl)ethanone, l-(3-propoxy-5,6,7,8-tetrahydro-5, 5,8,8- tetramethylnaphthalene-2-yl)ethanone, 3-(3-propoxy-5,6,7,8-tetrahydro-5, 5,8,8- tetramethylnaphthalene-2-yl)but-2-enenitrile, 3-(3-propoxy-5,6,7,8-tetrahydro-
• Vitamin D receptor antagonist include, without limitation: 1 alpha, 25-(OH)-D3-26,23 lactone; 1 alpha, 25-dihydroxyvitamin D (3); the 25-carboxylic ester ZK159222; (23S)- 25-dehydro-l alpha-OH-D (3); (23R)-25-dehydro-l alpha-OH-D (3); 1 beta, 25 (OH) 2 D 3 ; 1 beta, 25(OH) 2 -3-epi-D 3 ; (23S) 25-dehydro-l alpha(OH) D3-26,23-lactone; (23R) 25-dehydro-l alpha(OH)D3- 26,23-lactone and Butyl-(5Z,7E,22E-(lS,7E,22E-(lS,3R,24R)-l,3,24-trihydroxy- 26,27-cyclo-9,10-secocholesta-5,7,10(19),22-tetraene-25-carboxylate
• the method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• the method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• the method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and with a PI 3-kinase inhibitor, to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex- vivo.
• Final concentrations of the nicotinamide or the analogs, derivatives or metabolites thereof and of the PI 3-kinase inhibitor are preferably, depending on the specific application, in the millimolar ranges. For example, within about 0.1 mM to about 20 mM, preferably within about 1 mM to about 10 mM, more preferably within about 5 mM to about 10 mM.
• expansion of the hematopoietic stem cells population present in hematopoietic mononuclear cells can also be effected in the presence of copper chelators or caves.
• copper chelators or copper chelates As is discussed in detail in WO 00/18885 and in PCT/IL03/00062, addition of copper chelators or copper chelates to cells culturing media affects the cellular copper concentration, which in turn, affects signaling pathways associated with cells differentiation.
• addition of a copper chelate to the cells culturing media maintains the free copper concentration of the cells substantially unchanged during cell expansion, while addition of a copper chelator to the cells culturing media reduces the capacity ofthe cells in utilizing copper.
• copper chelator refers to a ligand that has at least two atoms capable of coordinating with copper or a copper ion, so as to form a ring.
• a copper chelator is free of, i.e., not complexed with, the copper ion. Additional features relating to chelating effects are described, for example, in PCT/IL03/00062.
• copper chelate refers to a copper chelator, as is defined hereinabove, which is complexed with a copper ion.
• the present invention there is provided another method of ex-vivo expanding a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• the method is effected by providing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, with ex-vivo culture conditions for ex-vivo cell proliferation and with one or more copper chelator(s) or copper chelate(s), to thereby expand a population of hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo.
• the copper chelate or chelators of the present invention is oftentimes capable of forming an organometallic complex with a transition metal other than copper.
• metals other than copper are typically present in the cells (e.g., zinc) or can be administered to cells during therapy (e.g., platinum), it was found that copper chelates or chelators that can also interact with other metals are highly effective.
• Representative examples of such transition metals include, without limitation, zinc, cobalt, nickel, iron, palladium, platinum, rhodium and ruthenium.
• the copper chelates ofthe present invention comprise a copper ion (e.g., Cu +1 ,
• Cu +2 copper chelator(s) and one or more copper chelator(s).
• Preferred copper chelators according to the present invention include polyamine molecules, which can form a cyclic complex with the copper ion via two or more amine groups present in the polyamine.
• the copper chelate or chelator used in the context of the different aspects and embodiments of the present invention preferably includes a polyamine chelator, namely a polymeric chain that is substituted and/or interrupted with 1-10 amine moieties, preferably 2-8 amine moieties, more preferably 4-6 amine moieties and most preferably 4 amine moieties.
• amine moiety amine group
• simply “amine” are used herein to describe a -NR'R” group or a -NR'- group, depending on its location within the molecule, where R' and R' ' are each independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclic, as these terms are defined hereinbelow.
• the polyamine chelator can be a linear polyamine, a cyclic polyamine or a combination thereof.
• a linear polyamine can be a polyamine that has a general formula I:
• the linear polyamine is preferably comprised of one or more alkylene chains (Am, Bp--Bn, in Formula I), is interrupted by one or more heteroatoms such as S, O and N (Yp--Yn in Formula I), and terminates with two such heteroatoms (X and Z in Formula I).
• Alkylene chain A includes 1-10 substituted or non-substituted carbon atoms and is connected, at least at one end thereof, to a heteroatom (e.g., X in Formula I). Whenever there are more than one alkylene chains A (in cases where m is greater than one), only the first alkylene chain A is connected to X. However, m is preferably 1 and hence the linear polyamine depicted in Formula I preferably includes only one alkylene chain A.
• Alkylene chain B includes between 1 and 20 substituted or non-substituted carbon atoms.
• the alkylene chain B is connected at its two ends to a heteroatom (Y
• the preferred linear polyamine delineated in Formula I comprises between 1 and 20 alkylene chains B, denoted as Bi •••• Bn, where "Bi • ⁇ •• Bn" is used herein to describe a plurality of alkylene chains B, namely, Bi, B 2 , B , ••••, Bn-1 and Bn, where n equals 0-20. These alkylene chains can be the same or different.
• Each of Bi •• • • Bn is connected to the respective heteroatom Yi ⁇ ••• Yn, and the last alkylene chain in the structure, Bn, is also connected to the heteroatom Z.
• n 2-10, more preferably 2-8 and most preferably 3-5.
• the linear polyamine depicted in Formula I preferably includes between 3 and 5 alkylene chains B, each connected to 3-5 heteroatoms Y.
• the linear polyamine depicted in Formula I must include at least one amine group, as this term is defined hereinabove, preferably at least two amine groups and more preferably at least four amine groups.
• the amine group can be present in the structure as the heteroatoms X, Z or Yi •••• Yn, such that at least one of X, Z and Yi — • Yn is a -NH- group, or as a substituent of one or more of the substituted carbon atoms in the alkylene chains A and Bi •••• Bn.
• the presence of these amine groups is required in order to form a stable chelate with the copper ion, as is discussed hereinabove.
• the alkylene chain A preferably has a general Formula II:
• the alkylene chain A is comprised of a plurality of carbon atoms C
• the alkylene chain A includes 2-10 carbon atoms, more preferably, 2-6 and most preferably 2-4 carbon atoms.
• the component CgH(Rg) is absent from the structure and hence the alkylene chain A comprises only 2 carbon atoms.
• Ri, R 2 and Rg are each a substituent attached to the carbon atoms in A.
• Each of Ri, R 2 and Rg can independently be a substituent such as, but not limited to, hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroalicyclic, heteroaryl, halo, amino, alkylamino, arylamino, cycloalkylamino, heteroalicyclic amino, heteroarylamino, hydroxy, alkoxy, aryloxy, azo, C-amido, N-amido, ammonium, thiohydroxy, thioalkoxy, thioaryloxy, sulfonyl, sulfinyl, N-sulfonamide, S- sulfonamide, phosphonyl, phosphinyl, phosphonium, carbonyl, thiocarbonyl, C- carboxy, O-carboxy, C-thiocarboxy, O-thiocarboxy, N-carbamate, O-carbamate, N-
• R 2 or Rg is hydrogen, its respective carbon atom in a non- substituted carbon atom.
• alkyl is a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
• the alkyl group has 1 to 20 carbon atoms. More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms.
• the alkyl group may be substituted or non-substituted.
• the substituent group can be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, O- carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, C-amido, N-amido, C- carboxy, O-carboxy, nitro, sulfonamide, silyl, guanidine, urea or amino, as these terms are defined hereinbelow.
• alkenyl describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon double bond.
• alkynyl describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
• cycloalkyl describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system.
• examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.
• a cycloalkyl group may be substituted or unsubstituted.
• the substituent group can be, for example, alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamate, N- carbamate, C-amido, N-amido, nitro, or amino, as these terms are defined hereinabove or hereinbelow.
• aryl describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system.
• aryl groups are phenyl, naphthalenyl and anthracenyl.
• the aryl group may be substituted or unsubstituted.
• the substituent group can be, for example, halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thiocarbonyl, C-carboxy, O-carboxy, O-carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, C-amido, N-amido, sulfinyl, sulfonyl or amino, as these terms are defined hereinabove or hereinbelow.
• heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
• heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
• the heteroaryl group may be substituted or unsubstituted.
• the substituent group can be, for example, alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thiocarbonyl, sulfonamide, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O- carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, C-amido, N-amido or amino, as these terms are defined hereinabove or hereinbelow.
• heteroalicyclic describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
• the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
• the heteroalicyclic may be substituted or unsubstituted.
• the substituted group can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamate, N-carbamate, O-thiocarbamate, N-thiocarbamate, sulfinyl, sulfonyl, C-amido, N-amido or amino, as these terms are defined hereinabove or hereinbelow.
• halo describes a fluorine, chlorine, bromine or iodine atom.
• amino as is defined hereinabove with respect to an "amine” or an
• amino group is used herein to describe an -NR'R", wherein R' and R" are each independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclic, as these terms are defined hereinabove.
• alkylamino alkylamino
• arylamino cycloalkylamino
• heteroalicyclic amino alkyl
• heteroarylamino alkylamino
• alkylamino alkylamino
• arylamino cycloalkylamino
• heteroalicyclic amino heteroarylamino
• hydroxy describes an -OH group.
• alkoxy describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
• aryloxy describes both an -O-aryl and an -O-heteroaryl group, as defined herein.
• An “ammonium” describes an -N + HR'R" group, where R' and R" are as defined hereinabove.
• thiohydroxy describes a -SH group.
• thioalkoxy describes both a -S-alkyl group and a -S-cycloalkyl group, as defined hereinabove.
• thioaryloxy describes both a -S-aryl and a -S-heteroaryl group, as defined hereinabove.
• a "phosphinyl” is a -PR'R" group, with R' and R" as defined hereinabove.
• a "phosphonium” is a -P + R'R"R'", where R' and R" are as defined hereinabove and R"' is defined as either R' or R".
• a “carboxylic acid” is a C-carboxy group in which R is hydrogen.
• R is as defined hereinabove with respect to the term "carbonyl”.
• borate describes an -O-B-(OR) group, with R as defined hereinabove.
• borane describes a -B-R'R" group, with R' and R" as defined hereinabove.
• boraza describes a -B(R')(NR"R' ") group, with R', R" and R' " as defined hereinabove.
• sil describes a -SiR'R"R' ", with R', R" and R"' as defined herein.
• sioxy is a -Si-(OR) 3 , with R as defined hereinabove.
• siaza describes a -Si-(NR'R") 3 , with R' and R" as defined herein.
• alcohol describes a ROH group, with R as defined hereinabove.
• peroxo describes an -OOR group, with R as defined hereinabove.
• a “hydrazine” is a -NR'-NR"R' “ group, with R', R" and R'" as defined herein.
• alkyl hydrazine and "aryl hydrazine” describe a hydrazine where R' is an alkyl or an aryl, respectively, and R" and R'" are as defined hereinabove.
• cyano is a -C ⁇ N group.
• a “cyanate” is an -O-C ⁇ N group.
• a “thiocyanate” is a "-S-C ⁇ N group.
• alkyl nitrile and "aryl nitrile” describe a -R-C ⁇ N group, where R is an alkyl or an aryl, respectively.
• a "nitrate” or “nitro” is a -NO 2 group.
• An “azido” is a N 3 + group.
• alkyl sulfonic acid and an “aryl sulfonic acid” describe a -R-SO 2 -OH group, with R being an alkyl or an aryl, respectively.
• alkyl sulfenic acid and aryl sulfenic acid describe a -R-S-OH group, where R is an alkyl or an aryl, respectively.
• a “sulfate” is a -O-SO 2 -OR' group, with R' as defined hereinabove.
• a “bisulfite” is a sulfite group, where R' is hydrogen.
• a “thiosulfate” is an -O-SO 2 -SR' group, with R' as defined hereinabove.
• alkyl/aryl phosphine describe a -R-PH 2 group, with R being an alkyl or an aryl, respectively, as defined above.
• alkyl/aryl phosphinic acid describes a -R'-P(OH) 2 group, with R' being an alkyl or an aryl as defined above.
• a “hydrogen phosphate” is a phosphate group, where R' is hydrogen.
• a “dihydrogen phosphate” is a phosphate group, where R' and R" are both hydrogen.
• a "phosphite” is an -O-P (OR') 2 group, with R' as defined hereinabove.
• a "pyrophosphite” is an -O-P-(OR')-O-P(OR") 2 group, with R' and R" as defined hereinabove.
• a “hypochlorite” is an -OC1 group.
• a “hypobromite” is an -OBr group.
• tetrahalomanganate describes MnCl 4 , MnBr 4 and Mnl 4 .
• tetrafluoroborate describes a -BF 4 group.
• a “tetrafluoroantimonate” is a SbF 6 group.
• a “hypophosphite” is a -P(OH) 2 group.
• tetraalkyl/tetraaryl borate describe a R'B " group, with R' being an alkyl or an aryl, respectively, as defined above.
• a “salycilate” is the group
• a “saccharirate” is an oxidized saccharide having two carboxylic acid group.
• amino acid as used herein includes natural and modified amino acids and hence includes the 21 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor- leucine and ornithine.
• amino acid includes both D- and L- amino acids which are linked via a peptide bond or a peptide bond analog to at least one addition amino acid as this term is defined herein.
• a “thiotosylate” is the group
• •••• Bn independently has a general formula III:
• p is an integer that equals 0 or g+1 and q is an integer from g+2 to g+20.
• ••• ⁇ Bn is comprised of a plurality of carbon atoms Cp, Cp+1, Cp+2 • ⁇ ••, Cq-1 and Cq, substituted by the respective Rp,
• Bn includes 2-20 carbon atoms, more preferably 2-10, and most preferably 2-6 carbon atoms.
• a preferred linear polyamine according to the present invention includes two or more alkylene chains.
• the alkylene chains are interrupted therebetween by a heteroatom and each is connected to a heteroatom at one end thereof.
• each of the alkylene chains include at least two carbon atoms, so as to enable the formation of a stable chelate between the heteroatoms and the copper ion.
• the linear polyamine delineated in Formula I preferably includes at least one chiral carbon atom.
• at least one of Ci, C 2 and Cg in the alkylene chain A and/or at least one of Cp, Cp+1 and Cq in the alkylene chain B is chiral.
• a preferred linear polyamine according to the present invention is tetraethylenepentamine.
• Other representative examples of preferred linear polyamines usable in the context of the present invention include, without limitation, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, aminoethylethanolamine, pentaethylenehexamine, triethylenetetramine, N,N'-bis(3- aminopropyl)-l,3-propanediamine, and N,N'-Bis(2-animoethyl)-l,3 propanediamine.
• the polyamine chelator is a cyclic polyamine
• the polyamine can have a general formula IV:
• U and V are each independently selected from the group consisting of substituted hydrocarbon chain and non-substituted hydrocarbon chain; and W is selected from the group consisting of amide, ether, ester, disulfide, thioether, thioester, imine and alkene, provided that at least one of said X, Z, Yi and Yn is a - NH group and/or at least one of said carbon atoms in said alkylene chains is substituted by an amine group.
• the cyclic polyamine has one of the general formulas VI-X:
• Formula X wherein m, n, X, Yi, Yn, Z, A, B and D are as described above and further wherein should the bridging group D is attached at one end to A (Formulas NI, VII and X), U or V are being attached to one carbon atom in the alkylene chain and should D is attached at one end to B 1 or Bn (Formulas VIII, IX and X), U or V are being attached to one carbon atom in the alkylene chain.
• a preferred cyclic polyamine according to the present invention includes two or more alkylene chains, A, Bi •••• Bn, as is detailed hereinabove with respect to the linear polyamine.
• the alkylene chains can form a cyclic structure by being connected, via the bridging group D, between the ends thereof, namely between the heteroatoms X and Z (Formula IV).
• the alkylene chains can form a conformationally restricted cyclic structure by being connected, via the bridging group D, therebetween (Formula X).
• a conformationally restricted cyclic structure can be formed by connecting one alkylene chain to one terminal heteroatom (X or Z, Formulas VI-IX).
• the bridging group D connects a terminal heteroatom, namely X or Z, and one carbon atom in the alkylene chains A and B
• This carbon atom can be anyone of Ci, C , Cg, Cp, Cp+1 and Cq described hereinabove.
• the cyclic structure is formed by the bridging group D, which connects two components in the structure.
• the bridging group D has a general formula U-W-V, where each of U and V is a substituted or non- substituted hydrocarbon chain.
• hydrocarbon chain describes a plurality of carbon atoms which are covalently attached one to another and are substituted, mter alia, by hydrogen atoms.
• the hydrocarbon chain can be saturated, unsaturated, branched or unbranched and can therefore include one or more alkyl, alkenyl, alkynyl, cycloalkyl and aryl groups and combinations thereof.
• the length of the hydrocarbon chains namely the number of carbon atoms in the chains, is preferably determined by the structure ofthe cyclic polyamine, such that on one hand, the ring tension of the formed cyclic structure would be minimized and on the other hand, an efficient chelation with the copper ion would be achieved.
• the substituents can be any one or combinations of the substituents described hereinabove with respect to Ri, R 2 and Rg in the linear polyamine.
• the two hydrocarbon chains are connected therebetween by the group W, which can be amide, ether, ester, disulfide, thioether, thioester, imine and alkene.
• ether is an -O- group.
• a “disulfide” is a -S-S- group.
• a “thioether” is a -S- group.
• the bridging group D is typically formed by connecting reactive derivatives of the hydrocarbon chains U and V, so as to produce a bond therebetween (W), via well- known techniques, as is described, for example, in U.S. Patent No. 5,811,392.
• the cyclic polyamine must include at least one amine group, preferably at least two amine groups and more preferably at least four amine groups, so as to form a stable copper chelate.
• a preferred cyclic polyamine according to the present invention is cyclam
• the polyamine chelator of the present invention can further include a multimeric combination of one or more linear polyamine(s) and one or more cyclic polyamine(s).
• a polyamine chelator can therefore be comprised of any combinations of the linear and cyclic polyamines described hereinabove.
• such a polyamine chelator has a general Formula XI:
• n is an integer greater than 1; each of f, g, h, i, j, k, 1, o and t is independently an integer from 0 to 10; each of Ei, E 2 and En is independently a linear polyamine, as is described hereinabove; each of Gi, G 2 and Gn is independently a cyclic polyamine as is described hereinabove; and each of Qi, Q 2 and Qn is independently a linker linking between two of said polyamines, provided that at least one of said Qi, Q 2 and
• Qn is an amine group and/or at least one of said linear polyamine and said cyclic polyamine has at least one free amine group.
• Each of Ei, E 2 and En in Formula XI represent a linear polyamine as is described in detail hereinabove, while each of Gi, G 2 and Gn represents a cyclic polyamine as is described in detail hereinabove.
• the polyamine described in Formula XI can include one or more linear polyamine(s), each connected to another linear polyamine or to a cyclic polyamine.
• Each of the linear or cyclic polyamines in Formula XI is connected to another polyamine via one or more linker(s), represented by Qi, Q 2 and Qn in Formula XI.
• Each of the linker(s) Qi, Q 2 and Qn can be, for example, alkylene, alkenylene, alkynylene, arylene, cycloalkylene, hetroarylene, amine, azo, amide, sulfonyl, sulfinyl, sulfonamide, phosphonyl, phosphinyl, phosphonium, ketoester, carbonyl, thiocarbonyl, ester, ether, thioether, carbamate, thiocarbamate, urea, thiourea, borate, borane, boroaza, silyl, siloxy and silaza.
• alkenylene describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon double bond.
• alkynylene describes an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
• cycloalkylene describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system.
• cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.
• arylene describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system.
• aryl groups are phenyl, naphthalenyl and anthracenyl.
• the aryl group may be substituted or unsubstituted.
• heteroaryl ene describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
• heteroaryl groups examples include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
• the heteroaryl group may be substituted or unsubstituted.
• amine describes an -NR'-, wherein R' can be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl or heterocyclic, as these terms are defined hereinabove.
• ammonium describes an -N + HR'- group, where R' is as defined hereinabove.
• phosphinyl describes a -PR'- group, with R' as defined hereinabove.
• phosphonium is a -P + R'R", where R' and R" are as defined hereinabove.
• borate describes an -O-B-(OR)- group, with R as defined hereinabove.
• borane describes a -B-R-'- group, with R as defined hereinabove.
• boraza describes a -B (NR'R")- group, with R' and R" as defined hereinabove.
• sil describes a -SiR'R"-, with R' and R" as defined herein.
• sioxy is a -Si-(OR) 2 -, with R as defined hereinabove.
• siaza describes a -Si-(NR'R") 2 -, with R' and R" as defined herein.
• the polyamine chelator is tetraethylenepentamine (TEPA).
• polyamine chelators include, without limitation, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, triethylenetetramine, captopril, penicilamine, N,N'-bis(3- aminopropyl)-l,3-propanediamine, N,N'-Bis(2-animoethyl)-l,3-propanediamine, 1,7- dioxa-4,10-diazacyclododecane, 1,4,8,1 l-tetraazacyclotetradecane-5,7-dione, 1,4,7- rriazacyclononane, l-oxa-4,7,10-triazacyclododecane, 1,4,8,12- tetraazacyclopentadecane and 1,4,7,10-tetraazacyclododecane.
• polyamine chelators described hereinabove can be either commercially obtained or can be synthesized using known procedures such as described, for example, in: T.W. Greene (ed.), 1999 ("Protective Groups in Organic Synthesis” 3ed Edition, John Wiley & Sons, Inc., New York 779 pp); or in: R.C. Larock and V.C.H. Wioley, "Comprehensive Organic Transformations - A Guide to Functional Group Preparations", (1999) 2 nd Edition.
• TEPA-Cu tetraethylenepentamine-copper chelate
• the copper chelate or chelator can be provided to the cell culture medium.
• the final concentrations of copper chelate may be, depending on the specific application, in the micromolar or millimolar ranges, for example, within about 0.1 ⁇ M to about 100 mM, preferably within about 4 ⁇ M to about 50 mM, more preferably within about 5 ⁇ M to about 40 mM.
• ex-vivo expanded populations of hematopoietic stem cells obtained by any of the methods described hereinabove.
• the expanded populations of hematopoietic stem cells according to the present invention comprise a plurality of cells characterized by
• CD34+ d i m i.e., fall below the median intensity in a FACS analysis, wherein, in the reselectable CD34+ cells, a majority of cells which are Lin " are also CD34+di m cells.
• the population of hematopoietic stem cells has a single genetic background.
• the ex-vivo expanded population of hematopoietic stem cells comprises at least N cells derived from a single donor, wherein N equals the average number of CD34+ cells derived from one sample of hematopoietic mononuclear cells, multiplied by 1,000.
• Cell surface expression of the CD34 and/or Lin markers can be determined, for example, via FACS analysis or immunohistological staining techniques.
• a self renewal potential of the hematopoietic stem cells can be determined in-vitro by long term colony formation (LTC-CFUc), as is further exemplified in the Examples section that follows.
• ex-vivo expansion of hematopoietic stem cells can be advantageously utilized in various applications such as, for example, hematopoietic cells transplantation or implantation, adoptive immunotherapy and gene therapy.
• the ability to practice the ex-vivo expansion of hematopoietic stem cells with hematopoietic mononuclear cells as the cells source substantially facilitates the utilization ofthe methods described hereinabove in these applications.
• a method of hematopoietic cells transplantation or implantation is provided.
• the method according to this aspect of the present invention is effected by (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a donor, (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing an expression and/or activity of CD38, so as to expand a population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells ex-vivo, and (c) transplanting or implanting the thus obtained hematopoietic stem cells to a recipient.
• various agents can be used in the context of the different aspects of the present invention for reducing an expression and/or activity of CD38.
• the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, as is described hereinabove.
• the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity of the hematopoietic mononuclear cells in responding to signaling pathways involving the retinoic acid receptor, the retinoid X receptor and/or the Vitamin D receptor, as is described hereinabove.
• the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, for reducing a capacity ofthe hematopoietic mononuclear cells in responding to signaling pathways involving PI 3-kinase, as is described hereinabove.
• the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite, as is described hereinabove.
• the method is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with a PI 3-kinase inhibitor, as is described hereinabove.
• the method of hematopoietic cells transplantation or implantation described above is effected by providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and with one or more ofthe copper chelator(s) or chelate(s) described hereinabove.
• the donor and the recipient can be a single individual or different individuals, for example, allogeneic or xenogeneic individuals.
• allogeneic transplantation is practiced, regimes for reducing implant rejection and/or graft vs. host disease, as well know in the art, should be undertaken. Such regimes are currently practiced in human therapy. Most advanced regimes are disclosed in publications by Slavin S. et al., e.g., J Clin Immunol (2002) 22: 64, and J Hematother Stem Cell Res (2002) 11 : 265), Gur H. et al. (Blood (2002) 99: 4174), and Martelli MF et al, (Semin Hematol (2002) 39: 48), which are incorporated herein by reference.
• transplantable hematopoietic cell preparation which comprises an expanded population of hematopoietic stem cells propagated ex- vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of an effective amount of an agent for reducing the expression and/or activity of CD38, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• various agents were found to reduce the expression and/or activity of CD38, while at the same time, substantially inhibit differentiation ofthe hematopoietic stem cells under these conditions.
• the agent described above is an agent that reduces a capacity of the hematopoietic mononuclear cells in responding to retinoic acid, retinoids and/or Vitamin D, while at the same time, substantially inhibits differentiation ofthe hematopoietic stem cells.
• the agent described above is an agent that reduces a capacity of the hematopoietic mononuclear cells in responding to retinoic acid receptor, retinoid X receptor and/or Vitamin D receptor signaling, while at the same time, substantially inhibits differentiation of the stem cells.
• the agent described above is an agent that reduces a capacity of the hematopoietic mononuclear cells in responding to PI 3-kinase signaling, while at the same time, substantially inhibits differentiation ofthe stem cells.
• the agent described above comprises an effective amount of an agent selected from the group consisting of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative and a nicotinamide or a nicotinamide analog metabolite.
• the agent described above comprises an effective amount of a PI 3-kinase inhibitor.
• a transplantable hematopoietic cell preparation which comprises an expanded population of hematopoietic stem cells propagated ex-vivo from hematopoietic mononuclear cells which comprise, prior to expansion, a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, in the presence of at least one copper chelate or chelator, as defined hereinabove, while at the same time, substantially inhibiting differentiation of said hematopoietic stem cells, and a pharmaceutically acceptable carrier.
• hematopoietic stem cells ofthe present invention can be utilized in adoptive immunotherapy.
• a method of adoptive therapy according to the present invention is effected by (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells from a recipient; (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, with each of the copper chelators or chelates described hereinabove and/or each of the agents for reducing the expression and/or activity of CD38 described hereinabove, so as to expand the population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells, as is detailed
• stem cells in general and hematopoietic stem cells in particular may serve to exert cellular gene therapy.
• Gene therapy refers to the transfer of genetic material (e.g., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition or phenotype.
• the genetic material of interest encodes a product (e.g., a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired.
• the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
• ex-vivo gene therapy Two basic approaches to gene therapy have evolved: (i) ex-vivo or cellular gene therapy; and (ii) in vivo gene therapy.
• ex-vivo gene therapy cells are removed from a patient, and while being cultured are treated in-vitro.
• a functional replacement gene is introduced into the cells via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
• These genetically re-implanted cells have been shown to express the transfected genetic material in situ.
• the method is effected by (a) obtaining hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells, (b) providing the hematopoietic mononuclear cells with ex-vivo culture conditions for cell proliferation and, at the same time, with each of the copper chelators or chelates described hereinabove and/or each of the agents for reducing the expression and/or activity of CD38 described hereinabove, so as to expand the population of the hematopoietic stem cells, while at the same time, substantially inhibiting differentiation of the hematopoietic stem cells, as is detailed hereinabove, and (c) genetically modifying the hematopoietic stem cells with the exogene.
• genetically modifying the cells is effected by a vector, which comprises the exogene or transgene, which vector is, for example, a viral vector or a nucleic acid vector.
• a vector which comprises the exogene or transgene
• which vector is, for example, a viral vector or a nucleic acid vector.
• viral vectors suitable for use in cellular gene therapy are known, examples are provided hereinbelow.
• nucleic acid vectors can be used to genetically transform the expanded cells of the invention, as is further described below.
• the expanded cells of the present invention can be modified to express a gene product.
• gene product refers to proteins, peptides and functional RNA molecules.
• the gene product encoded by the nucleic acid molecule is the desired gene product to be supplied to a subject. Examples of such gene products include proteins, peptides, glycoproteins and Hpoproteins normally produced by an organ of the recipient subject.
• gene products which may be supplied by way of gene replacement to defective organs in the pancreas include insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triaclyglycerol lipase, phospholipase A2, elastase, and amylase; gene products normally produced by the liver include blood clotting factors such as blood clotting Factor VIII and Factor IX, UDP glucuronyl transferae, ornithine transcarbanoylase, and cytochrome p450 enzymes, and adenosine deaminase, for the processing of serum adenosine or the endocytosis of low density Hpoproteins; gene products produced by the thymus include serum thymic factor, thymic humoral factor, thymopoietin
• the encoded gene product is one, which induces the expression ofthe desired gene product by the cell (e.g., the introduced genetic material encodes a transcription factor, which induces the transcription ofthe gene product to be supplied to the subject).
• the recombinant gene can provide a heterologous protein, e.g., not native to the cell in which it is expressed.
• a heterologous protein e.g., not native to the cell in which it is expressed.
• various human MHC components can be provided to non-human cells to support engraftment in a human recipient.
• the transgene is one, which inhibits the expression or action of a donor MHC gene product.
• a nucleic acid molecule introduced into a cell is in a form suitable for expression in the cell of the gene product encoded by the nucleic acid.
• the nucleic acid molecule includes coding and regulatory sequences required for transcription of a gene (or portion thereof) and, when the gene product is a protein or peptide, translation of the gene acid molecule include promoters, enhancers and polyadenylation signals, as well as sequences necessary for transport of an encoded protein or peptide, for example N-terminal signal sequences for transport of proteins or peptides to the surface ofthe cell or secretion.
• Nucleotide sequences which regulate expression of a gene product are selected based upon the type of cell in which the gene product is to be expressed and the desired level of expression of the gene product. For example, a promoter known to confer cell-type specific expression of a gene linked to the promoter can be used. A promoter specific for myoblast gene expression can be linked to a gene of interest to confer muscle-specific expression of that gene product. Muscle-specific regulatory elements, which are known in the art, include upstream regions from the dystrophin gene (Klamut et al., (1989) Mol. Cell Biol.9: 2396), the creatine kinase gene (Buskin and Hauschka, (1989) Mol. Cell Biol.
• Regulatory elements specific for other cell types are known in the art (e.g., the albumin enhancer for liver-specific expression; insulin regulatory elements for pancreatic islet cell-specific expression; various neural cell-specific regulatory elements, including neural dystrophin, neural enolase and A4 amyloid promoters).
• a regulatory element which can direct constitutive expression of a gene in a variety of different cell types, such as a viral regulatory element, can be used. Examples of viral promoters commonly used to drive gene expression include those derived from polyoma virus, Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
• a regulatory element which provides inducible expression of a gene linked thereto, can be used.
• an inducible regulatory element e.g., an inducible promoter
• examples of potentially useful inducible regulatory systems for use in eukaryotic cells include hormone-regulated elements (e.g., see Mader, S. and White, J.H. (1993) Proc. Natl. Acad. Sci. USA 90: 5603-5607), synthetic ligand-regulated elements (see, e.g., Spencer, D.M. et al. 1993) Science 262: 1019-1024) and ionizing radiation-regulated elements (e.g., see Manome, Y. Et al.
• tissue-specific or inducible regulatory systems which may be developed, can also be used in accordance with the invention.
• the nucleic acid is in the form of a naked nucleic acid molecule.
• the nucleic acid molecule introduced into a cell to be modified consists only of the nucleic acid encoding the gene product and the necessary regulatory elements.
• the nucleic acid encoding the gene product is contained within a plasmid vector.
• plasmid expression vectors include CDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman, et al. (1987) EMBO J. 6: 187-195).
• the nucleic acid molecule to be introduced into a cell is contained within a viral vector.
• the nucleic acid encoding the gene product is inserted into the viral genome (or partial viral genome).
• the regulatory elements directing the expression of the gene product can be included with the nucleic acid inserted into the viral genome (i.e., linked to the gene inserted into the viral genome) or can be provided by the viral genome itself.
• Naked nucleic acids can be introduced into cells using calcium phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome-mediated transfection, direct injection, and receptor-mediated uptake.
• Naked nucleic acid e.g., DNA
• a precipitate containing the nucleic acid and calcium phosphate For example, a HEPES-buffered saline solution can be mixed with a solution containing calcium chloride and nucleic acid to form a precipitate and the precipitate is then incubated with cells.
• a glycerol or dimethyl sulfoxide shock step can be added to increase the amount of nucleic acid taken up by certain cells.
• CaPO4-mediated transfection can be used to stably (or transiently) transfect cells and is only applicable to in vitro modification of cells. Protocols for CaPO4-mediated transfection can be found in Current Protocols in Molecular Biology, Ausubel, F.M.
• Naked nucleic acid can be introduced into cells by forming a mixture of the nucleic acid and DEAE-dextran and incubating the mixture with the cells.
• a dimethylsulfoxide or chloroquine shock step can be added to increase the amount of nucleic acid uptake.
• DEAE-dextran transfection is only applicable to in vitro modification of cells and can be used to introduce DNA transiently into cells but is not preferred for creating stably transfected cells. Thus, this method can be used for short-term production of a gene product but is not a method of choice for long-term production of a gene product. Protocols for DEAE-dextran-mediated transfection can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates (1989), Section 9.2 and in Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.41-16.46 or other standard laboratory manuals.
• Naked nucleic acid can also be introduced into cells by incubating the cells and the nucleic acid together in an appropriate buffer and subjecting the cells to a high-voltage electric pulse.
• the efficiency with which nucleic acid is introduced into cells by electroporation is influenced by the strength ofthe applied field, the length of the electric pulse, the temperature, the conformation and concentration of the DNA and the ionic composition of the media. Electroporation can be used to stably (or transiently) transfect a wide variety of cell types and is only applicable to in vitro modification of cells. Protocols for electroporating cells can be found in Current
• liposome-mediated transfection Another method by which naked nucleic acid can be introduced into cells includes liposome-mediated transfection (lipofection).
• the nucleic acid is mixed with a liposome suspension containing cationic lipids.
• the DNA/liposome complex is then incubated with cells.
• Liposome mediated transfection can be used to stably (or transiently) transfect cells in culture in vitro. Protocols can be found in Current Protocols in Molecular Biology, Ausubel F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.4 and other standard laboratory manuals. Additionally, gene delivery in vivo has been accomplished using liposomes. See for example Nicolau et al. (1987) Meth. Enz.
• Naked nucleic acid can also be introduced into cells by directly injecting the nucleic acid into the cells.
• DNA can be introduced by microinjection. Since each cell is microinjected individually, this approach is very labor intensive when modifying large numbers of cells.
• microinjection is a method of choice is in the production of transgenic animals (discussed in greater detail below).
• the DNA is stably introduced into a fertilized oocyte, which is then allowed to develop into an animal.
• the resultant animal contains cells carrying the DNA introduced into the oocyte.
• Direct injection has also been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al.
• a delivery apparatus e.g., a "gene gun" for injecting DNA into cells in vivo can be used.
• a delivery apparatus e.g., a "gene gun” for injecting DNA into cells in vivo
• a cation such as polylysine
• a ligand for a cell-surface receptor to be taken up by receptor-mediated endocytosis (see for example Wu, G. and Wu, CH. (1988) J. Biol. Chem. 263: 14621 ; Wilson et al. (1992) J Biol. Chem. 267: 963-967; and U.S. Patent No. 5,166,320).
• Binding of the nucleic acid-ligand complex to the receptor facilitates uptake of the
• Receptors to which a DNA-ligand complex has targeted include the transferrin receptor and the asialoglycoprotein receptor.
• DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al.
• Receptor-mediated DNA uptake can be used to introduce
• telomeres when naked DNA is introduced into cells in culture (e.g., by one of the transfection techniques described above, only a small fraction of cells (about 1 out of 10 ) typically integrate the transfected DNA into their genomes (i.e., the DNA is maintained in the cell episomally).
• a selectable marker in order to identify cells, which have taken up exogenous DNA, it is advantageous to transfect nucleic acid encoding a selectable marker into the cell along with the nucleic acid(s) of interest.
• selectable markers include those, which confer resistance to drugs such as G418, hygromycin and methotrexate. Selectable markers may be introduced on the same plasmid as the gene(s) of interest or may be introduced on a separate plasmid.
• a preferred approach for introducing nucleic acid encoding a gene product into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA, encoding the gene product.
• a viral vector containing nucleic acid e.g., a cDNA
• Infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid which can obviate the need for selection of cells which have received the nucleic acid.
• molecules encoded within the viral vector e.g., a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid and viral vector systems can be used either in vitro or in vivo.
• a recombinant retrovirus can be constructed having a nucleic acid encoding a gene product of interest inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.
• retroviruses include pLJ, pZIP, pWE and pEM, which are well known to those skilled in the art.
• suitable packaging virus lines include ⁇ Crip, ⁇ Crip, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danosand Mulligan (1988) Proc.
• Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
• the genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68: 143-155.
• Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
• Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl.
• adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
• the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol 57: 267).
• Most replication-defective adenoviral vectors currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material.
• Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
• another virus such as an adenovirus or a herpes virus
• helper virus for efficient replication and a productive life cycle.
• AAV Adeno-associated virus
• It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7: 349-356; Samulski et al. (1989) J. Virol.
• AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 can be used to introduce DNA into cells.
• a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466- 6470; Tratschin et al. (1985) Mol. Cell Biol.
• DNA introduced into a cell can be detected by a filter hybridization technique (e.g., Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
• the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product, such as an enzymatic assay.
• an expression system can first be optimized using a reporter gene linked to the regulatory elements and vector to be used.
• the reporter gene encodes a gene product, which is easily detectable and, thus, can be used to evaluate efficacy of the system.
• Standard reporter genes used in the art include genes encoding ⁇ - galactosidase, chloramphenicol acetyl transferase, luciferase and human growth hormone.
• the modified population of cells may be used without further isolation or subcloning of individual cells within the population. That is, there may be sufficient production of the gene product by the population of cells such that no further cell isolation is needed.
• Such a population of uniform cells can be prepared by isolating a single modified cell by limiting dilution cloning followed by expanding the single cell in culture into a clonal population of cells by standard techniques.
• providing the hematopoietic mononuclear cells with conditions for ex-vivo cell proliferation is effected by providing the cells with nutrients and with cytokines.
• the cytokines are early acting cytokines, such as, but not limited to, stem cell factor, FLT3 ligand, interleukin-1 , interleukin-2, interleukin-3, interleukin-6, interleukin-10, interleukin-12, tumor necrosis factor- ⁇ and thrombopoietin.
• stem cell factor FLT3 ligand
• interleukin-1 interleukin-2
• interleukin-3 interleukin-6
• interleukin-10 interleukin-12
• tumor necrosis factor- ⁇ tumor necrosis factor- ⁇
• thrombopoietin tumor necrosis factor- ⁇
• Late acting cytokines can also be used.
• granulocyte colony stimulating factor include, for example, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, erythropoietin, FGF, EGF, NGF, VEGF, LIF, Hepatocyte growth factor and macrophage colony stimulating factor.
• the ability of the agents of the present invention to inhibit differentiation of hematopoietic stem cells present in hematopoietic mononuclear cells can be further used in technical applications such as cells collection and cells culturing.
• a hematopoietic stem cells collection/culturing bag is supplemented with an effective amount of a retinoic acid receptor antagonist, a retinoid X receptor antagonist and/or a Vitamin D receptor antagonist, which substantially inhibits cell differentiation of a hematopoietic stem cells fraction of hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells.
• the hematopoietic stem cells collection/culturing bag of the present invention is supplemented with an effective amount of nicotinamide, a nicotinamide analog, a nicotinamide or a nicotinamide analog derivative or a nicotinamide or a nicotinamide analog metabolite.
• the hematopoietic stem cells collection/culturing bag of the present invention is supplemented with an effective amount of a PI 3-kinase inhibitor.
• the hematopoietic stem cells collection/culturing bag of the present invention is supplemented with an effective amount of one or more copper chelator(s) or chelate(s).
• an assay of determining whether a specific molecule/agent e.g., a retinoic acid receptor antagonist, a retinoid X receptor antagonist, a Vitamin D receptor antagonist, a CD38 inhibitor, a PI 3-kinase inhibitor, a copper chelator or a copper chelate, is an effective agent for expanding a population of hematopoietic stem cells that are present in a hematopoietic mononuclear cells fraction.
• a specific molecule/agent e.g., a retinoic acid receptor antagonist, a retinoid X receptor antagonist, a Vitamin D receptor antagonist, a CD38 inhibitor, a PI 3-kinase inhibitor, a copper chelator or a copper chelate
• the assay is performed by culturing hematopoietic mononuclear cells which comprise a major fraction of hematopoietic committed cells and a minor fraction of hematopoietic stem and progenitor cells in the presence of the tested agent/molecule and monitoring expansion of the hematopoietic stem cells over time, e.g., a few weeks to a few months. If increased expansion and decreased differentiation occurs, as compared to non-treated cells, the tested agent/molecule is an effective hematopoietic stem cell expansion agent.
• culturing the hematopoietic mononuclear cells is performed in a presence of an effective amount of a cytokine, preferably, an early acting cytokine or a combination of such cytokines, e.g., thrombopoietin (TPO), interleukin-6 (IL-6), an cytokine, a cytokine, preferably, thrombopoietin (TPO), interleukin-6 (IL-6),
• FLT-3 ligand and stem cell factor SCF
• This assay can be used, by one ordinarily skilled in the art, to determine, for example, which of the antagonists, inhibitors or copper chelators and chelates listed above is most efficient for the purpose of implementing the various methods and preparations ofthe present invention described hereinabove.
• the assay can be further used to determine most effective concentrations and exposure time for achieving optimal results with hematopoietic mononuclear cells of different origins.
• the hematopoietic mononuclear cells can be obtained from any multicellular organism including both animals and plants.
• the hematopoietic mononuclear cells are obtained from the bone marrow (Rowley SD et al. (1998) Bone Marrow Transplant 21: 1253), the peripheral blood (Koizumi K, (2000) Bone Marrow Transplant 26: 787, the liver (Petersen BE et al. (1998) Hepatology 27: 433) and neonatal umbilical cord blood.
• Sample collection and processing Samples were obtained from umbilical cord blood after a normal full-term delivery and were frozen within 24 hours pospartum. The blood cells were thawed in Dextran buffer and incubated for 15 hours in MEM (Biological Industries, Israel) supplemented with 10 % fetal calf serum
• FCS Ficoll-Hypaque
• the mononuclear cells in the interface layer were then collected, washed three times in phosphate -buffered saline (PBS; Biological Industries), and re-suspended in PBS containing 0.5 % human serum albumin (HSA).
• PBS phosphate -buffered saline
• HSA human serum albumin
• MiniMACS CD34+ progenitor cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's recommendations. The purity of the CD34 + cells obtained ranged between 95 % and 98 %, based on Flow Cytometry evaluation.
• alpha minimal essential medium ⁇ -MEM
• FBS fetal bovine serum
• the purified CD34 + cells were similarly plated or seeded in the Culture Bags, at a concentration of about 10 4 cells/ml.
• the media were supplemented with tetraethylpantamine (TEPA) chelator (obtained from Sigma) and/or with the following human recombinant cytokines (all obtained from Perpo Tech, Inc., Rocky Hill, NJ): Thrombopoietin (TPO), 50 ng/ml; interieukin 6 (IL-6), 50 ng/ml; FLT-3 ligand, 50 ng/ml and a stem cell factor (SCF), 50 ng/ml; occasionally SCF was replaced by IL-3, 20 ng/ml.
• TEPA tetraethylpantamine
• SCF stem cell factor
• Cells were harvested, washed with a PBS solution containing 1 % bovine sera albumin (BSA) and 0.1 % sodium azide (Sigma), and stained at 4 °C for 60 minutes with fluorescein isothiocyanate or phycoerythrin- conjugated antibodies (all from Immunoquality Products, the Netherlands). The cells were then washed with the same buffer and analyzed by FACS caliber or Facstarplus flow cytometers. Cells were passed at a rate of 1000 cells/second, using saline as the sheath fluid. A 488 nm argon laser beam served as the light source for excitation. Emission of ten thousand cells was measured using logarithmic amplification, and analyzed using CellQuest software.
• BSA bovine sera albumin
• sodium azide Sigma
• Reselected CD34+ cell subsets were stained for the following combination of antigens: CD34PE/CD38FITC and CD34PE/38-, 33-, 14-, 15-, 3, 4, 61, 19 (Lin) FITC.
• FACS analysis results are given as percentage values of cells. Absolute numbers of subsets are calculated from the absolute number of CD34+ cells.
• CD34+ cells were purified from 3 thawed cord blood units and stained for the above markers. The mean of these experiments was considered as the baseline value. Total cell counts, numbers of CD34+ cells and subsets, and CFU numbers are presented as cumulative numbers, with the assumption that the cultures had not been passaged; i.e., the number of cells per ml were multiplied by the number of passages performed.
• CFU Colony Forming Unit
• LTC-CFUc values The ability of the cultures to maintain self-renewal was measured by determination of the content of colony forming unit cells in the long and extended long-term cultures (LTC-CFUc), as described in the references hereinabove.
• MNC Mononuclear cells
• the MNC cultures were either treated or untreated (untreated controls) with various concentrations (5-10 ⁇ M) of TEPA chelator.
• the treated MNC cultures were supplemented with TEPA for only the first three weeks and from week three onward were topped with chelator-free media.
• the pre-purified CD34 + cultures were not supplemented with TEPA and served as positive controls.
• the cultures were analyzed weekly during a 12-week period for the number of cells, CFUc, CD34+ and CD34+CD38- cells. In order to precisely determine the CD34+ cell content, CD34+ cells were weekly reselected and enumerated from each of the experimental groups (treated and untreated MNC cultures) and the positive control (CD34+ cultures).
• stem and progenitor cells densities in the TEPA- treated MNC cultures, either equalized or surpassed the densities of stem and progenitor cells in pre-purified CD34+ cell cultures (not treated with TEPA, positive controls).
• stem and progenitor hematopoietic cells may be substantially expanded ex-vivo, continuously over at least 12 weeks period, in a culture of mixed (mononuclear fraction) blood cells, with no prior purification of CD34 + cells.
• the data also show that this effect resulted from supplementing the cells culture medium with TEPA chelator, only during the first three weeks of culturing.
• the chelator may also enable ex-vivo expansion of a small subset of cells that are not co-purified with the CD34+ cell fraction. This subset of cells, which is probably in nature CD34-, may support superior expansion of
• CD34+ cells and its subsets during the extended long-term cultures are CD34+ cells and its subsets during the extended long-term cultures.
• this Example illustrates a substantial ex-vivo expansion of stem and progenitor cells in a mixed mononuclear cells culture.
• This novel procedure circumvents the need of the laborious and costly enrichment of stem cells prior to initiation of cultures, which is currently used in the art.
• the use of a copper chelator, such as TEPA can substantially simplify, reduce cost and improve efficiency of procedures for an ex-vivo expansion of stem and/or progenitor cells.
• Copper-TEPA chelate was prepared as described, for example, in PCT/IL03/00062.
• Mononuclear cells (MNC) were seeded in culture bags and were provided with nutrients and cytokines as described in Example 1 above.
• the mononuclear cell cultures were either untreated (control) or treated with Cu-TEPA chelate.
• the treated MNC cultures were supplemented with Copper-TEPA chelate for the first three weeks and from week three onward were topped with chelator-free media. All cultures were analyzed eight weeks after an 8-week period.
• hematopoietic stem cells may be substantially expanded ex-vivo, over at least 8 weeks period, in a culture of mononuclear blood cells, with no prior purification of CD34 + cells, in the presence of a copper chelate such as Copper-TEPA.
• RAR retinoic acid receptor
• Example 1 MNC cultures were prepared and maintained as described above. AGN 194310 RAR antagonist was added to the tested cultures at concentrations ranging from 1 x 10 "3 - 1 x 10 " " M [or 410 ⁇ g/1 to 4.1 x 10 "5 ⁇ g/1]. The antagonist was added for a predetermined, limited period, for up to three weeks or continuously during the entire culture period.
• the MNC cells responded to the RAR antagonists and expanded an undifferentiated population, without prior purification of the CD34+ population.
• RAR antagonist treatment was sufficient to stimulate specific expansion of the stem/progenitor cell compartment, at 5 weeks post seeding. While control untreated MNCs had no detectable CD34+ population, RAR antagonist treated cultures revealed significant numbers of CD34+ cells, and those that were lineage marker deficient. Thus, any factors elaborated by the MNC culture cells that suppress CD34+ cell survival in control samples are insufficient to override the signal provided by the RAR antagonist to elaborate this compartment.

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