WO2013066368A1 - Use of mirna 126 to produce hematopoietic stem cells - Google Patents

Use of mirna 126 to produce hematopoietic stem cells Download PDF

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Publication number
WO2013066368A1
WO2013066368A1 PCT/US2012/000472 US2012000472W WO2013066368A1 WO 2013066368 A1 WO2013066368 A1 WO 2013066368A1 US 2012000472 W US2012000472 W US 2012000472W WO 2013066368 A1 WO2013066368 A1 WO 2013066368A1
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cells
nucleic acid
hspc
spc
acid molecule
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PCT/US2012/000472
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French (fr)
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Giovanna Tosato
Ombretta SALVUCCI
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The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services
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Publication of WO2013066368A1 publication Critical patent/WO2013066368A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/65—MicroRNA
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • HSPC hematopoietic stem/progenitor cells
  • the description relates to methods of using nucleic acid molecules having part or all of the sequence of miR126 (SEQ ID NOs: 1 or 2) to enhance mobilization of HSPC, particularly when harvesting of HSPC for transplantation would be beneficial in ameliorating the symptoms associated with hematopoietic diseases, disabilities or conditions.
  • the description also includes administering miR126 nucleic acids to treat such diseases, and for screening novel agents for their ability to affect expression of miR126.
  • HSPC hematopoietic stem/progenitor cells
  • Chemotherapy can cause myelosuppression and unacceptably low levels of white blood cells, making patients susceptible to infections and sepsis.
  • HSPC production is stimulated by G-CSF, which is used with certain cancer patients to accelerate recovery from neutropenia after chemotherapy, allowing higher-intensity treatment regimens.
  • G-CSF is also used to increase the number of transplantable HSPC in the blood of the donor before collection by leukapheresis for use in HSPC transplantation. It may also be given to the receiver, to compensate for conditioning regimens.
  • G-CSF has been used in treatment of hematological deficiencies involves isolating HSPC from blood of healthy donor by administering G-CSF, removing blood, purifying white cells, selecting CD34 + cells by immunopurification, storing the HSPC, and administering these cells to a histocompatible recipient patient.
  • HSPC reside in bone marrow niches that support their survival and function.
  • a variety of compounds can induce mobilization of HSPC from the bone marrow to the peripheral circulation.
  • G-CSF is the most commonly used inducer of HSPC mobilization, whereby HSPC are obtained for autologous and allogeneic hematopoietic reconstitution (Greenbaum et al., 201 1 , Leukemia 25:21 1-17; Pelus et al., 2008, Leukemia, 22:466-73).
  • G-CSFR G-CSF receptor
  • G-CSFR hematopoietic stem cells do not generally express G-CSFR (Ebihara et al., 2000, Br J Haematol, 109: 153-61). G-CSF mobilizes equally effectively HSPC that express or not express G-CSFR (Liu et al. 2000), pointing to other factors involved in G-CSF induced HSPC mobilization.
  • Neutrophils and/or monocytes are key intermediate regulators of HSPC mobilization by G-CSF (Pelus et al., 2004, Blood, 103: 1 10-19; Christopher et al., 201 1 , J Exp Med, 208:251-60).
  • CXCR4 and its unique ligand SDF1/CXCL12 are essential for the retention of granulocytes and other myeloid-lineage cells to the bone marrow, and that disruption of CXCR4 signaling is sufficient for mobilization of neutrophils and other myeloid-lineage cells to the peripheral circulation (Balabanian et al., 2005, Blood, 105:2449-57; Eash et al., 2009, Blood, 1 13:471 1-19; DiPersio et al., 2009, Blood, 1 13:5720-26; Semerad et al., 2005, Blood, 106:3020-27).
  • G-CSF rapidly inhibits expression of CXCR4 in myeloid cells (Kim et al., 2006,
  • G-CSF promotes the release of a variety of neutrophil proteases, including neutrophil elastase, cathepsin-G and MMP-9 generating a proteolytic environment in the bone marrow (Pelus et al. 2004; Levesque et al., 2001, Blood, 98:1289-97).
  • neutrophil elastase neutrophil elastase
  • MMP-9 a proteolytic environment in the bone marrow
  • CXCR4 is believed to be critical to mobilization of hematopoietic progenitors by G-CSF (Christopher et al. 2009), but other mechanisms have also been proposed and conclusive evidence is missing (Greenbaum et al. 201 1 ; Pelus et al. 2004, Levesque et al. 2001)
  • One aspect of the description is a method for modulating the phenotype of a stem progenitor cell (SPC), particularly hematopoietic SPC (HSPC), comprising contacting the SPC with a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least about 90% identical to the sequence of miR126 (SEQ ID NOs: 1 or 2), or the complements thereof.
  • the method may further comprise collecting and isolating the SPC after contact with the nucleic acid molecule.
  • Another aspect of the description is a method comprising administering G-CSF to a healthy subject, collecting the isolated SPC from the subject, and administering the SPC to a patient.
  • composition consisting of SPC that are produced by administering miR126 nucleic acids to a healthy subject, and collecting SPC from the subject.
  • Another aspect of the description is a method for treating hematopoietic deficiency or hematological failure, comprising administering SPC cells to a patient having hematopoietic deficiency or hematological failure, specifically SPC cells from blood of a healthy subject treated with miR126.
  • the SPC blood cells from a healthy subject may be administered to a patient following bone ablation therapy.
  • Another aspect of the description is a method for modulating the phenotype of a stem SPC, including HSPC, comprising administering miR126 nucleic acids to a patient having a hematopoietic deficiency or hematological failure.
  • Another aspect of the description is a method of treating the hematopoietic deficiency or hematological failure of the patient comprising administering miR126 nucleic acids to a patient having a hematopoietic deficiency or hematological failure.
  • the hematopoietic deficiency or hematological failure may be associated with a cancer.
  • Another aspect of the description is a method of using miR126 nucleic acid molecules to decrease VCAM-1 expression on the stem progenitor cells.
  • Another aspect of the description is a method for modulating the phenotype of SPC, including HSPC, comprising contacting SPC with miR126 performed ex vivo.
  • One embodiment consists of contacting isolated peripheral blood or bone marrow cells with miR126.
  • composition comprising SPC or HSPC.
  • Another aspect of the description is a method of screening a candidate substance for an effect on hematopoietic cell phenotype, comprising providing a SPC; contacting the SPC with the candidate substance; and assessing the effect of the candidate substance on the expression or stability miR126 miRNA.
  • Another aspect of the description is a method of treating cancer in a cancer patient in need thereof, comprising administering to a normal subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof, harvesting hematopoietic stem or progenitor cells (HSPC) from the blood of the subject, and administering the HSPC to the cancer patient.
  • HSPC hematopoietic stem or progenitor cells
  • Another aspect of the description is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • Another aspect of the description is a method of screening a candidate compound for an effect on mobilization of stem or progenitor cells (SPC), comprising providing a SPC; contacting the SPC with said candidate compound; and measuring the effect of said candidate substance on the expression or stability of miR126 miRNA.
  • SPC stem or progenitor cells
  • Another aspect of the description is a use of a chemotherapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises
  • HSPC allogenic hematopoietic stem or progenitor cells
  • a chemotherapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises administering the medicament and a composition comprising autologous hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • HSPC autologous hematopoietic stem or progenitor cells
  • Another aspect of the description is a use of a chemotherapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises
  • composition a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • Another aspect of the description is a chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the
  • chemotherapeutic agent and a composition comprising allogenic hematopoietic stem or progemtor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • HSPC allogenic hematopoietic stem or progemtor cells
  • Another aspect of the description is a chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the
  • chemotherapeutic agent and a composition comprising autologous hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • HSPC autologous hematopoietic stem or progenitor cells
  • Another aspect of the description is a chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the
  • chemotherapeutic agent and a composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • composition comprising allogenic hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • HSPC allogenic hematopoietic stem or progenitor cells
  • composition comprising autologous hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • HSPC autologous hematopoietic stem or progenitor cells
  • composition comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof, for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition.
  • composition comprising autologous hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
  • HSPC autologous hematopoietic stem or progenitor cells
  • FIG. 1 shows G-CSF mobilization modulated CXCR4 and CXCR2 expression in bone marrow myeloid cells, but not in HSPC.
  • Cell surface CXCR4 was evaluated by flow cytometry in all cells, Grl'° bone marrow cells (Fig. 1A) and Grl hl bone marrow cells and in Lin " Sca hi cKit hi bone marrow cells (Fig. 2B) in the presence and absence of G-CSF.
  • Cell surface CXCR2 was evaluated by flow cytometry in all cells, Grl'° bone marrow cells and Grl hl bone marrow cells (Fig. 1C) and in Lin " Sca h 'cKit hl bone marrow cells (Fig. ID) in the presence and absence of G-CSF. Representative experiment of 5 performed. Results are expressed as mean ⁇ SD (5-8/group). "* ** denotes p ⁇ 0.05.
  • FIG. 3 shows an analysis of relative levels of CXCR4 mRNA (Fig. 3A) and CXCR2 mRNA (Fig. 3B) in non-fractionated bone marrow cells ("All cells") and in sorted Lin " Sca hi cKit hi HSPC from mice mobilized with G-CSF or left untreated. Results are from
  • FIG. 4 shows G-CSF mobilization is associated with decreased percentage surface VCAM-1 expression on HSPC, LinTerl 19 " CD45 " and VE-cadherin + cells from the bone marrow.
  • Cell surface VCAM-1 in Lin “ Sca h 'cKit hl cells (representative individual results are shown as averages of the VCAM-1 expression) (Fig. 4A); LinTerl 19 " CD45 " (Fig. 4B) and VE- cadherin + cells (Fig. 4C) in bone marrow from mice mobilized with G-CSF or left untreated (5- 10/group).
  • FIG. 6 shows miR126 regulates VCAM-1 expression in bone marrow cells.
  • FIG. 7 shows G-CSF mobilization regulates miR126 distribution in the bone marrow.
  • Fig. 7A shows G-CSF mobilization reduces in bone marrow cells (left) and increased in the cell-free fraction (right) relative levels of miR126 (mean ⁇ SD; 5-8 mice/group untreated and G-CSF treated).
  • Fig. 7B shows relative levels of miR126 (miR126/miR16) in
  • FIG. 8 shows G-CSF modulates the relative content of miR126 in bone marrow cells and culture supernatant.
  • Unfractionated bone marrow cells were cultured (lxl0 6 /ml) for 18 hours with or without G-CSF (100 ng/ml) in complete culture medium; RNA was extracted from the cells and cell-free supernatant. The results are expressed as relative miR126/miR16 levels; representative experiment of 3 performed.
  • FIG. 9 shows G-CSF mobilization regulates levels of miR126 in bone marrow cell populations.
  • Bone marrows (3 combined) from untreated or G-CSF mobilized mice were tested for relative miR126/miR16 levels: (Fig. 9A) before and (Fig. 9B) after sorting the Sca hi cKit hi HPC; and (Fig. 9C) before and (Fig. 9D) after sorting the LinTerl 19 " CD45 " cells.
  • FIG. 10 shows uptake of microvesicles by bone marrow cells effects surface
  • FIG. 10 A shows uptake of PKH-labeled exosome preparations (Exo) from bone marrow of G-CSF mobilized or untreated mice (5 bone marrows each) by (from the top): unfractionated, Grl + , Sca-l hi cKit hi , Lin ' CD45 " and VE-cadherin + bone marrow cell populations.
  • FIG. 10B shows VCAM-1 MFI measured on Sca-l hi cKit hi cells from bone marrows incubated with PKH-labeled exosome preparations from untreated or G- CSF-mobilized bone marrows. VCAM-1 MFI was measured on Sca-l hl cKit hl cells with or without microvesicles-derived fluorescence (means ⁇ SD of 3 independent experiments). The percentage of VCAM1 + cells was measured in (Fig. IOC) LinTerl 19 " CD45 " and (Fig. 10D) VE- cadherin + bone marrow cells that had either acquired or not acquired microvesicles-derived PKH fluorescence after 18 hour incubation. Micro vescicles were from untreated and G-CSF-mobilized bone marrows. The results reflect the mean ⁇ SD of 3 independent experiments. "*" denotes p ⁇ 0.05.
  • FIG. 11 shows defective HSPC mobilization in G-CSF-treated miRl 26- deficient mice.
  • IIC shows schematic representations of HSPC and myeloid-lineage cell trafficking from the bone marrow to the blood; contribution of CXCR4, CXCR2, VCAM-1 and microvesicles containing miRl 26.
  • hematopoietic stem and progenitor cells are self-renewing precursors that regenerate myeloid, erythroid, and lymphoid cells throughout the life span of the subject or patient.
  • stem cell is meant to encompass stem cells and progenitor cells of various levels of pluripotency.
  • subject and patient are used interchangeably for the purpose of this description, wherein either a subject or a patient refers to a living mammal, which includes humans and other mammals that persons of ordinary skill in the art commonly use.
  • hematopoietic stem and progenitor cells refers to the recruitment of HSPC into the blood.
  • HSPC are found in bone marrow, spleen, umbilical cord blood, and the blood and liver of fetuses and newborns.
  • cells obtained from bone marrow, cord blood, or mobilized peripheral blood of healthy donors are clinically useful for transplantation into a recipient subject.
  • Modulation or “modulates” or “modulating” refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
  • Treatment refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted.
  • the treatments using the agents described may be provided to treat patients suffering from a cancerous condition or hyperproliferative disease, whereby the treatment of the disease with chemotherapy or irradiation therapy results in a decrease in bone marrow cellularity, thus making the patient more prone to acquiring infectious agents or diseases.
  • the administration of any of the agents of the description allows for the mobilization of hematopoietic stem cells or progenitor cells from the bone marrow to the peripheral blood.
  • the treating is for the purpose of reducing or diminishing the symptoms or progression of a cancerous disease or disorder by allowing for the use of accelerated doses of chemotherapy or irradiation therapy.
  • Subject or “patient” refers to a mammal, preferably a human, in need of treatment for a condition, disorder or disease.
  • prophylactic or therapeutic treatment refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • a "mobilizer of hematopoietic stem cells or progenitor cells” or “mobilizer”, (used interchangeably) as described herein refers to any compound, whether it is a small organic molecule, synthetic or naturally derived, or a polypeptide, such as a growth factor or colony stimulating factor or an active fragment or mimic thereof, a nucleic acid, a carbohydrate, an antibody, or any other agent that acts to enhance the migration of stem cells from the bone marrow into the peripheral blood.
  • Such a “mobilizer” may increase the number of hematopoietic stem cells or hematopoietic progenitor/precursor cells in the peripheral blood, thus allowing for a more accessible source of stem cells for use in transplantation.
  • Stem Cells are cells, which are not terminally differentiated and are therefore able to produce cells of other types. Stem cells are divided into three types, including totipotent, pluripotent, and multipotent. "Totipotent stem cells” can grow and differentiate into any cell in the body, and thus can grow into an entire organism. These cells are not capable of self-renewal. In mammals, only the zygote and early embryonic cells are totipotent. "Pluripotent stem cells” are true stem cells, with the potential to make any differentiated cell in the body, but cannot contribute to making the extraembryonic membranes (which are derived from the trophoblast).
  • Multipotent stem cells are clonal cells that self-renew as well as differentiate to regenerate adult tissues, and can give rise to cells of multiple lineages. In contrast, “unipotent stem cells” can only give rise to cells of one lineage, such as blood cells or bone cells.
  • stem cells refers to pluripotent stem cells capable of self-renewal.
  • Cancer stem cells refers to a small population of cells that are quiescent, which are capable of self-renewal, and which appear to be the sources of cells comprising a malignant and/or metastatic tumor.
  • Hematopoiesis refers to the highly orchestrated process of blood cell development and homeostasis. Prenatally, hematopoiesis occurs in the yolk sack, then liver, and eventually the bone marrow. In normal adults it occurs in bone marrow and lymphatic tissues. All blood cells develop from pluripotent stem cells. Pluripotent cells differentiate into stem cells that are committed to three, two or one hematopoietic differentiation pathway. None of these stem cells are morphologically distinguishable, however.
  • hematopoietic stem cells as used in the present description means multipotent stem cells that are capable of differentiating into all blood cells including
  • hematopoietic stem cells are contained not only in bone marrow but also in umbilical cord blood derived cells.
  • hematopoietic progenitors which is used interchangeably with the term “hematopoietic precursors”, refers to those progenitor or precursor cells which are differentiated further than hematopoietic stem cells but have yet to differentiate into progenitors or precursors of respective blood cell lineages (unipotent precursor cells).
  • progenitor cell(s) or “precursor cell(s)” are defined as cells that are lineage-committed, i.e., an individual cell can give rise to progeny limited to a single lineage such as the myeloid or lymphoid lineage or two lineages. They do not have self-renewal properties. They can also be stimulated by lineage-specific growth factors to proliferate. If activated to proliferate, progenitor cells have life-spans limited to 50-70 cell doublings before programmed cell senescence and death occurs.
  • the "hematopoietic progenitors” as used in the present description include granulocyte/macrophage associated progenitors (colony-forming unit granulocyte, macrophage, CFU-GM), erythroid associated progenitors (burst forming unit erythroid, BFU-E),
  • megakaryocyte associated progenitors colony-forming unit megakaryocyte, CFU-Mk
  • myeloid associated stem cells colony-forming unit mixed, CFU-Mix.
  • Hematopoietic progenitor cells possess the ability to differentiate into a final cell type directly or indirectly through a particular developmental lineage. Undifferentiated, plunpotent progenitor cells that are not committed to any lineage are referred to herein as "stem cells.” All hematopoietic cells can in theory be derived from a single stem cell, which is also able to perpetuate the stem cell lineage, as daughter cells become differentiated.
  • the term "differentiation" of hematopoietic stem cells and/or hematopoietic progenitors as used in the description means both the change of hematopoietic stem cells into hematopoietic progenitors and the change of hematopoietic progenitors into unipotent hematopoietic progenitors and/or cells having characteristic functions, namely mature cells including erythrocytes, leukocytes and megakaryocytes.
  • Differentiation of hematopoietic stem cells into a variety of blood cell types involves sequential activation or silencing of several sets of genes. Hematopoietic stem cells choose either a lymphoid or myeloid lineage pathway at an early stage of differentiation.
  • CXCL12 also known as stromal cell-derived factor-1 or "SDF-1" refers to a
  • CXC chemokine that demonstrates in vitro activity with respect to lymphocytes and monocytes but not neutrophils. It is highly potent in vivo as a chemoattractant for mononuclear cells. SDF-1 has been shown to induce intracellular actin polymerization in lymphocytes, and to induce a transient elevation of cytoplasmic calcium in some cells.
  • function of a chemokine, CXCL12 is meant the binding of the chemokine to its receptor and the subsequent effects on signaling.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Cell used herein may be a naturally occurring cell or a transformed cell that may contain a vector and may support replication of the vector.
  • Cells may be cultured cells, explants, cells in vivo, and the like.
  • Cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, such as CHO and HeLa.
  • a host cell is a hematopoietic cell, for example, a hematopoietic stem-progenitor cell, a long-term repopulating hematopoietic stem cell, a short- term repopulating hematopoietic stem cell, a multipotent progenitor cell, a common lymphoid progenitor cell, a pro-T cell, a T cell, an NK progenitor cell, an NK cell, a pro-B cell, a B cell, a common myeloid progenitor cell, a B-cell/macrophage bipotential cell, a granulocyte- macrophage progenitor cell, a megakaryocyte-erythrocyte progenitor cell, a macrophage, a granulocyte, a megakaryocyte progenitor cell, a megakaryocyte, or an erythrocyte. It is contemplated that the
  • nucleic acids or polypeptide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • Thymine (T) and uracil (U) may be considered equivalent when comparing DNA and RNA.
  • Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
  • nucleic acid or "oligonucleotide” or “polynucleotide” used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the
  • nucleic acid is a single stranded nucleic acid.
  • nucleic acid may be used for the same purpose as a given nucleic acid.
  • a single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions.
  • a nucleic acid may also be a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
  • a nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S.
  • nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids.
  • the modified nucleotide analog may be located for example at the 5'-end and/or the 3 '-end of the nucleic acid molecule.
  • Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase- modified ribonucleotides, i.e.
  • ribonucleotides containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5- position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N- alkylated nucleotides, e.g. N6-methyl adenosine are suitable.
  • uridines or cytidines modified at the 5- position e.g. 5-(2-amino)propyl uridine, 5-bromo uridine
  • adenosines and guanosines modified at the 8-position e.g. 8-bromo guanosine
  • the 2 -OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is Cj-Cg alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
  • Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Publication No. 20050107325.
  • Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 200201 15080. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005.
  • LNA locked nucleic acids
  • Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • miR126 consists of RNA encoded by a family of 22 gene sequences found in different locations of the human genome. One of these is located within the 7 th intron of the EFGL7 gene on chromosome 9. Its expression there is regulated by ETS1 and ETS2
  • the mature miR126 molecule has the following sequence (SEQ ID NO:2):
  • operably linked used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected.
  • a promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control.
  • the distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • Stringent hybridization conditions used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence- dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Tm thermal melting point
  • Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization.
  • Exemplary stringent hybridization conditions include the following: 50% formamide, 5xSSC, and 1% SDS, incubating at 42° C, or, 5xSSC, 1% SDS, incubating at 65° C, with wash in 0.2xSSC, and 0.1% SDS at 65° C.
  • substantially complementary used herein may mean that a first sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.
  • substantially identical used herein may mean that a first and second sequence are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22,23,24,25,30,35,40,45, 50, 55, 60, 65,70,75, 80, 85, 90, 95,100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
  • Variant used herein to refer to a nucleic acid may mean (i) a portion of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
  • Vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • the miRNAs of the present description may be desirable to express in a vector.
  • exogenous means that the vector or entity referred to is foreign to the cell into which it is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., BACs, YACs).
  • BACs BACs, YACs
  • Expression vector refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell.
  • control sequences refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • a gene coding for an miRNA may be transcribed leading to production of an miRNA precursor known as the pri-miRNA.
  • the pri- miRNA may be part of a polycistronic RNA comprising multiple pri-miRNAs.
  • the pri-miRNA may form a hairpin with a stem and loop.
  • the stem may comprise mismatched bases.
  • the hairpin structure of the pri-miRNA may be recognized by Drosha, which is an RNase III endonuclease. Drosha may recognize terminal loops in the pri-miRNA and cleave approximately two helical turns into the stem to produce a 60-70 nt precursor known as the pre- miRNA. Drosha may cleave the pri-miRNA with a staggered cut typical of RNase III
  • pre-miRNA stem loop with a 5' phosphate and about a 2 nucleotide 3' overhang.
  • Approximately one helical turn of the stem (about 10 nucleotides) extending beyond the Drosha cleavage site may be essential for efficient processing.
  • the pre-miRNA may then be actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex- portin-5.
  • the pre-miRNA may be recognized by Dicer, which is also an RNase III endonuclease. Dicer may recognize the double-stranded stem of the pre-miRNA. Dicer may also recognize the 5' phosphate and 3' overhang at the base of the stem loop. Dicer may cleave off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and about a 2 nucleotide 3' overhang. The resulting siRNA-like duplex, which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*. The miRNA and miRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA. miRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
  • RISC RNA-induced silencing complex
  • miRNA/miRNA* duplex is loaded in to the RISC.
  • the miRNA* When the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* may be removed and degraded.
  • the strand of the miRNA:miRNA* duplex that is loaded into the RISC may be the strand whose 5' end is less tightly paired. In cases where both ends of the miRNA: miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA* may have gene silencing activity.
  • the RISC may identify target nucleic acids based on high levels of
  • Base-pairing between miRNA and its mRNA target is required for achieving efficient inhibition of translation.
  • the first 8 nucleotides of the miRNA may be important.
  • other parts of the miRNA may also participate in mRNA binding.
  • the target sites in the mRNA may be in the 5' UTR, the 3' UTR or within the coding region.
  • Multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites.
  • the presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
  • a single miRNA may regulate multiple mRNA targets by recognizing the same or similar sites on the different targets.
  • miRNAs may direct the RISC to downregulate gene expression by several mechanisms: mRNA cleavage, translational repression, or chromatin remodeling.
  • the miRNA may specify cleavage of the mRNA if the miRNA has a certain degree of complementarity to the mRNA. When an miRNA guides cleavage, the cut may be between the nucleotides pairing to residues 10 and 1 1 of the miRNA.
  • the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
  • any pair of miRNA and miRNA* there may be variability in the 5' and 3' ends of any pair of miRNA and miRNA*. This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the site of cleavage. Variability at the 5' and 3' ends of miRNA and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a population of different hairpin structures. Variability in the stem structures may also lead to variability in the products of cleavage by Drosha and Dicer.
  • Nucleic acids for modulating the level or activity of miRNAs may, in addition to a sequence that is identical or homologous to that of the miRNA or complement thereof, also comprise additional, unrelated nucleotides, provided that they do not interfere with the mimicking or inhibition, respectively, of the miRNA. They may comprise, e.g., about 1,2,3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides that are located either 5' and/or 3' (or internal) to the sequence for modulating the level or activity of the miRNA.
  • An agonist of a miRNA is any agent, e.g., a nucleic acid comprising a nucleotide sequence, that is identical or homologous to that of the miRNA, and that mimics the action of the miRNA.
  • An antagonist of a miRNA (understood herein to also include agonists of the related molecules, e.g., pre- or pri-miRNA) is any agent, e.g., a nucleic acid comprising a nucleotide sequence, that is identical or homologous to that of the complement of the miRNA, and that inhibits the action of the miRNA.
  • a nucleic acid comprises at least about 2, 3, 5, 10, 15, 20, 25, 30, or more nucleotide sequences that are identical or homologous to that of a miRNA or the complement thereof.
  • the nucleotide sequences may be mimicking or inhibiting the level or activity of one miRNA or alternatively of at least about 2, 3, 5, 10, 15, 20, 25, 30, 32, 33, or more different miRNAs, e.g., those having SEQ ID NOs: 1 or 2.
  • nucleic acids controlling stem progenitor cell e.g., hematopoietic stem- progenitor, cell differentiation are provided herein.
  • the nucleic acid may comprise the sequence of SEQ ID NOs: 1 or 2, or variants thereof.
  • the variant may be a complement of the referenced nucleotide sequence.
  • the variant may also be a nucleotide sequence that is substantially identical to the referenced nucleotide sequence or the complement thereof.
  • the variant may also be a nucleotide sequence that hybridizes under stringent conditions to the referenced nucleotide sequence, complements thereof, or nucleotide sequences substantially identical thereto.
  • the nucleic acid may have a length of from 10 to 250 nucleotides.
  • the nucleic acid may have a length of at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,20,21 ,22,23,24,25,26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 nucleotides.
  • the nucleic acid may be synthesized or expressed in a cell (in vitro or in vivo) using a synthetic gene described herein.
  • the nucleic acid may be synthesized as a single stranded molecule and hybridized to a substantially complementary nucleic acid to form a duplex.
  • the nucleic acid may be introduced to a cell, tissue or organ in a single- or double-stranded form or capable of being expressed by a synthetic gene using methods well known to those skilled in the art. In certain embodiments, it may be useful to incorporate the nucleic acids into a vector, as described supra.
  • the nucleic acid may comprise a sequence of a pri-miRNA or a variant thereof.
  • the pri-miRNA sequence may comprise from 45-250, 55-200, 70-150 or 80-100 nucleotides.
  • the sequence of the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof.
  • the sequence of the pri-miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of an miRNA binding site on VCAM-1 , e.g., VCAM-1 3' UTR (Harris et al., 2008, PNAS, 105:1516-21), or variants thereof.
  • the pri-miRNA may form a hairpin structure.
  • the hairpin may comprise a first and second nucleic acid sequence that are substantially complementary.
  • the first and second nucleic acid sequence may be from 37-50 nucleotides.
  • the first and second nucleic acid sequence may be separated by a third sequence of from 8-12 nucleotides.
  • the hairpin structure may have a free energy less than -25 Kcal/mole as calculated by the Vienna algorithm with default parameters, as described in Hofacker et al., 1994, Monatshefte f. Chemie 125: 167-188.
  • the hairpin may comprise a terminal loop of 4-20, 8-12 or 10 nucleotides.
  • the pri-miRNA may comprise at least 19% adenosine nucleotides, at least 16% cytosine nucleotides, at least 23% thymine nucleotides and at least 19% guanine nucleotides.
  • the nucleic acid may also comprise a sequence of a pre-miRNA or a variant thereof.
  • the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
  • the sequence of the pre-miRNA may comprise a miRNA and a miRNA* as set forth herein.
  • the sequence of the pre-miRNA may also be that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA.
  • the sequence of the pri-miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of a miRNA binding site on VCAM-1, e.g., VCAM-1 3' UTR, or variants thereof.
  • the nucleic acid may also comprise a sequence of a miRNA (including miRNA*) or a variant thereof.
  • the miRNA sequence may comprise from 13-33, 18-24 or 21-23 nucleotides.
  • the miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26,27,28, 29,30,31 ,32, 33, 34,35,36,37, 38, 39 or 40 nucleotides.
  • the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
  • the sequence of the pri-miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of a miRNA binding site on VCAM-1 , e.g., VCAM-1 3' UTR, or variants thereof.
  • the nucleic acid may also comprise a sequence of an anti-miRNA that is capable of blocking the activity of a miRNA or miRNA*, such as by binding to the pri-miRNA, pre-miRNA, miRNA or miRNA* (e.g. antisense or RNA silencing), or by binding to the target binding site.
  • the anti-miRNA may comprise a total of 5-100 or 10-60 nucleotides.
  • the anti- miRNA may also comprise a total of at least 5, 6, 7, 8, 9,, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the sequence of the anti-miRNA may comprise (a) at least 5 nucleotides that are substantially complementary to the 5' of a miRNA and at least 5-12 nucleotides that are substantially identical to the flanking regions of the target site from the 5 * end of the miRNA, for the purposes of binding to a miRNA and repressing its activity; or (b) at least 5-12 nucleotides that are substantially identical to the 3' of a miRNA and at least 5 nucleotide that are substantially complementary to the flanking region of the target site from the 3' end of the miRNA, for the purposes of inhibiting the ability of a miR A to bind to its target.
  • the sequence of the pri- miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of an miRNA binding site on VCAM-1 , e.g., VCAM-1 3' UTR, or variants thereof.
  • Morpholinos comprise standard nucleic acid bases bound to
  • morpholino rings instead of deoxyribose rings, and linked through phosphorodiamidate groups, instead of phosphates.
  • multi-blocking morpholinos which may inhibit the activity of a targeted miRNA by blocking several steps of its maturation, may be used.
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • Ligand refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • the vectors or constructs of the present description will generally comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase.
  • a termination signal that ends the production of an RNA transcript is contemplated.
  • a terminator may be necessary in vivo to achieve desirable message levels.
  • the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a
  • polyadenylation site This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
  • terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.
  • the terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • Terminators contemplated for use in the description include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • polyadenylation signal to effect proper polyadenylation of the transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the description, and any such sequence may be employed.
  • Preferred embodiments include the SV40
  • polyadenylation signal or the bovine growth hormone polyadenylation signal convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • cells containing a nucleic acid construct of the present description may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • a plasmid vector is contemplated for use to transform a host cell.
  • plasmid vectors containing replicon and control sequences, which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences, which are capable of providing phenotypic selection in transformed cells.
  • E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • the phage lambda GEM-1 1 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.
  • Further useful plasmid vectors include ⁇ vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase-soluble fusion proteins for later purification and separation or cleavage.
  • Other suitable fusion proteins are those with beta- galactosidase, ubiquitin, and the like.
  • Bacterial host cells for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB.
  • suitable media for example, LB.
  • the expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.
  • viruses The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells).
  • cells e.g., mammalian cells.
  • virus vectors that may be used to deliver a nucleic acid of the present description are described below.
  • Adenoviral Vectors A particular method for delivery of nucleic acids involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.
  • AAV Vectors The nucleic acid may be introduced into a cell using adenovirus- assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus-coupled systems.
  • Adeno-associated virus (AAV) is an attractive vector system as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture or in vivo.
  • AAV has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797, 368.
  • Retroviral Vectors have promise as delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).
  • a nucleic acid is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed.
  • a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media.
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells.
  • Lenti viruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldinietal. 1996; Zufferey etal. 1997; Blomeretal. 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136).
  • Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV -2 and the Simian
  • Immunodeficiency Virus SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vifi vpr, vpu and nef are deleted making the vector biologically safe.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
  • recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136.
  • One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type.
  • a sequence (including a regulatory region) of interest into the viral vector, along with another gene that encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
  • viral vectors may be employed as vaccine constructs in the present description.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
  • a nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand.
  • the virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
  • a novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
  • Suitable methods for nucleic acid delivery to cells for use with the current description are believed to include virtually any method by which a nucleic acid (e.g., DNA), as known to one of ordinary skill in the art.
  • a nucleic acid e.g., DNA
  • Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859), including by microinjection (Harland and Weintraub, 1985; U.S. Pat. No.
  • WO 94/09699 and 95/06128 U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods.
  • Methods for transfecting cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art.
  • canine endothelial cells have been genetically altered by retroviral gene transfer in vitro and have been transplanted into a canine (Wilson et al., 1989).
  • Yucatan minipig endothelial cells were transfected by retrovirus in vitro and transplanted into an artery using a double-balloon catheter ' (Nabel et al., 1989).
  • the transplanted cells or tissues may be placed into an organism.
  • a nucleic acid is expressed in the transplanted cells or tissues.
  • a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, etc.
  • injections i.e., a needle injection
  • Methods of injection of vaccines are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution).
  • Further embodiments of the present description include the introduction of a nucleic acid by direct microinjection. Direct
  • nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985).
  • the amount of nucleic acid used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used
  • a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation.
  • Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
  • certain cell wall-degrading enzymes such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384, 253).
  • recipient cells can be made more susceptible to transformation by mechanical wounding.
  • a nucleic acid is introduced to the cells using calcium phosphate precipitation.
  • Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
  • mouse L(A9), mouse CI 27, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
  • DEAE-Dextran In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
  • Sonication Loading Additional embodiments of the present description include the introduction of a nucleic acid by direct sonic loading. LTK.sup.-fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).
  • a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
  • a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989).
  • HVJ hemagglutinating virus
  • a liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991).
  • HMG-1 nuclear nonhistone chromosomal proteins
  • a liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • a delivery vehicle may comprise a ligand and a liposome.
  • a nucleic acid may be delivered to a target cell via receptor mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present description.
  • Certain receptor-mediated gene targeting vehicles comprise a cell receptor-specific ligand and a nucleic acid binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached.
  • Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique.
  • Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993).
  • a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.
  • a nucleic acid delivery vehicle component of a cell- specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome.
  • the nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell.
  • the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
  • lipids or glycoproteins that direct cell-specific binding.
  • lactosyl-ceramide, a galactose-terminal asialoganglioside have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is
  • tissue specific transforming constructs of the present description can be specifically delivered into a target cell in a similar manner.
  • a synthetic gene is also provided comprising a nucleic acid described herein operably linked to a transcriptional and/or translational regulatory sequence.
  • the synthetic gene may be capable of modifying the expression of a target gene with a binding site for a nucleic acid described herein. Expression of the target gene may be modified in a cell, tissue or organ.
  • the synthetic gene may be synthesized or derived from naturally-occurring genes by standard recombinant techniques.
  • the synthetic gene may also comprise terminators at the 3'-end of the transcriptional unit of the synthetic gene sequence.
  • the synthetic gene may also comprise a selectable marker.
  • a subject may receive an agonist of one or more of the miRNAs having any one of SEQ ID NOs: 1 or 2 or of one of the stem cell miRNAs to block cell differentiation in situations in which an excessive cell differentiation is present and/or insufficient amounts of undifferentiated cells are present in the subject.
  • Such conditions may include those in which a subject has been treated, e.g., by irradiation and has lost a considerable amount of undifferentiated cells or HSPC.
  • a subject may receive one or more agonists to thereby increase the number of stem progenitor cells.
  • a treatment may comprise administering an agonist of one or more stem cell miRNA or HSPC miRNA.
  • a subject having cancer may be treated by receiving one or more antagonists of one or stem cell miRNA or HSPC miRNA. This treatment could stimulate the differentiation of an undifferentiated cancer cell and/or reduce the number of cancer cells.
  • Other conditions in which one may want to induce the differentiation of a stem-progenitor cell include those associated with an insufficient number of a differentiated cell, e.g., a differentiated or partially differentiated hematopoietic cell.
  • Exemplary conditions or diseases that may be treated with antagonists include those associated with insufficient leukocytes (leukopenia, e.g., granulocytosis, neutropenia such as drug or chemotherapy induced neutropenia, and congenital neutropenia); insufficient lymphocytes, insufficient red blood cells (e.g., anemias, such as aplastic anemia, nutritional deficiency anemia, hemolytic anemia);
  • diseases include immunodeficiency diseases, e.g., inherited immunodeficiency diseases, drug or therapy induced immunodeficiency diseases, HIV-immunodeficiency diseases.
  • diseases in which differentiation of cells may be beneficial include blood cancers in which undifferentiated or partially differentiated cells accumulate, e.g., leukemias and lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphomas, acute leukemias, e.g., lymphoblastic (ALL) and myelogenous (AML) and chronic leukemias.
  • leukemias and lymphomas e.g., Hodgkin's and non-Hodgkin's lymphomas
  • acute leukemias e.g., lymphoblastic (ALL) and myelogenous (AML) and chronic leukemias.
  • ALL lymphoblastic
  • AML myelogenous
  • stem cell transplantation graft versus host disease
  • graft versus host disease graft versus host disease
  • transplantation e.g., bone marrow transplantation.
  • Methods described herein may also be used for stem cell treatments.
  • Diseases that would benefit from stem cell treatments include Parkinson's disease, spinal cord injuries (as well as other neuromuscular or neurological degenerative diseases), cancer, muscle damage, bone marrow transplants, leukemias and lymphomas.
  • disorders that can be treated by infusion of stem cells include but are not limited to five broad categories. The first group are diseases resulting from a failure or dysfunction of normal blood cell production and maturation
  • the second group are neoplastic, malignant diseases in the hematopoietic organs (e.g., leukemias, lymphomas, myelomas).
  • the third group of disorders comprises those of patients with a broad spectrum of malignant solid tumors of non-hematopoietic origin. Stem cell infusion in these patients serves as a bone marrow rescue procedure, which is provided to a patient following otherwise lethal or strongly
  • the fourth group of diseases consists of autoimmune conditions, where the stem cells serve as a source of replacement of an abnormal immune system.
  • the fifth group of diseases comprises a number of genetic disorders which can be corrected by infusion of hematopoietic stem cells, preferably syngeneic, which prior to transplantation have undergone gene therapy.
  • diseases and disorders which can be treated by hematopoietic reconstitution with substantially enriched population of hematopoietic stem cells include but are not limited to those listed here: diseases resulting from a failure or dysfunction of normal blood (cell production and maturation, hyperproliferative stem cell disorders, aplastic anemia, pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia, Blackfan-Diamond syndrome, due to drugs, radiation, or infection, idiopathic); hematopoietic malignancies (acute
  • lymphoblastic leukemia chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute malignant myelosclerosis, multiple myeloma, polycythemia vera, agnogenic myelometaplasia, Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, non-Hodgkins's lymphoma); malignant, solid tumors (malignant melanoma, carcinoma of the stomach, ovarian carcinoma, breast carcinoma, small cell lung carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, lymphoma); autoimmune diseases (rheumatoid arthritis, diabetes type I, chronic hepatitis, multiple sclerosis, systemic lupus erythematosus); genetic (congenital) disorders (an
  • dyskeratosis congenita Blackfan-Diamond syndrome, congenital dyserythropoietic syndromes I-IV, Schwachmann-Diamond syndrome, dihydrofolate reductase deficiencies, formamino transferase deficiency, Lesch-Nyhan syndrome, congenital spherocytosis, congenital elliptocytosis, congenital stomatocytosis, congenital Rh null disease, paroxysmal nocturnal hemoglobinuria, G6PD (glucose6-phosphate dehydrogenase) variants 1, 2, 3, pyruvate kinase deficiency, congenital erythropoietin sensitivity deficiency, sickle cell disease and trait, thalassemia alpha, beta, gamma, met-hemoglobinemia, congenital disorders of immunity, severe combined immunodeficiency disease (SCID), bare lymphocyte syndrome, ionophore-responsive
  • compositions e.g., pharmaceutical compositions are also provided herein.
  • a composition may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33 or 34 agonists or antagonists or combinations thereof of miRNAs described herein.
  • therapeutic compositions and methods may include the use of modified miRNAs or miRNA antagonists, including 2-O-methyl oligoribonucleotides (2- O-Me-RNAs).
  • 2- O-Me-RNAs 2-O-methyl oligoribonucleotides
  • Antagomirs have been successfully used to down-regulate mi-RNAs in mouse tissue, in vivo, after intravenous injection (Krutzfeldt et al., 2005, Nature, 438: 685-689).
  • Other modifications may include the introduction of phosphorothioate linkages, the addition of 2-0- methoxyethyl groups, and the addition of a cholesteryl or cholesterol moiety.
  • Locked nucleic acid molecules wherein the 2'-0 oxygen of a 2'-0 modified RNA is bridged to the 4' position via a methylene linker, to form a rigid bicycle locked into a C3'-endo (RNA) sugar conformation are also contemplated.
  • a pharmaceutical composition is also provided.
  • the composition may comprise a nucleic acid described herein and optionally a pharmaceutically acceptable carrier and/or excipient.
  • the compositions may be used for diagnostic or therapeutic applications.
  • the pharmaceutical composition may be administered by known methods, including wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, viral methods and cationic liposomes.
  • the compositions may include the use of synthetic miRNAs, which are more potent than naturally occurring miRNAs (Chang, 2006, Clin Cancer Res, 12: 6557-64).
  • miR126 may be used in allogeneic transplantation.
  • a composition comprising oligonucleotides comprising miR126 sequence or compatible sequence is administered to a healthy subject, or to isolated cells of a healthy subject. Four to ten hours later blood is drawn from the subject or isolated cells are harvested. The isolated blood cells are administered to a recipient, preferably a histocompatible patient. G-CSF may be administered to the recipient after administering the cells.
  • mice C57BL/6; 4-8 weeks of age were housed in the animal facilities at the National Institutes of Health; animal studies were approved by the NCI/Bethesda Institutional Animal Care and Use Committee, and were conducted in accordance with institutional guidelines. G-CSF mobilization experiments were performed as described (De La Luz Sierra et al., 2007, Blood, 1 10:2276-85).
  • Bone marrow cells were obtained by flushing femurs and tibias; Lin “ cells were derived with Mouse Lineage Cell Depletion Kit (Miltenyi Biotech) as described by the
  • Progenitor cells were derived from Lin " cells by positive sorting of live (D API- negative or identified with LIVE/DEAD kit, Invitrogen/Molecular Probes) APC-labeled Sca- 1 bright ApCeFluor 780 . Iabeled c Kit bright cells after gating out cells expressing CD45, TER1 19, 7-4, CD1 lb, CD 19, Ly-6G/C and CD5 (all biotin-labeled antibodies followed by FITC-streptavin).
  • LinTerl 15 " CD45 " cells were sorted (FACSVantage SE; BD Biosciences) from Lin " cells after staining with CD45 and TER1 19 biotin-labeled antibodies and FITC-streptavin.
  • VE-cadherin + cells were derived from Lin " cells by positive sorting of live, APC-labeled VE-cadherin + cells. All antibodies and FITC-streptavidin were from BD Pharmingen.
  • Bone marrow-derived microvesicles were prepared essentially as described (Raposo et al., 1996, J Exp Med, 183:1 161- 72) from extracellular fluid derived from flushing femurs and tibias with 10 ml ice-cold PBS, cell removal through centrifugation (10 min at 300 g), filtration through 0.45 mm filters followed by two sequential ultracentrifugation steps: 30 min at 10,000 g and 60 min at 70,000 g.
  • Bone marrow cells (lxloVml) were cultured (18-24 hour) in Iscove's DMEM with 10% FBS with or without G-CSF (25 ng/ml) alone or with labeled microvesicle preparations.
  • Bone marrow cells were cultured (18-24 hour) in Iscove's DMEM with 10% FBS alone or with labeled microvesicle preparations. The ratio between cells and microvesicles was chosen to reflect the expected ratios in bone marrow (typically 50x10 6 bone marrow cells generated microvesicles were suspended in 100 ⁇ PBS; thus, lxlO 6 cells were cultured with 2 ⁇ microvesicle preparations).
  • methylcellulose medium supplemented with a cocktail of recombinant growth factors (Methocult M3434, Stem Cell Technologies). Heparinized blood (10 and 20 ⁇ ) and bone marrow cells (2xl0 4 ) were plated in duplicate on methylcellulose medium (2.5 ml) and incubated (5% C0 2 , 37° C) for 7-12 days of culture. Colonies (at least 50 cells) were counted.
  • a precursor to miR126 pre-miR126, Ambion PM12841
  • a precursor control Ambion 171
  • a CyTM3-labeled pre-miR control AMI 7120
  • Real-time PCR was performed using Assay-on-Demand Taqman probes for mouse CXCR4, CXCR2, VCAM-1, and GAPDH (Applied Biosystems).
  • a miRVANA isolation kit was used; for amplification, Taqman microRNA reverse transcription kit was used; and for real-time PCR miR126 and miR16 Taqman probes were used (all from
  • Example 4 Flow cytometry and immunocytochemistry.
  • biotin anti-human/mouse CXCR7 (clone 8F1 l -M-16, Biolegend); PE or biotin rat anti-mouse CD106/VCAM-1 , biotin rat anti-mouse CD49d (a4 integrin); PE, biotin or PECy5
  • rat anti-mouse Sca-1 (Ly-6A-E, AbD Serotec); biotin anti-mouse Sca-1 (Ly-6A/E); FITC, PE or APC rat anti-mouse c it CDl 17; PE-Cy5 rat anti-mouse CD45 (LCA, Ly5); PE anti-mouse VE- cadherin/CD144 (BioLegend), Lineage markers (CD45, TERl 19, 7-4 clone/neutrophils, CDl lb, CD 19, Ly-6G/C and CD5, all biotin-labeled from StemCell Technologies), FITC rat anti-mouse F4/80 antigen (AbD Serotec); APC rat anti-mouse CD45R/B220, APC rat anti-mouse CDl lb.
  • Protein extracts prepared in NP-40 lysis buffer with protease inhibitor cocktail setlll (Calbiochem), 50 mM NaF, and 1 raM sodium ortho vanadate were resolved through 4- 12% Bis Tris Gels (Invitrogen). After protein transfer, the nitrocellulose membranes were immunoblotted with specific rat anti-mouse antibodies to VCAM-1/CD106 (R&D Systems) and re-probed with goat IgG anti-actin antibodies (Santa Cruz Biotechnology).
  • Example 7 G-CSF induced changes in hematopoietic stem/progenitor cells (HSPC)
  • G-CSF mobilization significantly reduced surface CXCR4 levels in unselected bone marrow cells ("All cells"), in Grl' 0 (i.e., immature neutrophils and monocytes) and Grl hl cells (mature neutrophils do not stain for the monocyte marker CDl 15/M-CSFR) (p ⁇ 0.05) (see
  • Figs. 1 A and 2A In contrast, G-CSF mobilization was not associated with significant changes in the percentage and mean fluorescence intensity (MFI) of surface CXCR4 + cells within the Lin " cKit hi Sca-l hi cell population(Fig. IB), which includes HSPC.
  • Lin * cKit hi Sca-l hi cells were sorted from both control bone marrow cells and from G-CSF-mobilized bone marrow cells.
  • levels of CXCR4 mRNA were similar in Lin " cKit h 'Sca-l hl cells from untreated and G- CSF-mobilized bone marrows, although they markedly differed from unsorted bone marrow cells (Fig.3A).
  • Lin " cKit h 'Sca-l hl cells from either control or G-CSF-mobilized mice responded similarly to SDF1 in trans- well migration assays over a wide range of chemokine concentrations (i.e., 1-100 ng/ml).
  • G-CSF mobilization induced a significant increase in surface CXCR2 expression in unfractionated bone marrow cells, including the Grl'° and Grl hl cell populations (p ⁇ 0.05 all comparisons) (Fig. 1C).
  • Fig. 1C the percentage and MFI of surface CXCR2 + cells within the Lin " cKit h 'Sca- l hi cell population (Fig. ID).
  • Significant changes in the levels of CXCR2 mRNA after G-CSF mobilization were observed in unfractionated bone marrow cells, but these changes were not seen in sorted Lin " cKit hi Sca-l hi cells (Fig. 3B).
  • G-CSF regulation of CXCR4 is a critical pathway for HSPC release by G-CSF. While the role of CXCR4 in the mobilization of myeloid cells, particularly with a contribution of CXCR2, is known by others, its role in the mobilization of HSPC is not. Others have shown that G-CSF mobilization induces a gradual increase in the expression of CXCR4 on bone marrow progenitors (CD34 + or CD38 + cells) over a five-day treatment. In contrast, in the present measurements CXCR4 and CXCR2 were not found to change significantly on bone marrow HSPC after G-CSF mobilization (Figs.
  • CXCR4 antagonists are known to promote HSPC mobilization. Based on the observation that G-CSF was ineffective at enhancing HSPC mobilization in blood and spleen of mice with CXCR4 7" bone marrows, but an antagonist of VLA4 VCAM-1 induced a 2-fold increase in HSPC mobilization, it is possible that G-CSF is ineffective at mobilizing CXCR4- null HSPC. Alternatively, it is possible that the absence of CXCR4 on myeloid lineage cells prevents G-CSF-induced neutrophil/monocyte mobilization, which is indirectly required for HSPC mobilization by G-CSF.
  • Example 8 G-CSF modulates surface VCAM-1 in bone marrow HSPC and non- hematopoietic stromal and endothelial cells.
  • VCAM* cells A marked reduction in the proportion of VCAM* cells was found within the non-hematopoietic bone marrow Lin erl 19 CD45 “ cells (stromal cells) (Fig. 4B) and the Lin " VE-cadherin + cells (endothelial cells) (Fig. 4C).
  • VCAM-1 -deficient mice mobilize normally when transplanted in normal recipients. Accordingly, reduction in VCAM-1 in bone marrow stromal cells may not be required for mobilization of HSPC by G-CSF. However, mice deficient of VCAM-1 in hematopoietic, endothelial and stromal cells mobilized poorly HSPC in response to G-CSF as judged by the abnormally low number of progenitors found in the blood and spleen combined, in spite of having a normal number of bone marrow progenitors.
  • Example 9 miR126 regulates VCAM-1 protein expression in hematopoietic and non- hematopoietic cells.
  • VCAM-1 /VLA4 interactions may play a role in the retention and mobilization of HSPC from the bone marrow.
  • hematopoietic, stromal and endothelial cells by a Tie2-driven ere transgene is known to increase the release of hematopoietic progenitors from the bone marrow to the circulation.
  • Antibodies to VCAM-1 , to a4 integrin, and BI05192, a small molecule inhibitor of VLA4 ( ⁇ 4 ⁇ 1 integrin) binding to VCAM-1 are all know to promote the mobilization of HSPC in mice and/or humans, indicating that the disruption of VLA4 interaction with VCAM-1 promotes the mobilization of HSPC.
  • a link between G-CSF mobilization of HSPC and modulation of VCAM- 1 /VLA4 in the bone marrow has previously been missing.
  • VCAM-1 mR A (Fig. 6 A, right), but caused a decrease in the proportion of surface VCAM-1 positive Sca-l hl cKit hl cells (Fig. 6A, right).
  • Expression of miR126 reduced the percentage of Sca- l hi cKit hi cells that express surface VCAM-1 (Fig. 6B).
  • Expression of miR126 also reduced the percent surface VCAM-1 + cells in bone marrow VE-cadherin-expressing cells compared to the control (Fig. 6C). Similar effects were observed in MS5 cells transfected (mean 76% transfection efficiency) with the miR126 precursor (Fig. 6D).
  • miR126 reduced VCAM-1 protein surface expression in HSPC and non-hematopoietic bone marrow cells without significantly affecting VCAM-1 mRNA levels.
  • VCAM-1 protein expression is modulated by miR126. It is known that miR126 inhibits VCAM-1 expression in endothelial cells. However, in endothelial cells, miR126 only blocked TNF-a-induced expression of VCAM- 1. The present results are surprising in they show that miR126 regulates constitutive elevated expression of VCAM-1 in bone marrow cells, in contrast to endothelial cells. In addition these results show that miR126 can reduce VCAM-1 in hematopoietic cells and bone marrow stromal cells, which could not have been predicted due to cell selectivity of miR functions.
  • Example 10 Regulation of miR126 in bone marrow following G-CSF mobilization.
  • G-CSF G-CSF mobilization significantly reduced the levels of miR126 expression in these cells (Fig. 7A left).
  • G-CSF increased levels of miR126 in the cell-free fraction of flushed bone marrows (obtained after removal of cells, filtration through 0.45 ⁇ filters,
  • the levels of miR126 expression were measured in bone marrow HSPC (Sca- l hi cKit hi cells) and non-hematopoietic cells.
  • the unfractionated bone marrow cells from which the Sca-l h, cKit hl cells were sorted showed G- CSF-mobilization inhibited miR126 expression compared to untreated bone marrows (Fig. 7B, left).
  • VCAM-1 mRNA levels in the sorted bone marrow HSPC or LinTerl 19 " CD45 " cells did not decrease in response to G-CSF, whereas a decrease was observed in the unsorted populations (Fig. 9).
  • G-CSF mobilization is associated with a complex redistribution of miR126 in the bone marrow, an overall reduction in cells with a relative increase in HSPC and LinTerl 19 " CD45 " cells, and an increase of miR126 found released from the cells in the extracellular bone marrow compartment.
  • Example 11 Microvesicle production and transfer to HSPC and non-hematopoietic cell subsets.
  • miRNAs are released in the extracellular compartment through "exosomes” and apoptotic bodies, which play a role in intracellular communication.
  • microvesicles were purified following protocols used for exosome preparation from G-CSF-mobilized and non-mobilized bone marrow cells. A red (PKH26) or green (PKH67) fluorescent lipid dye was used. After washing, the isolated microvesicles were incubated for 18 hours with cells from control or G-CSF mobilized bone marrows. A low proportion (2.8%-6.7%) of all bone marrow cells acquired red fluorescence, showing low-level microvesicle uptake by bone marrow cells.
  • VCAM-1 mean fluorescence intensity was significantly reduced when the exosomes were derived from G-CSF-mobilized bone marrows, by analyzing bone marrow Sca-l h, cKit hl bone marrow progenitors that had incorporated the fluorescent microvesicles. In contrast, after incubation with the microvesicles, VCAM-1 MFI minimally changed in non-fluorescent Sca-l hl cKit hl (Fig.
  • Apoptotic bodies and exosomes are known to be an important conduit for intercellular communication as they can transfer microRNAs, mRNAs and proteins to other cells and have thus been implicated in many cell functions. Exosomes known to be present in many tissues, including the bone marrow and blood, and apoptotic bodies are abundant in the bone marrow as they are largely a physiologic product of neutrophil death at this site. Endothelium- derived apoptotic bodies enriched in miR126 were found to trigger the incorporation Sca-1 + progenitors to the atherosclerotic plaques, in part through stimulation of SDF-1 expression. In addition, human CD34 + peripheral blood HSPC mobilized by G-CSF contained significantly higher levels of miR126 compared to peripheral blood mononuclear cells.
  • G-CSF indirectly promotes the mobilization of HSPC from the bone marrow to the peripheral blood by inducing the release of miR126-containing microvesicles in the bone marrow cavity.
  • Bone marrow microvesicles can deliver miR126 to bone marrow HSPC, endothelial and stromal cells, and miR126 reduces cell surface expression of VCAM-1 , a receptor molecule that regulates the trafficking of HSPC from the bone marrow to the blood through its major integrin ligand VLA4 ( ⁇ 4 ⁇ 1 integrin).
  • Example 12 Defective mobilization of HSPC by G-CSF in miRl 26-deficient mice.
  • miR126-null mice are known to have a vascular phenotype with leaky vessels and hemorrhaging; -40-50% of mice die embryonically or perinatally. If miRl 26 is important to
  • miRl 26-deficient mice may be defective at mobilizing HSPC with G-CSF.
  • G-CSF-mobilized groups of 13-week old miR126 +/+ and miR126 "A littermates were compared to non-mobilized mice. Blood cell counts showed that G-CSF treatment induced an increase in circulating white blood cells and neutrophils in miR.126 7" mice, albeit lower than that displayed by the miR126 +/+ littermates (Fig. 1 1 A).
  • miR126-deficient mice mobilize poorly in response to G-CSF. These present results identify miR126 as a regulator of HSPC trafficking from the bone marrow to the peripheral blood, and clarify the role of VCAM-1 in G-CSF mobilization. A schematic representation of this process is shown in Fig. 1 1 C.

Abstract

What is described is a method for modulating the phenotype of a stem progenitor cell (SPC), including hematopoietic SPC (HSPC), comprising contacting the stem-progenitor cell with one or more nucleic acid molecules, in which each of the nucleic acid molecules comprises a nucleotide sequence that is at least about 90% identical to a sequence of miR126 (e.g., SEQ ID NOs: 1 or 2), or the complements thereof. Upon such contact, expression VCAM-1 is inhibited, resulting in mobilization of SPC. The miR126 nucleic acids may be administered to a healthy subject, and blood cells may be collected from the subject and used for allogeneic transplantation with greater ease than current procedures.

Description

USE OF MIR A126 TO PRODUCE HEMATOPOIETIC STEM CELLS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of United States Provisional Application No. 61/542,468 filed October 3, 201 1 which is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] What is described relates to methods and compositions for modulating the mobilization of stem cells, particularly for promoting or increasing the mobilization of hematopoietic stem/progenitor cells (HSPC) from the bone marrow to the peripheral blood, or alternatively, for preventing the movement of cancer stem cells from their niche in the microenvironment to distant organs and tissues. More specifically, the description relates to methods of using nucleic acid molecules having part or all of the sequence of miR126 (SEQ ID NOs: 1 or 2) to enhance mobilization of HSPC, particularly when harvesting of HSPC for transplantation would be beneficial in ameliorating the symptoms associated with hematopoietic diseases, disabilities or conditions. The description also includes administering miR126 nucleic acids to treat such diseases, and for screening novel agents for their ability to affect expression of miR126.
BACKGROUND
[0003] A number of diseases or conditions can be treated by transplantation of hematopoietic stem/progenitor cells (HSPC), including leukemias, lymphomas, myelomas solid tumor cancers, hematologic diseases, metabolic diseases, radiation poisoning, viral diseases, lysosomal storage diseases, and immunodeficiencies. Chemotherapy can cause myelosuppression and unacceptably low levels of white blood cells, making patients susceptible to infections and sepsis. HSPC production is stimulated by G-CSF, which is used with certain cancer patients to accelerate recovery from neutropenia after chemotherapy, allowing higher-intensity treatment regimens. G-CSF is also used to increase the number of transplantable HSPC in the blood of the donor before collection by leukapheresis for use in HSPC transplantation. It may also be given to the receiver, to compensate for conditioning regimens.
[0004] In addition to protecting cancer patients in chemotherapy, G-CSF has been used in treatment of hematological deficiencies involves isolating HSPC from blood of healthy donor by administering G-CSF, removing blood, purifying white cells, selecting CD34+ cells by immunopurification, storing the HSPC, and administering these cells to a histocompatible recipient patient.
[0005] HSPC reside in bone marrow niches that support their survival and function. A variety of compounds can induce mobilization of HSPC from the bone marrow to the peripheral circulation. In the clinical setting, G-CSF is the most commonly used inducer of HSPC mobilization, whereby HSPC are obtained for autologous and allogeneic hematopoietic reconstitution (Greenbaum et al., 201 1 , Leukemia 25:21 1-17; Pelus et al., 2008, Leukemia, 22:466-73). However, G-CSF mobilizes committed myeloid cells in vast excess of HSPC, which are mobilized only after 5 to 7 days of treatment (Ryan et al., 2010, Nat Med, 16: 1 141-46).
[0006] Expression of G-CSF receptor (G-CSFR) in hematopoietic cells is required for G-CSF-induced HSPC mobilization (Liu et al., 2000, Blood, 95:3025-31). However,
hematopoietic stem cells do not generally express G-CSFR (Ebihara et al., 2000, Br J Haematol, 109: 153-61). G-CSF mobilizes equally effectively HSPC that express or not express G-CSFR (Liu et al. 2000), pointing to other factors involved in G-CSF induced HSPC mobilization.
Neutrophils and/or monocytes are key intermediate regulators of HSPC mobilization by G-CSF (Pelus et al., 2004, Blood, 103: 1 10-19; Christopher et al., 201 1 , J Exp Med, 208:251-60). Further CXCR4 and its unique ligand SDF1/CXCL12 are essential for the retention of granulocytes and other myeloid-lineage cells to the bone marrow, and that disruption of CXCR4 signaling is sufficient for mobilization of neutrophils and other myeloid-lineage cells to the peripheral circulation (Balabanian et al., 2005, Blood, 105:2449-57; Eash et al., 2009, Blood, 1 13:471 1-19; DiPersio et al., 2009, Blood, 1 13:5720-26; Semerad et al., 2005, Blood, 106:3020-27). Patients with WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) are neutropenic due to reduced neutrophil mobilization attributable in most cases to a "gain-of-function" mutation of CXCR4, which exhibits an abnormally prolonged signaling to SDF1 (Gorlin et al., 2000, Am J Med Genet, 91 :368-76; McCormick et al., 2009, PLoS One, 4:e8102). Selective deletion of CXCR4 in myeloid cells causes a spontaneous redistribution of neutrophils from the bone marrow to the blood resulting in neutrophilia (Eash et al. 2009). AMD3100, a synthetic inhibitor of SDF1 binding to CXCR4, promotes rapid neutrophil mobilization when injected alone in mice and man (DiPersio et al. 2009).
[0007] G-CSF rapidly inhibits expression of CXCR4 in myeloid cells (Kim et al., 2006,
Blood, 108:812-20; Levesque et al., 2003, J Clin Invest, 1 1 1 : 187-96) and expression of SDF1 in bone marrow stromal cells, osteoblasts and endothelial cells (Semerad et al. 2005; Christopher et al., 2009, Blood, 1 14: 1331-39; Petit et al., 2002, Nat Immunol, 3:687-94; Ponomaryov et al.,
2000, J Clin Invest, 106: 1331-39). In addition, G-CSF promotes the release of a variety of neutrophil proteases, including neutrophil elastase, cathepsin-G and MMP-9 generating a proteolytic environment in the bone marrow (Pelus et al. 2004; Levesque et al., 2001, Blood, 98:1289-97). Yet, the linkage between those changes induced directly by G-CSF in neutrophils, monocyte/macrophages and potentially other cells, and the indirect HSPC mobilizing effect of G-CSF is unclear. CXCR4 is believed to be critical to mobilization of hematopoietic progenitors by G-CSF (Christopher et al. 2009), but other mechanisms have also been proposed and conclusive evidence is missing (Greenbaum et al. 201 1 ; Pelus et al. 2004, Levesque et al. 2001)
[0008] A significant number of donors and recipients are not amenable to G-CSF- induced mobilization of HSPC cells. There remains an unmet need to simplify and provide more reliability to methods for treating hematological deficiencies by allogeneic transplantation.
SUMMARY
[0009] One aspect of the description is a method for modulating the phenotype of a stem progenitor cell (SPC), particularly hematopoietic SPC (HSPC), comprising contacting the SPC with a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence that is at least about 90% identical to the sequence of miR126 (SEQ ID NOs: 1 or 2), or the complements thereof. The method may further comprise collecting and isolating the SPC after contact with the nucleic acid molecule.
[0010] Another aspect of the description is a method comprising administering G-CSF to a healthy subject, collecting the isolated SPC from the subject, and administering the SPC to a patient.
[0011] Another aspect of the description is a composition consisting of SPC that are produced by administering miR126 nucleic acids to a healthy subject, and collecting SPC from the subject.
[0012] Another aspect of the description is a method for treating hematopoietic deficiency or hematological failure, comprising administering SPC cells to a patient having hematopoietic deficiency or hematological failure, specifically SPC cells from blood of a healthy subject treated with miR126. The SPC blood cells from a healthy subject may be administered to a patient following bone ablation therapy.
[0013] Another aspect of the description is a method for modulating the phenotype of a stem SPC, including HSPC, comprising administering miR126 nucleic acids to a patient having a hematopoietic deficiency or hematological failure.
[0014] Another aspect of the description is a method of treating the hematopoietic deficiency or hematological failure of the patient comprising administering miR126 nucleic acids to a patient having a hematopoietic deficiency or hematological failure. The hematopoietic deficiency or hematological failure may be associated with a cancer.
[0015] Another aspect of the description is a method of using miR126 nucleic acid molecules to decrease VCAM-1 expression on the stem progenitor cells.
[0016] Another aspect of the description is a method for modulating the phenotype of SPC, including HSPC, comprising contacting SPC with miR126 performed ex vivo. One embodiment consists of contacting isolated peripheral blood or bone marrow cells with miR126.
[0017] Another aspect of the description is a composition comprising SPC or HSPC.
[0018] Another aspect of the description is a method of screening a candidate substance for an effect on hematopoietic cell phenotype, comprising providing a SPC; contacting the SPC with the candidate substance; and assessing the effect of the candidate substance on the expression or stability miR126 miRNA.
[0019] Another aspect of the description is a method of treating cancer in a cancer patient in need thereof, comprising administering to a normal subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof, harvesting hematopoietic stem or progenitor cells (HSPC) from the blood of the subject, and administering the HSPC to the cancer patient.
[0020] Another aspect of the description is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0021] Another aspect of the description is a method of screening a candidate compound for an effect on mobilization of stem or progenitor cells (SPC), comprising providing a SPC; contacting the SPC with said candidate compound; and measuring the effect of said candidate substance on the expression or stability of miR126 miRNA.
[0022] Another aspect of the description is a use of a chemotherapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises
administering the medicament and a composition comprising allogenic hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0023] Another aspect of the description is a use of a chemotherapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises administering the medicament and a composition comprising autologous hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0024] Another aspect of the description is a use of a chemotherapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises
administering the medicament and a composition a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0025] Another aspect of the description is a chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the
chemotherapeutic agent and a composition comprising allogenic hematopoietic stem or progemtor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0026] Another aspect of the description is a chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the
chemotherapeutic agent and a composition comprising autologous hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0027] Another aspect of the description is a chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the
chemotherapeutic agent and a composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0028] Another aspect of the description is a composition comprising allogenic hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0029] Another aspect of the description is a composition comprising autologous hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
[0030] Another aspect of the description is a composition comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof, for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition.
[0031] Another aspect of the description is a composition comprising autologous hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows G-CSF mobilization modulated CXCR4 and CXCR2 expression in bone marrow myeloid cells, but not in HSPC. Cell surface CXCR4 was evaluated by flow cytometry in all cells, Grl'° bone marrow cells (Fig. 1A) and Grlhl bone marrow cells and in Lin"ScahicKithi bone marrow cells (Fig. 2B) in the presence and absence of G-CSF. Cell surface CXCR2 was evaluated by flow cytometry in all cells, Grl'° bone marrow cells and Grlhl bone marrow cells (Fig. 1C) and in Lin"Scah'cKithl bone marrow cells (Fig. ID) in the presence and absence of G-CSF. Representative experiment of 5 performed. Results are expressed as mean ± SD (5-8/group). "*** denotes p<0.05.
[0033] FIG. 2 shows G-CSF mobilization effected the distribution of bone marrow cell populations. Percentage of cells with no surface Grl (Grlneg), low levels (Grl'°) or high levels Grlhl8h are expressed as mean ± SD (n=5-8).
[0034] FIG. 3 shows an analysis of relative levels of CXCR4 mRNA (Fig. 3A) and CXCR2 mRNA (Fig. 3B) in non-fractionated bone marrow cells ("All cells") and in sorted Lin" ScahicKithi HSPC from mice mobilized with G-CSF or left untreated. Results are from
quantitative PCR and are presented as mean ± SD (n=5-6).
[0035] FIG. 4 shows G-CSF mobilization is associated with decreased percentage surface VCAM-1 expression on HSPC, LinTerl 19"CD45" and VE-cadherin+ cells from the bone marrow. Cell surface VCAM-1 in Lin"Scah'cKithl cells (representative individual results are shown as averages of the VCAM-1 expression) (Fig. 4A); LinTerl 19"CD45" (Fig. 4B) and VE- cadherin+ cells (Fig. 4C) in bone marrow from mice mobilized with G-CSF or left untreated (5- 10/group). Relative levels of VCAM-1 mRNA (measured by quantitative PCR; n=6), cell- associated protein and cell surface in bone marrow cells from mice mobilized with G-CSF or untreated are shown in Fig. 4D. Bar graphs reflect the mean ± SD. "*" denotes p<0.05.
[0036] FIG. 5 shows relative levels of VCAM-1 mRNA measured by quantitative PCR in unfractionated bone marrow cells and in Lin"Sca+cKit+ cells sorted from bone marrow from mice mobilized with G-CSF or untreated. The results are expressed as group means ± SD (n=6).
[0037] FIG. 6 shows miR126 regulates VCAM-1 expression in bone marrow cells. (Fig. 6 A) shows transfection of pre-miR126 or a pre-miR control in Lin" bone marrow cells relative levels of miR126 (miR126/miR16; left) and VCAM-1 mRNA (right) measured 18 hours after transfection. Also shown are the percentage surface levels of VCAM-1 on Lin"Scah'cKithl (Fig. 6B), VE-cadherin+ (Fig. 6C) and MS5 cells (Fig. 6D) 18 hours after transfection with pre- miR126 or control. The results are expressed as mean ± SD (n=3). "*" denotes p<0.05.
[0038] FIG. 7 shows G-CSF mobilization regulates miR126 distribution in the bone marrow. (Fig. 7A) shows G-CSF mobilization reduces in bone marrow cells (left) and increased in the cell-free fraction (right) relative levels of miR126 (mean ±SD; 5-8 mice/group untreated and G-CSF treated). (Fig. 7B) shows relative levels of miR126 (miR126/miR16) in
unfractionated bone marrow cells (Pre-sort) and in Sca-lhlcKithl cells sorted from these cells. (Fig. 7C) shows relative levels of miR126 in bone marrow cells and in Lin erl 19"CD45" cells sorted from these cells. The results in B and C reflect relative mean miR126 levels ± SD measured in 9 untreated and 9 G-CSF-mobilized bone marrows (3 each combined prior to sorting). "*" denotes p<0.05.
[0039] FIG. 8 shows G-CSF modulates the relative content of miR126 in bone marrow cells and culture supernatant. Unfractionated bone marrow cells were cultured (lxl06/ml) for 18 hours with or without G-CSF (100 ng/ml) in complete culture medium; RNA was extracted from the cells and cell-free supernatant. The results are expressed as relative miR126/miR16 levels; representative experiment of 3 performed.
[0040] FIG. 9 shows G-CSF mobilization regulates levels of miR126 in bone marrow cell populations. Bone marrows (3 combined) from untreated or G-CSF mobilized mice were tested for relative miR126/miR16 levels: (Fig. 9A) before and (Fig. 9B) after sorting the ScahicKithi HPC; and (Fig. 9C) before and (Fig. 9D) after sorting the LinTerl 19"CD45" cells.
[0041] FIG. 10 shows uptake of microvesicles by bone marrow cells effects surface
VCAM-1 levels. (Fig. 10 A) shows uptake of PKH-labeled exosome preparations (Exo) from bone marrow of G-CSF mobilized or untreated mice (5 bone marrows each) by (from the top): unfractionated, Grl+, Sca-lhicKithi, Lin'CD45" and VE-cadherin+ bone marrow cell populations.
Results reflect means ± SD. (Fig. 10B) shows VCAM-1 MFI measured on Sca-lhicKithi cells from bone marrows incubated with PKH-labeled exosome preparations from untreated or G- CSF-mobilized bone marrows. VCAM-1 MFI was measured on Sca-lhlcKithl cells with or without microvesicles-derived fluorescence (means ±SD of 3 independent experiments). The percentage of VCAM1+ cells was measured in (Fig. IOC) LinTerl 19"CD45"and (Fig. 10D) VE- cadherin+ bone marrow cells that had either acquired or not acquired microvesicles-derived PKH fluorescence after 18 hour incubation. Micro vescicles were from untreated and G-CSF-mobilized bone marrows. The results reflect the mean ± SD of 3 independent experiments. "*" denotes p<0.05.
[0042] FIG. 11 shows defective HSPC mobilization in G-CSF-treated miRl 26- deficient mice. (Fig. 11 A) shows control miR126+ + and miR126"A littermates were mobilized with G-CSF or left untreated. Blood white blood cells, neutrophil, lymphocytes, monocytes and immature bone marrow cells were counted; the results reflect the group mean ±SD (n=3). (Fig.
IIB) shows CFU-c measured in the peripheral blood of control miR126+/+ and miR126"/" littermates untreated or mobilized with G-CSF. The results represent group means ±SD (n=3) for the mobilized mice; no colonies developed from blood of untreated mice. * denotes PO.05. (Fig.
IIC) shows schematic representations of HSPC and myeloid-lineage cell trafficking from the bone marrow to the blood; contribution of CXCR4, CXCR2, VCAM-1 and microvesicles containing miRl 26.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0043] Results are presented herein demonstrating the novel discovery that G-CSF, the primary means by which HSPC are mobilized to obtain stem cell graphs for hematopoietic cell transplantation, promotes the accumulation of microRNA-126 (miR126)-containing
microvesicles in the bone marrow extracellular compartment, and that miRl 26 delivered by these microvesicles reduces VCAM-1 surface expression in bone marrow HSPC and non- hematopoietic cells. VCAM-1 is critical to the retention of HSPC to the bone marrow. These results point to miRl 26 as a factor involved in HSPC trafficking between the bone marrow and peripheral sites by inhibiting VCAM-1 expression, triggering selective mobilization of HSPC. These results identify the important role of miRl 26 class as a potentially important new therapeutic.
[0044] As used in the context of the present description, the term "hematopoietic stem and progenitor cells," i.e., HSPC, are self-renewing precursors that regenerate myeloid, erythroid, and lymphoid cells throughout the life span of the subject or patient. The term "stem cell" is meant to encompass stem cells and progenitor cells of various levels of pluripotency. [0045] The terms "subject" and "patient" are used interchangeably for the purpose of this description, wherein either a subject or a patient refers to a living mammal, which includes humans and other mammals that persons of ordinary skill in the art commonly use.
[0046] In the context of the present description, the term "mobilization" of
hematopoietic stem and progenitor cells, i.e., HSPC, refers to the recruitment of HSPC into the blood. Generally, HSPC are found in bone marrow, spleen, umbilical cord blood, and the blood and liver of fetuses and newborns. Generally, cells obtained from bone marrow, cord blood, or mobilized peripheral blood of healthy donors, are clinically useful for transplantation into a recipient subject.
[0047] "Modulation" or "modulates" or "modulating" refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
[0048] "Treatment" or "treating" refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted. In the present description, the treatments using the agents described may be provided to treat patients suffering from a cancerous condition or hyperproliferative disease, whereby the treatment of the disease with chemotherapy or irradiation therapy results in a decrease in bone marrow cellularity, thus making the patient more prone to acquiring infectious agents or diseases. Thus, the administration of any of the agents of the description allows for the mobilization of hematopoietic stem cells or progenitor cells from the bone marrow to the peripheral blood. Most preferably, the treating is for the purpose of reducing or diminishing the symptoms or progression of a cancerous disease or disorder by allowing for the use of accelerated doses of chemotherapy or irradiation therapy.
[0049] "Subject" or "patient" refers to a mammal, preferably a human, in need of treatment for a condition, disorder or disease.
[0050] "Prophylactic" or "therapeutic" treatment refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom). [0051] A "mobilizer of hematopoietic stem cells or progenitor cells" or "mobilizer", (used interchangeably) as described herein refers to any compound, whether it is a small organic molecule, synthetic or naturally derived, or a polypeptide, such as a growth factor or colony stimulating factor or an active fragment or mimic thereof, a nucleic acid, a carbohydrate, an antibody, or any other agent that acts to enhance the migration of stem cells from the bone marrow into the peripheral blood. Such a "mobilizer" may increase the number of hematopoietic stem cells or hematopoietic progenitor/precursor cells in the peripheral blood, thus allowing for a more accessible source of stem cells for use in transplantation.
[0052] "Stem Cells" are cells, which are not terminally differentiated and are therefore able to produce cells of other types. Stem cells are divided into three types, including totipotent, pluripotent, and multipotent. "Totipotent stem cells" can grow and differentiate into any cell in the body, and thus can grow into an entire organism. These cells are not capable of self-renewal. In mammals, only the zygote and early embryonic cells are totipotent. "Pluripotent stem cells" are true stem cells, with the potential to make any differentiated cell in the body, but cannot contribute to making the extraembryonic membranes (which are derived from the trophoblast). "Multipotent stem cells" are clonal cells that self-renew as well as differentiate to regenerate adult tissues, and can give rise to cells of multiple lineages. In contrast, "unipotent stem cells" can only give rise to cells of one lineage, such as blood cells or bone cells. The term "stem cells", as used herein, refers to pluripotent stem cells capable of self-renewal.
[0053] "Cancer stem cells" refers to a small population of cells that are quiescent, which are capable of self-renewal, and which appear to be the sources of cells comprising a malignant and/or metastatic tumor.
[0054] "Hematopoiesis" refers to the highly orchestrated process of blood cell development and homeostasis. Prenatally, hematopoiesis occurs in the yolk sack, then liver, and eventually the bone marrow. In normal adults it occurs in bone marrow and lymphatic tissues. All blood cells develop from pluripotent stem cells. Pluripotent cells differentiate into stem cells that are committed to three, two or one hematopoietic differentiation pathway. None of these stem cells are morphologically distinguishable, however.
[0055] The term "hematopoietic stem cells" as used in the present description means multipotent stem cells that are capable of differentiating into all blood cells including
erythrocytes, myeloid cells, leukocytes and platelets. For instance, the "hematopoietic stem cells" as used in the description are contained not only in bone marrow but also in umbilical cord blood derived cells. [0056] The term "hematopoietic progenitors", which is used interchangeably with the term "hematopoietic precursors", refers to those progenitor or precursor cells which are differentiated further than hematopoietic stem cells but have yet to differentiate into progenitors or precursors of respective blood cell lineages (unipotent precursor cells). Thus, "progenitor cell(s)" or "precursor cell(s)" are defined as cells that are lineage-committed, i.e., an individual cell can give rise to progeny limited to a single lineage such as the myeloid or lymphoid lineage or two lineages. They do not have self-renewal properties. They can also be stimulated by lineage-specific growth factors to proliferate. If activated to proliferate, progenitor cells have life-spans limited to 50-70 cell doublings before programmed cell senescence and death occurs. For example, the "hematopoietic progenitors" as used in the present description include granulocyte/macrophage associated progenitors (colony-forming unit granulocyte, macrophage, CFU-GM), erythroid associated progenitors (burst forming unit erythroid, BFU-E),
megakaryocyte associated progenitors (colony-forming unit megakaryocyte, CFU-Mk), and myeloid associated stem cells (colony-forming unit mixed, CFU-Mix). Hematopoietic progenitor cells possess the ability to differentiate into a final cell type directly or indirectly through a particular developmental lineage. Undifferentiated, plunpotent progenitor cells that are not committed to any lineage are referred to herein as "stem cells." All hematopoietic cells can in theory be derived from a single stem cell, which is also able to perpetuate the stem cell lineage, as daughter cells become differentiated. The isolation of populations of mammalian bone marrow cell populations, which are enriched to a greater or lesser extent in pluripotent stem cells has been reported (see for example, Verfaillie et al., 1990, J. Exp. Med., 172, 509).
[0057] The term "differentiation" of hematopoietic stem cells and/or hematopoietic progenitors as used in the description means both the change of hematopoietic stem cells into hematopoietic progenitors and the change of hematopoietic progenitors into unipotent hematopoietic progenitors and/or cells having characteristic functions, namely mature cells including erythrocytes, leukocytes and megakaryocytes. Differentiation of hematopoietic stem cells into a variety of blood cell types involves sequential activation or silencing of several sets of genes. Hematopoietic stem cells choose either a lymphoid or myeloid lineage pathway at an early stage of differentiation.
[0058] "CXCL12", also known as stromal cell-derived factor-1 or "SDF-1 " refers to a
CXC chemokine that demonstrates in vitro activity with respect to lymphocytes and monocytes but not neutrophils. It is highly potent in vivo as a chemoattractant for mononuclear cells. SDF-1 has been shown to induce intracellular actin polymerization in lymphocytes, and to induce a transient elevation of cytoplasmic calcium in some cells. By "function of a chemokine, CXCL12" is meant the binding of the chemokine to its receptor and the subsequent effects on signaling.
[0059] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
[0060] "Cell" used herein may be a naturally occurring cell or a transformed cell that may contain a vector and may support replication of the vector. Cells may be cultured cells, explants, cells in vivo, and the like. Cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, such as CHO and HeLa.
[0061] In certain embodiments, a host cell is a hematopoietic cell, for example, a hematopoietic stem-progenitor cell, a long-term repopulating hematopoietic stem cell, a short- term repopulating hematopoietic stem cell, a multipotent progenitor cell, a common lymphoid progenitor cell, a pro-T cell, a T cell, an NK progenitor cell, an NK cell, a pro-B cell, a B cell, a common myeloid progenitor cell, a B-cell/macrophage bipotential cell, a granulocyte- macrophage progenitor cell, a megakaryocyte-erythrocyte progenitor cell, a macrophage, a granulocyte, a megakaryocyte progenitor cell, a megakaryocyte, or an erythrocyte. It is contemplated that the miRNAs described here may play a role in the differentiation pathways of other cells. The cell may also be a bacterial, fungal, plant, insect or other type of animal cell.
[0062] "Identical" or "identity" as used herein in the context of two or more nucleic acids or polypeptide sequences may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. Thymine (T) and uracil (U) may be considered equivalent when comparing DNA and RNA. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.
[0063] "Nucleic acid" or "oligonucleotide" or "polynucleotide" used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the
complementary strand of a depicted single strand, unless the nucleic acid is a single stranded nucleic acid. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid may also be a probe that hybridizes under stringent hybridization conditions.
[0064] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
[0065] A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S.
Pat. Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids. The modified nucleotide analog may be located for example at the 5'-end and/or the 3 '-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase- modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5- position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N- alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2 -OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is Cj-Cg alkyl, alkenyl or alkynyl and halo is F, CI, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Publication No. 20050107325. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 200201 15080. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005.
Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
[0066] "miR126" consists of RNA encoded by a family of 22 gene sequences found in different locations of the human genome. One of these is located within the 7th intron of the EFGL7 gene on chromosome 9. Its expression there is regulated by ETS1 and ETS2
transcription factors. It is identified as RF00701 on the Rfam database, 61 1767 on the OMIM database, and MI0000471 on the miRBase database. It has the following sequence (SEQ ID NO:l):
10 20 30 40
GGCUG GCGAC GGGAC AUUAU UACUU TJTJGGU ACGCG . CUGUG
50 60 70 80
ACACU UCAAA CUCGU ACCGU GAGUA AUAAU GCGCC GUCCA
CGGCA .
The mature miR126 molecule has the following sequence (SEQ ID NO:2):
52 73
ucgua ccgug aguaa uaaug eg .
[0067] "Operably linked" used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
[0068] "Stringent hybridization conditions" used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence- dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5xSSC, and 1% SDS, incubating at 42° C, or, 5xSSC, 1% SDS, incubating at 65° C, with wash in 0.2xSSC, and 0.1% SDS at 65° C.
[0069] "Substantially complementary" used herein may mean that a first sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.
[0070] "Substantially identical" used herein may mean that a first and second sequence are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22,23,24,25,30,35,40,45, 50, 55, 60, 65,70,75, 80, 85, 90, 95,100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
[0071] "Variant" used herein to refer to a nucleic acid may mean (i) a portion of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. [0072] "Vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. In accordance with the present description, it may be desirable to express the miRNAs of the present description in a vector. The term "exogenous," means that the vector or entity referred to is foreign to the cell into which it is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., BACs, YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994).
[0073] "Expression vector" refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
MicroRNA
[0074] While not being bound by any theory, a gene coding for an miRNA may be transcribed leading to production of an miRNA precursor known as the pri-miRNA. The pri- miRNA may be part of a polycistronic RNA comprising multiple pri-miRNAs. The pri-miRNA may form a hairpin with a stem and loop. The stem may comprise mismatched bases.
[0075] The hairpin structure of the pri-miRNA may be recognized by Drosha, which is an RNase III endonuclease. Drosha may recognize terminal loops in the pri-miRNA and cleave approximately two helical turns into the stem to produce a 60-70 nt precursor known as the pre- miRNA. Drosha may cleave the pri-miRNA with a staggered cut typical of RNase III
endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and about a 2 nucleotide 3' overhang. Approximately one helical turn of the stem (about 10 nucleotides) extending beyond the Drosha cleavage site may be essential for efficient processing. The pre-miRNA may then be actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex- portin-5.
[0076] The pre-miRNA may be recognized by Dicer, which is also an RNase III endonuclease. Dicer may recognize the double-stranded stem of the pre-miRNA. Dicer may also recognize the 5' phosphate and 3' overhang at the base of the stem loop. Dicer may cleave off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and about a 2 nucleotide 3' overhang. The resulting siRNA-like duplex, which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*. The miRNA and miRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA. miRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
[0077] Although initially present as a double-stranded species with miRNA*, the miRNA may eventually become incorporated as a single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). Various proteins can form the RISC, which can lead to variability in specificity for miRNA/miRNA* duplexes, binding site of the target gene, activity of miRNA (repress or activate), and which strand of the
miRNA/miRNA* duplex is loaded in to the RISC.
[0078] When the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* may be removed and degraded. The strand of the miRNA:miRNA* duplex that is loaded into the RISC may be the strand whose 5' end is less tightly paired. In cases where both ends of the miRNA: miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA* may have gene silencing activity.
[0079] The RISC may identify target nucleic acids based on high levels of
complementarity between the miRNA and the mRNA, especially by nucleotides 2-8 of the miRNA. A case has been reported in animals where the interaction between the miRNA and its target was along the entire length of the miRNA.
[0080] Base-pairing between miRNA and its mRNA target is required for achieving efficient inhibition of translation. In mammalian cells, the first 8 nucleotides of the miRNA may be important. However, other parts of the miRNA may also participate in mRNA binding.
Moreover, sufficient base pairing at the 3' can compensate for insufficient pairing at the 5'. Computational studies analyzing miRNA binding on whole genomes have suggested a specific role for bases 2-7 at the 5' of the miRNA in target binding but a role for the first nucleotide, usually "A", is also recognized. Similarly, nucleotides 1-7 or 2-8 were used to identify and validate targets.
[0081] The target sites in the mRNA may be in the 5' UTR, the 3' UTR or within the coding region. Multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites. The presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition. Similarly, a single miRNA may regulate multiple mRNA targets by recognizing the same or similar sites on the different targets.
[0082] Without being bound by theory, miRNAs may direct the RISC to downregulate gene expression by several mechanisms: mRNA cleavage, translational repression, or chromatin remodeling. The miRNA may specify cleavage of the mRNA if the miRNA has a certain degree of complementarity to the mRNA. When an miRNA guides cleavage, the cut may be between the nucleotides pairing to residues 10 and 1 1 of the miRNA. Alternatively, the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
[0083] It should be noted that there may be variability in the 5' and 3' ends of any pair of miRNA and miRNA*. This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the site of cleavage. Variability at the 5' and 3' ends of miRNA and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a population of different hairpin structures. Variability in the stem structures may also lead to variability in the products of cleavage by Drosha and Dicer.
[0084] Nucleic acids for modulating the level or activity of miRNAs may, in addition to a sequence that is identical or homologous to that of the miRNA or complement thereof, also comprise additional, unrelated nucleotides, provided that they do not interfere with the mimicking or inhibition, respectively, of the miRNA. They may comprise, e.g., about 1,2,3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides that are located either 5' and/or 3' (or internal) to the sequence for modulating the level or activity of the miRNA.
[0085] An agonist of a miRNA (understood herein to also include agonists of the related molecules, e.g., pre- or pri-miRNA) is any agent, e.g., a nucleic acid comprising a nucleotide sequence, that is identical or homologous to that of the miRNA, and that mimics the action of the miRNA. An antagonist of a miRNA (understood herein to also include agonists of the related molecules, e.g., pre- or pri-miRNA) is any agent, e.g., a nucleic acid comprising a nucleotide sequence, that is identical or homologous to that of the complement of the miRNA, and that inhibits the action of the miRNA.
[0086] In certain embodiments, a nucleic acid comprises at least about 2, 3, 5, 10, 15, 20, 25, 30, or more nucleotide sequences that are identical or homologous to that of a miRNA or the complement thereof. The nucleotide sequences may be mimicking or inhibiting the level or activity of one miRNA or alternatively of at least about 2, 3, 5, 10, 15, 20, 25, 30, 32, 33, or more different miRNAs, e.g., those having SEQ ID NOs: 1 or 2.
Nucleic Acids Controlling Stem-Progenitor Cell Differentiation
[0087] Nucleic acids controlling stem progenitor cell, e.g., hematopoietic stem- progenitor, cell differentiation are provided herein. The nucleic acid may comprise the sequence of SEQ ID NOs: 1 or 2, or variants thereof. The variant may be a complement of the referenced nucleotide sequence. The variant may also be a nucleotide sequence that is substantially identical to the referenced nucleotide sequence or the complement thereof. The variant may also be a nucleotide sequence that hybridizes under stringent conditions to the referenced nucleotide sequence, complements thereof, or nucleotide sequences substantially identical thereto.
[0088] The nucleic acid may have a length of from 10 to 250 nucleotides. The nucleic acid may have a length of at least 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,20,21 ,22,23,24,25,26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 nucleotides. The nucleic acid may be synthesized or expressed in a cell (in vitro or in vivo) using a synthetic gene described herein. The nucleic acid may be synthesized as a single stranded molecule and hybridized to a substantially complementary nucleic acid to form a duplex. The nucleic acid may be introduced to a cell, tissue or organ in a single- or double-stranded form or capable of being expressed by a synthetic gene using methods well known to those skilled in the art. In certain embodiments, it may be useful to incorporate the nucleic acids into a vector, as described supra.
Pri-miRNA
[0089] The nucleic acid may comprise a sequence of a pri-miRNA or a variant thereof. The pri-miRNA sequence may comprise from 45-250, 55-200, 70-150 or 80-100 nucleotides. The sequence of the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof. The sequence of the pri-miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of an miRNA binding site on VCAM-1 , e.g., VCAM-1 3' UTR (Harris et al., 2008, PNAS, 105:1516-21), or variants thereof.
[0090] The pri-miRNA may form a hairpin structure. The hairpin may comprise a first and second nucleic acid sequence that are substantially complementary. The first and second nucleic acid sequence may be from 37-50 nucleotides. The first and second nucleic acid sequence may be separated by a third sequence of from 8-12 nucleotides. The hairpin structure may have a free energy less than -25 Kcal/mole as calculated by the Vienna algorithm with default parameters, as described in Hofacker et al., 1994, Monatshefte f. Chemie 125: 167-188. The hairpin may comprise a terminal loop of 4-20, 8-12 or 10 nucleotides. The pri-miRNA may comprise at least 19% adenosine nucleotides, at least 16% cytosine nucleotides, at least 23% thymine nucleotides and at least 19% guanine nucleotides.
Pre-miRNA
[0091] The nucleic acid may also comprise a sequence of a pre-miRNA or a variant thereof. The pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides. The sequence of the pre-miRNA may comprise a miRNA and a miRNA* as set forth herein. The sequence of the pre-miRNA may also be that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA. The sequence of the pri-miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of a miRNA binding site on VCAM-1, e.g., VCAM-1 3' UTR, or variants thereof.
[0092] The nucleic acid may also comprise a sequence of a miRNA (including miRNA*) or a variant thereof. The miRNA sequence may comprise from 13-33, 18-24 or 21-23 nucleotides. The miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26,27,28, 29,30,31 ,32, 33, 34,35,36,37, 38, 39 or 40 nucleotides. The sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA. The sequence of the pri-miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of a miRNA binding site on VCAM-1 , e.g., VCAM-1 3' UTR, or variants thereof.
Anti-mi RN A
[0093] The nucleic acid may also comprise a sequence of an anti-miRNA that is capable of blocking the activity of a miRNA or miRNA*, such as by binding to the pri-miRNA, pre-miRNA, miRNA or miRNA* (e.g. antisense or RNA silencing), or by binding to the target binding site. The anti-miRNA may comprise a total of 5-100 or 10-60 nucleotides. The anti- miRNA may also comprise a total of at least 5, 6, 7, 8, 9,, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the anti-miRNA may comprise (a) at least 5 nucleotides that are substantially complementary to the 5' of a miRNA and at least 5-12 nucleotides that are substantially identical to the flanking regions of the target site from the 5* end of the miRNA, for the purposes of binding to a miRNA and repressing its activity; or (b) at least 5-12 nucleotides that are substantially identical to the 3' of a miRNA and at least 5 nucleotide that are substantially complementary to the flanking region of the target site from the 3' end of the miRNA, for the purposes of inhibiting the ability of a miR A to bind to its target. The sequence of the pri- miRNA may comprise the sequence of SEQ ID NOs: 1 or 2, the complement of an miRNA binding site on VCAM-1 , e.g., VCAM-1 3' UTR, or variants thereof.
[0094] In some embodiments, morpholino-based oligonucleotides may be used to block the activity of a miRNA. Morpholinos comprise standard nucleic acid bases bound to
morpholino rings, instead of deoxyribose rings, and linked through phosphorodiamidate groups, instead of phosphates. In other embodiments, "multi-blocking morpholinos," which may inhibit the activity of a targeted miRNA by blocking several steps of its maturation, may be used.
Vectors
[0095] Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
[0096] The vectors or constructs of the present description will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
[0097] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a
polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
[0098] Terminators contemplated for use in the description include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
[0099] In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the description, and any such sequence may be employed. Preferred embodiments include the SV40
polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
[0100] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.
[0101] In certain embodiments of the description, cells containing a nucleic acid construct of the present description may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
[0102] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
[0103] In certain embodiments, a plasmid vector is contemplated for use to transform a host cell. In general, plasmid vectors containing replicon and control sequences, which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences, which are capable of providing phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.
[0104] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, the phage lambda GEM-1 1 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.
[0105] Further useful plasmid vectors include ρΓΝ vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S-transferase-soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with beta- galactosidase, ubiquitin, and the like.
[0106] Bacterial host cells, for example, E. coli, comprising the expression vector, are grown in any of a number of suitable media, for example, LB. The expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.
Viral Vectors
[0107] The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of the present description are described below.
[0108] Adenoviral Vectors. A particular method for delivery of nucleic acids involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell-specific construct that has been cloned therein. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.
[0109] AAV Vectors. The nucleic acid may be introduced into a cell using adenovirus- assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus-coupled systems. Adeno-associated virus (AAV) is an attractive vector system as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture or in vivo. AAV has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797, 368.
[0110] Retroviral Vectors. Retroviruses have promise as delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller, 1992).
[0111] In order to construct a retroviral vector, a nucleic acid is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells. [0112] Lenti viruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldinietal. 1996; Zufferey etal. 1997; Blomeretal. 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV -2 and the Simian
Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vifi vpr, vpu and nef are deleted making the vector biologically safe.
[0113] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136. One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type. By inserting a sequence (including a regulatory region) of interest into the viral vector, along with another gene that encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
[0114] Other Viral Vectors. Other viral vectors may be employed as vaccine constructs in the present description. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0115] Delivery Using Modified Viruses. A nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
[0116] Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin
(Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro.
Delivering Nucleic Acids to Cells
[0117] Suitable methods for nucleic acid delivery to cells for use with the current description are believed to include virtually any method by which a nucleic acid (e.g., DNA), as known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection (Wilson et al., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859), including by microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215); by electroporation (U.S. Pat. No. 5,384,253; Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van DerEb, 1973; Chen and Okayama, 1987; Rippeetal., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al, 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kanedaetal., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods.
[0118] Methods for transfecting cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art. For example, canine endothelial cells have been genetically altered by retroviral gene transfer in vitro and have been transplanted into a canine (Wilson et al., 1989). In another example, Yucatan minipig endothelial cells were transfected by retrovirus in vitro and transplanted into an artery using a double-balloon catheter ' (Nabel et al., 1989). Thus, it is contemplated that cells or tissues may be removed and transfected ex vivo using the nucleic acids of the present description. In some embodiments, the transplanted cells or tissues may be placed into an organism. In some embodiments, a nucleic acid is expressed in the transplanted cells or tissues.
[0119] Injection. In certain embodiments, a nucleic acid may be delivered to an organelle, a cell, a tissue or an organism via one or more injections (i.e., a needle injection), such as, for example, subcutaneously, intradermally, intramuscularly, intravenously, intraperitoneally, etc. Methods of injection of vaccines are well known to those of ordinary skill in the art (e.g., injection of a composition comprising a saline solution). Further embodiments of the present description include the introduction of a nucleic acid by direct microinjection. Direct
microinjection has been used to introduce nucleic acid constructs into Xenopus oocytes (Harland and Weintraub, 1985). The amount of nucleic acid used may vary upon the nature of the antigen as well as the organelle, cell, tissue or organism used
[0120] Electroporation. In certain embodiments of the present description, a nucleic acid is introduced into an organelle, a cell, a tissue or an organism via electroporation.
Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In some variants of this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384, 253). Alternatively, recipient cells can be made more susceptible to transformation by mechanical wounding.
[0121] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.
[0122] To effect transformation by electroporation in cells such as, for example, plant cells, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Examples of some species which have been transformed by electroporation of intact cells include maize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco (Lee et al., 1989).
[0123] Calcium Phosphate. In other embodiments of the present description, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse CI 27, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
[0124] DEAE-Dextran. In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985). [0125] Sonication Loading. Additional embodiments of the present description include the introduction of a nucleic acid by direct sonic loading. LTK.sup.-fibroblasts have been transfected with the thymidine kinase gene by sonication loading (Fechheimer et al., 1987).
[0126] Liposome-Mediated Transfection. In a further embodiment of the description, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).
[0127] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
[0128] In certain embodiments of the description, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, a liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.
[0129] Receptor Mediated Transfection. Still further, a nucleic acid may be delivered to a target cell via receptor mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell. In view of the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present description.
[0130] Certain receptor-mediated gene targeting vehicles comprise a cell receptor- specific ligand and a nucleic acid binding agent. Others comprise a cell receptor-specific ligand to which the nucleic acid to be delivered has been operatively attached. Several ligands have been used for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishes the operability of the technique. Specific delivery in the context of another mammalian cell type has been described (Wu and Wu, 1993). In certain aspects of the present description, a ligand will be chosen to correspond to a receptor specifically expressed on the target cell population.
[0131] In other embodiments, a nucleic acid delivery vehicle component of a cell- specific nucleic acid targeting vehicle may comprise a specific binding ligand in combination with a liposome. The nucleic acid(s) to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the liposome membrane. The liposome will thus specifically bind to the receptor(s) of a target cell and deliver the contents to a cell.
[0132] In still further embodiments, the nucleic acid delivery vehicle component of a targeted delivery vehicle may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactosyl-ceramide, a galactose-terminal asialoganglioside, have been incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes (Nicolau et al., 1987). It is
contemplated that the tissue specific transforming constructs of the present description can be specifically delivered into a target cell in a similar manner.
[0133] Synthetic Gene. A synthetic gene is also provided comprising a nucleic acid described herein operably linked to a transcriptional and/or translational regulatory sequence. The synthetic gene may be capable of modifying the expression of a target gene with a binding site for a nucleic acid described herein. Expression of the target gene may be modified in a cell, tissue or organ. The synthetic gene may be synthesized or derived from naturally-occurring genes by standard recombinant techniques. The synthetic gene may also comprise terminators at the 3'-end of the transcriptional unit of the synthetic gene sequence. The synthetic gene may also comprise a selectable marker.
[0134] Therapeutics. Modulating the differentiation of stem cells, e.g., hematopoietic stem cells, may be used for treating or preventing a disease or condition. For example, a subject may receive an agonist of one or more of the miRNAs having any one of SEQ ID NOs: 1 or 2 or of one of the stem cell miRNAs to block cell differentiation in situations in which an excessive cell differentiation is present and/or insufficient amounts of undifferentiated cells are present in the subject. Such conditions may include those in which a subject has been treated, e.g., by irradiation and has lost a considerable amount of undifferentiated cells or HSPC. A subject may receive one or more agonists to thereby increase the number of stem progenitor cells.
[0135] In other embodiments, in which it is necessary to increase the number of differentiated cells in a subject, a treatment may comprise administering an agonist of one or more stem cell miRNA or HSPC miRNA. For example, a subject having cancer may be treated by receiving one or more antagonists of one or stem cell miRNA or HSPC miRNA. This treatment could stimulate the differentiation of an undifferentiated cancer cell and/or reduce the number of cancer cells. Other conditions in which one may want to induce the differentiation of a stem-progenitor cell include those associated with an insufficient number of a differentiated cell, e.g., a differentiated or partially differentiated hematopoietic cell. Exemplary conditions or diseases that may be treated with antagonists include those associated with insufficient leukocytes (leukopenia, e.g., granulocytosis, neutropenia such as drug or chemotherapy induced neutropenia, and congenital neutropenia); insufficient lymphocytes, insufficient red blood cells (e.g., anemias, such as aplastic anemia, nutritional deficiency anemia, hemolytic anemia);
insufficient neutrophils (neutropenia), or insufficient platelets (thrombocytopenia). Other diseases include immunodeficiency diseases, e.g., inherited immunodeficiency diseases, drug or therapy induced immunodeficiency diseases, HIV-immunodeficiency diseases. Yet other diseases in which differentiation of cells may be beneficial include blood cancers in which undifferentiated or partially differentiated cells accumulate, e.g., leukemias and lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphomas, acute leukemias, e.g., lymphoblastic (ALL) and myelogenous (AML) and chronic leukemias.
[0136] Other conditions that may benefit from methods of modulating stem-progenitor cell differentiation include stem cell transplantation, graft versus host disease, and
transplantation, e.g., bone marrow transplantation.
[0137] Methods described herein may also be used for stem cell treatments. Diseases that would benefit from stem cell treatments include Parkinson's disease, spinal cord injuries (as well as other neuromuscular or neurological degenerative diseases), cancer, muscle damage, bone marrow transplants, leukemias and lymphomas. Generally, disorders that can be treated by infusion of stem cells include but are not limited to five broad categories. The first group are diseases resulting from a failure or dysfunction of normal blood cell production and maturation
(i.e., aplastic anemia and hypoproliferative stem cell disorders). The second group are neoplastic, malignant diseases in the hematopoietic organs (e.g., leukemias, lymphomas, myelomas). The third group of disorders comprises those of patients with a broad spectrum of malignant solid tumors of non-hematopoietic origin. Stem cell infusion in these patients serves as a bone marrow rescue procedure, which is provided to a patient following otherwise lethal or strongly
myelosuppressive chemotherapy or irradiation of the patient, designed to eliminate malignant tumor cells. The fourth group of diseases consists of autoimmune conditions, where the stem cells serve as a source of replacement of an abnormal immune system. The fifth group of diseases comprises a number of genetic disorders which can be corrected by infusion of hematopoietic stem cells, preferably syngeneic, which prior to transplantation have undergone gene therapy. Particular diseases and disorders which can be treated by hematopoietic reconstitution with substantially enriched population of hematopoietic stem cells include but are not limited to those listed here: diseases resulting from a failure or dysfunction of normal blood (cell production and maturation, hyperproliferative stem cell disorders, aplastic anemia, pancytopenia, agranulocytosis, thrombocytopenia, red cell aplasia, Blackfan-Diamond syndrome, due to drugs, radiation, or infection, idiopathic); hematopoietic malignancies (acute
lymphoblastic (lymphocytic) leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute malignant myelosclerosis, multiple myeloma, polycythemia vera, agnogenic myelometaplasia, Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, non-Hodgkins's lymphoma); malignant, solid tumors (malignant melanoma, carcinoma of the stomach, ovarian carcinoma, breast carcinoma, small cell lung carcinoma, retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, lymphoma); autoimmune diseases (rheumatoid arthritis, diabetes type I, chronic hepatitis, multiple sclerosis, systemic lupus erythematosus); genetic (congenital) disorders (anemias, familial aplastic, Fanconi's syndrome, Bloom's syndrome, pure red cell aplasia
(PRCA), dyskeratosis congenita, Blackfan-Diamond syndrome, congenital dyserythropoietic syndromes I-IV, Schwachmann-Diamond syndrome, dihydrofolate reductase deficiencies, formamino transferase deficiency, Lesch-Nyhan syndrome, congenital spherocytosis, congenital elliptocytosis, congenital stomatocytosis, congenital Rh null disease, paroxysmal nocturnal hemoglobinuria, G6PD (glucose6-phosphate dehydrogenase) variants 1, 2, 3, pyruvate kinase deficiency, congenital erythropoietin sensitivity deficiency, sickle cell disease and trait, thalassemia alpha, beta, gamma, met-hemoglobinemia, congenital disorders of immunity, severe combined immunodeficiency disease (SCID), bare lymphocyte syndrome, ionophore-responsive combined immunodeficiency, combined immunodeficiency with a capping abnormality, nucleoside phosphorylase deficiency, granulocyte actin deficiency, infantile agranulocytosis, Gaucher's disease, adenosine deaminase deficiency, Kostmann's syndrome, reticular dysgenesis, congenital leukocyte dysfunction syndromes) and others (osteopetrosis, myelosclerosis, acquired hemolytic anemias, acquired immunodeficiencies, infectious disorders causing primary or secondary, immunodeficiencies, bacterial infections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy), parasitic infections (e.g., malaria, Leishmaniasis), fungal infections, disorders involving disproportions in lymphoid cell sets and, impaired immune functions due to aging, phagocyte disorders, Kostmann's agranulocytosis, chronic granulomatous disease, Chediak-Higachi syndrome, neutrophil actin deficiency, neutrophil membrane GP-180 deficiency, metabolic storage diseases, mucopolysaccharidoses, mucolipidoses, miscellaneous disorders involving immune mechanisms, Wiskott Aldrich Syndrome, alpha 1 -antitrypsin deficiency).
[0138] Any of the disorders set forth in.the previous paragraph may also be diagnosed as further described herein.
[0139] Compositions, e.g., pharmaceutical compositions are also provided herein. A composition may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 31, 32, 33 or 34 agonists or antagonists or combinations thereof of miRNAs described herein.
[0140] In some embodiments, therapeutic compositions and methods may include the use of modified miRNAs or miRNA antagonists, including 2-O-methyl oligoribonucleotides (2- O-Me-RNAs). Cell permeable forms of these 2-0' Me-RNAs, known as antagomirs, are also contemplated. Antagomirs have been successfully used to down-regulate mi-RNAs in mouse tissue, in vivo, after intravenous injection (Krutzfeldt et al., 2005, Nature, 438: 685-689). Other modifications may include the introduction of phosphorothioate linkages, the addition of 2-0- methoxyethyl groups, and the addition of a cholesteryl or cholesterol moiety. Locked nucleic acid molecules, wherein the 2'-0 oxygen of a 2'-0 modified RNA is bridged to the 4' position via a methylene linker, to form a rigid bicycle locked into a C3'-endo (RNA) sugar conformation are also contemplated.
Compositions
[0141] A pharmaceutical composition is also provided. The composition may comprise a nucleic acid described herein and optionally a pharmaceutically acceptable carrier and/or excipient. The compositions may be used for diagnostic or therapeutic applications. The pharmaceutical composition may be administered by known methods, including wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, viral methods and cationic liposomes. In some embodiments, the compositions may include the use of synthetic miRNAs, which are more potent than naturally occurring miRNAs (Chang, 2006, Clin Cancer Res, 12: 6557-64).
Allogeneic transplantation
[0142] miR126 may be used in allogeneic transplantation. A composition comprising oligonucleotides comprising miR126 sequence or compatible sequence is administered to a healthy subject, or to isolated cells of a healthy subject. Four to ten hours later blood is drawn from the subject or isolated cells are harvested. The isolated blood cells are administered to a recipient, preferably a histocompatible patient. G-CSF may be administered to the recipient after administering the cells.
[0143] The content of all publications cited herein are incorporated by referencing in their entirety. The following examples are non-limiting embodiments of the description.
EXAMPLES
Example 1: Mice
[0144] All mice (C57BL/6; 4-8 weeks of age) were housed in the animal facilities at the National Institutes of Health; animal studies were approved by the NCI/Bethesda Institutional Animal Care and Use Committee, and were conducted in accordance with institutional guidelines. G-CSF mobilization experiments were performed as described (De La Luz Sierra et al., 2007, Blood, 1 10:2276-85).
Example 2: In vitro cell culture
[0145] Bone marrow cells were obtained by flushing femurs and tibias; Lin" cells were derived with Mouse Lineage Cell Depletion Kit (Miltenyi Biotech) as described by the
Manufacturer. Progenitor cells were derived from Lin" cells by positive sorting of live (D API- negative or identified with LIVE/DEAD kit, Invitrogen/Molecular Probes) APC-labeled Sca- 1 brightApCeFluor 780.Iabeled cKitbright cells after gating out cells expressing CD45, TER1 19, 7-4, CD1 lb, CD 19, Ly-6G/C and CD5 (all biotin-labeled antibodies followed by FITC-streptavin). LinTerl 15"CD45" cells were sorted (FACSVantage SE; BD Biosciences) from Lin" cells after staining with CD45 and TER1 19 biotin-labeled antibodies and FITC-streptavin. VE-cadherin+ cells were derived from Lin" cells by positive sorting of live, APC-labeled VE-cadherin+ cells. All antibodies and FITC-streptavidin were from BD Pharmingen. Bone marrow-derived microvesicles were prepared essentially as described (Raposo et al., 1996, J Exp Med, 183:1 161- 72) from extracellular fluid derived from flushing femurs and tibias with 10 ml ice-cold PBS, cell removal through centrifugation (10 min at 300 g), filtration through 0.45 mm filters followed by two sequential ultracentrifugation steps: 30 min at 10,000 g and 60 min at 70,000 g.
Individual pellets were suspended in 0.1 ml PBS, fixed (2% gluteraldehyde in 0.1 M cacodylate buffer) for electron microscopy (NCI core Electron Microscopy Laboratory, SAIC Frederick, MD) or labeled with PKH26 (red) or PKH67 (green) fluorescent dyes (Sigma-Aldrich) following manufacturer's instructions. Labeled microvesicles were washed twice in PBS (10 ml) by ultracentrifugation (60 min at 70,000 g). Bone marrow cells (lxloVml) were cultured (18-24 hour) in Iscove's DMEM with 10% FBS with or without G-CSF (25 ng/ml) alone or with labeled microvesicle preparations.
[0146] Bone marrow cells were cultured (18-24 hour) in Iscove's DMEM with 10% FBS alone or with labeled microvesicle preparations. The ratio between cells and microvesicles was chosen to reflect the expected ratios in bone marrow (typically 50x106 bone marrow cells generated microvesicles were suspended in 100 μΐ PBS; thus, lxlO6 cells were cultured with 2 μΐ microvesicle preparations). HUVEC or MS-5 cells cultured according to Nakayama et al., 2007, J Natl Cancer Inst, 99:223-35, were incubated (6-10 hours) alone or with labeled microvesicles (at 1/10th the ratio used for bone marrow cells). Colony assays were performed using
methylcellulose medium supplemented with a cocktail of recombinant growth factors (Methocult M3434, Stem Cell Technologies). Heparinized blood (10 and 20 μΐ) and bone marrow cells (2xl04) were plated in duplicate on methylcellulose medium (2.5 ml) and incubated (5% C02, 37° C) for 7-12 days of culture. Colonies (at least 50 cells) were counted.
Example 3: Transfection and RNA analysis
[0147] A precursor to miR126 (pre-miR126, Ambion PM12841), a precursor control (Ambion 171 10) or a Cy™3-labeled pre-miR control (AMI 7120) were transfected into freshly derived Lin" bone marrow cells (5xl06 cells/transfection) or MS-5 cells (after
trypsinization/washing 5xl06 cells /transfection) using the Amaxa system (following the
Manufacturer's 4D-Nucleofector™ protocol for mouse T cells). After 18 hour culture (bone marrow cells: 2x106 cell/ml, in DMEM-supplemented with 10% serum, 25 ng/ml Stem Cell Factor, 25 ng/ml cKit Ligand and 10 ng/ml IL-3; MS-5 cells: lxlO6 cell/ml in aMEM
supplemented with 10% FBS), transfection efficiency was assessed by Cy™3 (premiR control) and DAPI fluorescence detected by flow cytometry. Total RNA was isolated using TRIsol reagent (Invitrogen). Real-time PCR was performed using Assay-on-Demand Taqman probes for mouse CXCR4, CXCR2, VCAM-1, and GAPDH (Applied Biosystems). For isolation of miR, a miRVANA isolation kit was used; for amplification, Taqman microRNA reverse transcription kit was used; and for real-time PCR miR126 and miR16 Taqman probes were used (all from
Applied Biosystems), as described in Taqman microRNA Protocols (Applied Biosystems).
Example 4: Flow cytometry and immunocytochemistry.
[0148] Cells were stained with the antibodies: FITC, PE or APC anti-mouse Grl (LY-
6G and Ly-6C); biotin rat anti-mouse CXCR4/CD184; PE-labeled rat anti-mouse CXCR-2
(R&D Systems); biotin anti-human/mouse CXCR7 (clone 8F1 l -M-16, Biolegend); PE or biotin rat anti-mouse CD106/VCAM-1 , biotin rat anti-mouse CD49d (a4 integrin); PE, biotin or PECy5
(rat anti-mouse Sca-1 (Ly-6A-E, AbD Serotec); biotin anti-mouse Sca-1 (Ly-6A/E); FITC, PE or APC rat anti-mouse c it CDl 17; PE-Cy5 rat anti-mouse CD45 (LCA, Ly5); PE anti-mouse VE- cadherin/CD144 (BioLegend), Lineage markers (CD45, TERl 19, 7-4 clone/neutrophils, CDl lb, CD 19, Ly-6G/C and CD5, all biotin-labeled from StemCell Technologies), FITC rat anti-mouse F4/80 antigen (AbD Serotec); APC rat anti-mouse CD45R/B220, APC rat anti-mouse CDl lb. Unless otherwise specified, all antibodies and appropriate isotype control antibodies were from BD Pharmingen. Streptavin-FITC (Invitrogen) -APC (Biolegend) and -PE (BD Biosciences) was used for detection of biotin-labeled antibodies. Live cells were identified using DAPI or
LIVE/DEAD Fixable Far Red Dead Cell Stain Kit (Invitrogen/Molecular Probes). Results acquired by flow cytometry were analyzed with CELLQuest software (BD Biosciences) after acquisition of data from 104 live cells. Fluorescence and bright-field images were acquired through a Nikon Eclipse E600 microscope equipped with Plan Apo 40X/0.95 DIC M, 60X/1.40 oil DIC H, and l OOX/1.40 oil DIC H lenses, and photographed with a digital camera (Retiga 1300, Qimaging). Images obtained with IPLab for Windows software (Scanalytics) were imported into Adobe Photoshop.
Example 5: Western blotting
[0149] Protein extracts prepared in NP-40 lysis buffer with protease inhibitor cocktail setlll (Calbiochem), 50 mM NaF, and 1 raM sodium ortho vanadate were resolved through 4- 12% Bis Tris Gels (Invitrogen). After protein transfer, the nitrocellulose membranes were immunoblotted with specific rat anti-mouse antibodies to VCAM-1/CD106 (R&D Systems) and re-probed with goat IgG anti-actin antibodies (Santa Cruz Biotechnology).
Example 6: Statistical analysis
[0150] Group differences were evaluated by two-tailed Student t test. P values less than 0.05 were considered significant.
Example 7: G-CSF induced changes in hematopoietic stem/progenitor cells (HSPC)
[0151] The association of G-CSF mobilization to with G-CSF-induced changes in HSPC expression was measured. Specifically, expression of CXCR4 and CXCR2 was measured in parallel in bone marrow cells that express the myeloid marker Grl and in the Lin"cKith'Sca- lhl HSPC. These cells account for <0.5% of bone marrow cells and are the cell fraction with hematopoietic reconstitution potential (Fig. 1).
[0152] G-CSF mobilization significantly reduced surface CXCR4 levels in unselected bone marrow cells ("All cells"), in Grl'0 (i.e., immature neutrophils and monocytes) and Grlhl cells (mature neutrophils do not stain for the monocyte marker CDl 15/M-CSFR) (p<0.05) (see
Figs. 1 A and 2A). In contrast, G-CSF mobilization was not associated with significant changes in the percentage and mean fluorescence intensity (MFI) of surface CXCR4+ cells within the Lin" cKithiSca-lhi cell population(Fig. IB), which includes HSPC. Lin*cKithiSca-lhi cells were sorted from both control bone marrow cells and from G-CSF-mobilized bone marrow cells. By realtime PCR, levels of CXCR4 mRNA were similar in Lin"cKith'Sca-lhl cells from untreated and G- CSF-mobilized bone marrows, although they markedly differed from unsorted bone marrow cells (Fig.3A).
[0153] Surface expression of CXCR7, the alternative SDF-1 receptor, was low and minimally changed in Lin"c ithlSca-lhl after G-CSF mobilization. Consistently, Lin"cKith'Sca-lhl cells from either control or G-CSF-mobilized mice responded similarly to SDF1 in trans- well migration assays over a wide range of chemokine concentrations (i.e., 1-100 ng/ml).
[0154] G-CSF mobilization induced a significant increase in surface CXCR2 expression in unfractionated bone marrow cells, including the Grl'° and Grlhl cell populations (p<0.05 all comparisons) (Fig. 1C). However, G-CSF mobilization was not associated with a significant change in the percentage and MFI of surface CXCR2+ cells within the Lin"cKith'Sca- lhi cell population (Fig. ID). Significant changes in the levels of CXCR2 mRNA after G-CSF mobilization were observed in unfractionated bone marrow cells, but these changes were not seen in sorted Lin"cKithiSca-lhi cells (Fig. 3B).
[0155] G-CSF regulation of CXCR4 is a critical pathway for HSPC release by G-CSF. While the role of CXCR4 in the mobilization of myeloid cells, particularly with a contribution of CXCR2, is known by others, its role in the mobilization of HSPC is not. Others have shown that G-CSF mobilization induces a gradual increase in the expression of CXCR4 on bone marrow progenitors (CD34+ or CD38+ cells) over a five-day treatment. In contrast, in the present measurements CXCR4 and CXCR2 were not found to change significantly on bone marrow HSPC after G-CSF mobilization (Figs. IB and 1C), while significant changes in the levels of CXCR2 mRNA and CXCR4 mRNA after G-CSF mobilization were observed in unfractionated bone marrow cells, but not in sorted Lin-cKithlSca-lhl cells (Figs. 3 A and 3B).
[0156] CXCR4 antagonists are known to promote HSPC mobilization. Based on the observation that G-CSF was ineffective at enhancing HSPC mobilization in blood and spleen of mice with CXCR47" bone marrows, but an antagonist of VLA4 VCAM-1 induced a 2-fold increase in HSPC mobilization, it is possible that G-CSF is ineffective at mobilizing CXCR4- null HSPC. Alternatively, it is possible that the absence of CXCR4 on myeloid lineage cells prevents G-CSF-induced neutrophil/monocyte mobilization, which is indirectly required for HSPC mobilization by G-CSF.
[0157] The results described herein demonstrate that G-CSF mobilization is not associated with changes in surface CXCR4 and CXCR2 levels in bone marrow HSPC. Further, the results show that changes in surface CXCR4 and CXCR2 expression are neither typical nor required for HSPC mobilization by G-CSF. This result is surprising and unexpected because G- CSF mobilization of bone marrow myeloid cells was previously shown to be associated with G- CSF-induced changes in the expression of CXCR4 and CXCR2, and these changes were believed to be critical for HSPC mobilization (Eash et al., 2010, J Clin Invest, 120:2423-31).
Example 8: G-CSF modulates surface VCAM-1 in bone marrow HSPC and non- hematopoietic stromal and endothelial cells.
[0158] Potential changes induced by G-CSF in HSPC were examined, particularly with respect to integrin a4 and VCAM-1. Similar levels of integrin a4 surface expression were found in both Lin"cKithiSca-lhi cells from untreated and G-CSF-mobilized mice. All Lin"cKithiSca-l hi cells expressed surface integrin a4 with similar MFI within non-mobilized and G-CSF-mobilized bone marrows.
[0159] In contrast, G-CSF mobilization was associated with a significant (PO.05) decrease in the proportion of surface VCAM-1 positive Lin"cKith'Sca-lhl cells (Fig. 4A). VCAM- 1 MFI was also reduced on Lin"cKithlSca-lhl cells from a mean of 572 units to a mean of 304 units. However, no differences in the levels of VCAM-1 mRNA were found in in sorted Lin" cKithiSca-lhi cells from non-mobilized and G-CSF-mobilized mice (Fig. 5).
[0160] The reduction in the proportion of surface VCAM-1 -positive HSPC is surprising and unexpected. This reduction is comparable in magnitude to the reduction found previously in the number of bone marrow HSPC induced by G-CSF (Uchida et al., 1997, Blood, 89:465-72). This suggests a link between reduction of surface VCAM levels on HSPC and their mobilization out of the bone marrow to the circulation.
[0161] A marked reduction in the proportion of VCAM* cells was found within the non-hematopoietic bone marrow Lin erl 19 CD45" cells (stromal cells) (Fig. 4B) and the Lin" VE-cadherin+ cells (endothelial cells) (Fig. 4C).
[0162] A global reduction of VCAM-1 expression induced by G-CSF was evident by analysis of mRNA, protein and surface expression in unselected bone marrow cells from G-CSF- mobilized and non-mobilized bone marrows (Fig. 4D).
[0163] These results raise the possibility that modulation of surface VCAM-1 contributes to HSPC mobilization by G-CSF. Others have shown that G-CSF inhibits VCAM-1 expression in bone marrow stromal cells. It is possible that enzymatic cleavage of VCAM-1 on these stromal cells removes a critical retention ligand for HSPC that express VLA4, However, it is known that enzyme-deficient mice mobilize HSPC normally with G-CSF, and that HSPC from
VCAM-1 -deficient mice mobilize normally when transplanted in normal recipients. Accordingly, reduction in VCAM-1 in bone marrow stromal cells may not be required for mobilization of HSPC by G-CSF. However, mice deficient of VCAM-1 in hematopoietic, endothelial and stromal cells mobilized poorly HSPC in response to G-CSF as judged by the abnormally low number of progenitors found in the blood and spleen combined, in spite of having a normal number of bone marrow progenitors.
[0164] In contrast to these earlier suggestions, the results described herein demonstrate that VCAM-1 expression is required for G-CSF mobilization, but cleavage of cell surface VCAM-1 is not involved. These results are consistent with modulation of surface VCAM-1 in bone marrow hematopoietic and non-hematopoietic cells as a contributing factor to HSPC mobilization by G-CSF.
Example 9: miR126 regulates VCAM-1 protein expression in hematopoietic and non- hematopoietic cells.
[0165] VCAM-1 /VLA4 interactions may play a role in the retention and mobilization of HSPC from the bone marrow. Conditional deletion of VCAM-1 or a4-integrin in
hematopoietic, stromal and endothelial cells by a Tie2-driven ere transgene is known to increase the release of hematopoietic progenitors from the bone marrow to the circulation. Antibodies to VCAM-1 , to a4 integrin, and BI05192, a small molecule inhibitor of VLA4 (α4β1 integrin) binding to VCAM-1, are all know to promote the mobilization of HSPC in mice and/or humans, indicating that the disruption of VLA4 interaction with VCAM-1 promotes the mobilization of HSPC. However, a link between G-CSF mobilization of HSPC and modulation of VCAM- 1 /VLA4 in the bone marrow has previously been missing.
[0166] The ability of miR126 to regulate VCAM-1 expression was tested in HSPC, in VE-cadherin+ bone marrow cells, and in the MS-5 bone marrow stromal cells line. Lin" bone marrow cells and MS-5 were transfected with a precursor RNA to miR126, a precursor control, or with a Cy™3-labeled pre-miR control. After 18 hour, 36-96% of the bone marrow cells and >62% of MS-5 cells transfected with the fluorescent pre-miR control were fluorescent. Pre- miR126 transfection of primary bone marrow cells increased levels of miR126 (relative to miR16 used as a control) by >35-fold in comparison to control (Fig. 6A, left).
[0167] Forcing expression of miR126 induced minimal change in the relative levels of
VCAM-1 mR A (Fig. 6 A, right), but caused a decrease in the proportion of surface VCAM-1 positive Sca-lhlcKithl cells (Fig. 6A, right). Expression of miR126 reduced the percentage of Sca- lhicKithi cells that express surface VCAM-1 (Fig. 6B). Expression of miR126 also reduced the percent surface VCAM-1+ cells in bone marrow VE-cadherin-expressing cells compared to the control (Fig. 6C). Similar effects were observed in MS5 cells transfected (mean 76% transfection efficiency) with the miR126 precursor (Fig. 6D). Thus, miR126 reduced VCAM-1 protein surface expression in HSPC and non-hematopoietic bone marrow cells without significantly affecting VCAM-1 mRNA levels.
[0168] The results described herein demonstrate that VCAM-1 protein expression is modulated by miR126. It is known that miR126 inhibits VCAM-1 expression in endothelial cells. However, in endothelial cells, miR126 only blocked TNF-a-induced expression of VCAM- 1. The present results are surprising in they show that miR126 regulates constitutive elevated expression of VCAM-1 in bone marrow cells, in contrast to endothelial cells. In addition these results show that miR126 can reduce VCAM-1 in hematopoietic cells and bone marrow stromal cells, which could not have been predicted due to cell selectivity of miR functions.
Example 10: Regulation of miR126 in bone marrow following G-CSF mobilization.
[0169] The ability of G-CSF to modulate levels of miR126 in bone marrow cells was tested. G-CSF mobilization significantly reduced the levels of miR126 expression in these cells (Fig. 7A left). However, G-CSF increased levels of miR126 in the cell-free fraction of flushed bone marrows (obtained after removal of cells, filtration through 0.45 μηι filters,
ultracentrifugation and pellet suspension in l/lO"1 the original volume) (Fig. 7A right). Similarly, G-CSF decreased levels of miR126 in bone marrow cells in vitro, and promoted miR126 release in the culture supernatant after 24 hour culture (Fig. 8).
[0170] The levels of miR126 expression were measured in bone marrow HSPC (Sca- lhicKithi cells) and non-hematopoietic cells. The Lin'Sca-lhicKithi cells sorted from G-CSF- mobilized bone marrows (n=9; 3 bone marrows combined for 3 sorting) contained significantly more miR126 compared to the Lin"Sca-lhlcKithl cells from untreated mice (n=9) (Fig. 7B, right). The unfractionated bone marrow cells from which the Sca-lh,cKithl cells were sorted showed G- CSF-mobilization inhibited miR126 expression compared to untreated bone marrows (Fig. 7B, left).
[0171] The Lin" Terl 19"CD45" cells sorted from G-CSF-mobilized bone marrows (n=9; 3 bone marrows combined for 3 sorting) contained significantly more miR 126 than the Lin" Terl 19"CD45" cells from untreated mice (n=9) (Fig. 7C). VCAM-1 mRNA levels in the sorted bone marrow HSPC or LinTerl 19"CD45" cells did not decrease in response to G-CSF, whereas a decrease was observed in the unsorted populations (Fig. 9). Thus, G-CSF mobilization is associated with a complex redistribution of miR126 in the bone marrow, an overall reduction in cells with a relative increase in HSPC and LinTerl 19"CD45" cells, and an increase of miR126 found released from the cells in the extracellular bone marrow compartment. Example 11: Microvesicle production and transfer to HSPC and non-hematopoietic cell subsets.
[0172] It is known that that miRNAs are released in the extracellular compartment through "exosomes" and apoptotic bodies, which play a role in intracellular communication.
[0173] Electron microscopy of the cell-free fraction from G-CSF-mobilized bone marrows revealed large numbers of microvesicles resembling exosomes and others
morphologically similar to neutrophil-derived apoptotic bodies described in the bone marrow. Fewer of the microvesicles were present in the medium of non-mobilized bone marrows.
[0174] To test if miR126 might be delivered by microvesicles to bone marrow HSPC and to subsets of non-hematopoietic cells, microvesicles were purified following protocols used for exosome preparation from G-CSF-mobilized and non-mobilized bone marrow cells. A red (PKH26) or green (PKH67) fluorescent lipid dye was used. After washing, the isolated microvesicles were incubated for 18 hours with cells from control or G-CSF mobilized bone marrows. A low proportion (2.8%-6.7%) of all bone marrow cells acquired red fluorescence, showing low-level microvesicle uptake by bone marrow cells. Only a minority (0.5%- 1.6%) of Grl+ cells picked up the dye, regardless the source of the microvesicle preparations (bone marrow from G-CSF-treated or untreated mice) or the derivation of the Grl+ cells from G-CSF- mobilized or not mobilized marrows (Fig. 10A).
[0175] In contrast, a greater proportion of bone marrow Sca-1 h,cKithl HSPC (14%- 24%), Lin"CD45" cells (14%-17%) and VE-cadherin+ cells (55%-70%; likely endothelial) acquired the microvesicle fluorescence in comparison to the Grl+ cells (Fig. 10A). This result demonstrated preferential uptake by HSPC cells. The source of the microvesicle preparations (from G-CSF mobilized or non-mobilized bone marrows) and the source of cells incubated with the microvesicles minimally affected the magnitude of uptake (Fig. 1 OA).
[0176] After incubation with fluorescent dye-labeled "exosome" preparations and imaging, a proportion of bone marrow Sca-lhicKithi cells, primary human umbilical vein endothelial cells (HUVEC) and the mouse MS-5 cells exhibited a dot-like cytoplasmic fluorescence attributable to endocytosis of the labeled exosomes.
[0177] Microvesicles from G-CSF-mobilized bone marrow cells were examined for their ability to reduce VCAM-1 cell surface expression. These cells contained more miR126 than those from non-mobilized bone marrows. VCAM-1 mean fluorescence intensity (MFI) was significantly reduced when the exosomes were derived from G-CSF-mobilized bone marrows, by analyzing bone marrow Sca-lh,cKithl bone marrow progenitors that had incorporated the fluorescent microvesicles. In contrast, after incubation with the microvesicles, VCAM-1 MFI minimally changed in non-fluorescent Sca-lhlcKithl (Fig. 10B), cells that did not take up the exosomes. The degree of VCAM-1 MFI reduction in Sca-lhlcKithl cell induced by wild-type "exosome" preparations was similar in magnitude to that induced by G-CSF after an 18 hour culture.
. [0178] A significant reduction in the percentage of VCAM-1+ cells was found after incubation with microvesicles from G-CSF-mobilized in comparison to non-mobilized bone marrow, again measuring Lin erl 19"CD45" cells that had acquired the fluorescent microvesicles (Fig. IOC). VCAM-1 reduction was not observed in the LinTerl 19"CD45" cells that had not acquired the fluorescent microvesicles (Fig. IOC). A similar reduction of surface VCAM-1 was observed in VE-cadherin+ bone marrow cells that had acquired the fluorescent microvesicles (Fig. 10D). Thus, miR126-containing microvesicles from G-CSF-mobilized bone marrows can reduce levels of VCAM-1 surface expression in HSPC, LinTerl 19"CD45" and in VE-cadherin+ cells.
[0179] Apoptotic bodies and exosomes are known to be an important conduit for intercellular communication as they can transfer microRNAs, mRNAs and proteins to other cells and have thus been implicated in many cell functions. Exosomes known to be present in many tissues, including the bone marrow and blood, and apoptotic bodies are abundant in the bone marrow as they are largely a physiologic product of neutrophil death at this site. Endothelium- derived apoptotic bodies enriched in miR126 were found to trigger the incorporation Sca-1+ progenitors to the atherosclerotic plaques, in part through stimulation of SDF-1 expression. In addition, human CD34+ peripheral blood HSPC mobilized by G-CSF contained significantly higher levels of miR126 compared to peripheral blood mononuclear cells.
[0180] The results described herein show that G-CSF indirectly promotes the mobilization of HSPC from the bone marrow to the peripheral blood by inducing the release of miR126-containing microvesicles in the bone marrow cavity. Bone marrow microvesicles can deliver miR126 to bone marrow HSPC, endothelial and stromal cells, and miR126 reduces cell surface expression of VCAM-1 , a receptor molecule that regulates the trafficking of HSPC from the bone marrow to the blood through its major integrin ligand VLA4 (α4β1 integrin).
Example 12: Defective mobilization of HSPC by G-CSF in miRl 26-deficient mice.
[0181] miR126-null mice are known to have a vascular phenotype with leaky vessels and hemorrhaging; -40-50% of mice die embryonically or perinatally. If miRl 26 is important to
HSPC mobilization by G-CSF, miRl 26-deficient mice may be defective at mobilizing HSPC with G-CSF. G-CSF-mobilized groups of 13-week old miR126+/+ and miR126"A littermates were compared to non-mobilized mice. Blood cell counts showed that G-CSF treatment induced an increase in circulating white blood cells and neutrophils in miR.1267" mice, albeit lower than that displayed by the miR126+/+ littermates (Fig. 1 1 A). Colony assays showed that blood from the G- CSF-mobilized miR126"A mice contained significantly fewer CFU-c compared to the miR126+/+ controls, whereas blood from untreated miR126+/+ and miR126"A mice contained no colony precursors (Fig. 1 IB). Variability was found within the mobilized miR126 " mice, consistent with the variability of other defective phenotypes in these mice. These results indicate that miR126-null mice are defective at mobilizing HSPC after G-CSF treatment.
[0182] Consistent with miR126 playing an important role in G-CSF HSPC
mobilization, miR126-deficient mice mobilize poorly in response to G-CSF. These present results identify miR126 as a regulator of HSPC trafficking from the bone marrow to the peripheral blood, and clarify the role of VCAM-1 in G-CSF mobilization. A schematic representation of this process is shown in Fig. 1 1 C.

Claims

What is claimed:
1. A method for modulating the phenotype of stem or progenitor cells (SPC), comprising contacting the SPC with a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
2. The method of claim 1, further comprising collecting and isolating the SPC after contact with the nucleic acid molecule.
3. The method of claim 2, further comprising administering the collected SPC to a patient.
4. The method of claim 1, wherein the nucleic acid molecule is administered to a healthy subject.
5. The method of claim 4, further comprising collecting the SPC from said subject.
6. The SPC produced according to the method of claim 5.
7. A method for treating hematopoietic deficiency or hematological failure, comprising administering the SPC of claim 6 to a patient having hematopoietic deficiency or hematological failure.
8. The method of claim 7, wherein the SPC are administered to the patient following bone ablation therapy.
9. The method of claim 1, wherein the nucleic acid molecule is administered to a patient having hematopoietic deficiency or hematological failure
10. The method of claim 9, wherein said method treats the hematopoietic deficiency or
hematological failure.
1 1. The method of claim 9, wherein the hematopoietic deficiency or hematological failure is associated with a cancer.
12. The method of claim 9, wherein the hematopoietic deficiency or hematological failure is associated with a treatment of cancer.
13. The method of claim 1, wherein contacting the SPC with the nucleic acid molecule
causes a decrease in VCAM-1 protein expression by the SPC.
14. The method of claim 1, wherein the nucleic acid molecule contacts the SPC in vitro.
15. The method of claim 13, further comprising isolating the SPC.
16. The isolated SPC of claim 14, wherein the SPC comprises the nucleic acid molecule, and wherein the nucleic acid molecule is a heterologous to the SPC.
17. The isolated SPC of claim 14, wherein the SPC comprise a hematopoietic cell.
18. A method of increasing mobilization of hematopoietic stem or progenitor cells (HSPC), comprising administering to a subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
19. The method of claim 18, further comprising harvesting said HSPC from the peripheral blood of the subject.
20. A method of treating cancer in a cancer patient in need thereof, comprising administering to a normal subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof, harvesting hematopoietic stem or progenitor cells (HSPC) from the blood of the subject, and administering the HSPC to the cancer patient.
21. A method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
22. A method of screening a candidate compound for an effect on mobilization of stem or progenitor cells (SPC), comprising providing SPC; contacting the SPC with said candidate compound; and measuring the effect of said candidate substance on the expression or stability of miR126 miRNA.
23. Use of a chemo therapeutic agent for the manufacture of a medicament for treating a cancer wherein said treatment comprises administering the medicament and a
composition comprising allogenic hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
24. Use of a chemotherapeutic agent for the manufacture of a medicament for treating a
cancer wherein said treatment comprises administering the medicament and a
composition comprising autologous hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
25. Use of a chemotherapeutic agent for the manufacture of a medicament for treating a
cancer wherein said treatment comprises administering the medicament and a
composition a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
26. A chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the chemotherapeutic agent and a composition comprising allogenic hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
27. A chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the chemotherapeutic agent and a composition comprising autologous hematopoietic stem or progenitor cells (HSPC), wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
28. A chemotherapeutic agent for use in a method for treating a cancer wherein the treatment comprises administering the chemotherapeutic agent and a composition comprising a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
29. A composition comprising allogenic hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a
chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
30. A composition comprising autologous hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a
chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
31. A composition comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof, for use in treating a cancer, wherein the treatment comprises administering a chemotherapeutic agent and said composition.
32. A composition comprising autologous hematopoietic stem or progenitor cells (HSPC) for use in treating a cancer, wherein the treatment comprises administering a
chemotherapeutic agent and said composition, wherein the HSPC are produced by administering a nucleic acid molecule comprising a nucleotide sequence that is at least about 90% identical to SEQ ID NOS: 1 or 2, or a complement thereof.
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