WO2019050841A1 - Composition de cellules souches hématopoïétiques - Google Patents

Composition de cellules souches hématopoïétiques Download PDF

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WO2019050841A1
WO2019050841A1 PCT/US2018/049355 US2018049355W WO2019050841A1 WO 2019050841 A1 WO2019050841 A1 WO 2019050841A1 US 2018049355 W US2018049355 W US 2018049355W WO 2019050841 A1 WO2019050841 A1 WO 2019050841A1
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hematopoietic stem
stem cells
human
population
cells
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PCT/US2018/049355
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Ilya Shestopalov
Sean BENDALL
Luciene BORGES
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Bluebird Bio, Inc.
The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO2019050841A1 publication Critical patent/WO2019050841A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/14—Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
    • C12Y304/14009—Tripeptidyl-peptidase I (3.4.14.9)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01076—L-Iduronidase (3.2.1.76)
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Definitions

  • the present invention generally relates, in part, to hematopoietic stem cell compositions and methods for using the same in gene therapy.
  • HCT has become the most successful and widely used form of stem cell therapy.
  • HCT is currently the standard of care for otherwise incurable hematological malignancies, such as leukemia and lymphoma; genetic diseases; hemoglobinopathies, and immune diseases.
  • therapeutic applications of HCT have been extended to cell-based gene therapies, where autologous stem cells are modified ex vivo in and subsequently re-infused into the patient.
  • CB umbilical cord blood
  • a population of human hematopoietic cells comprises isolated human CD34+CD84- hematopoietic stem cells.
  • the human CD34+CD84- hematopoietic stem cells are also CD90+.
  • the human CD34+CD84- hematopoietic stem cells are also CD45RA-.
  • the human CD34+CD84- hematopoietic stem cells are also CD49f+.
  • the human CD34+CD84- hematopoietic stem cells are also CD38Lo/.
  • the human CD34+CD84- hematopoietic stem cells are also CD164+.
  • the human CD34+CD84- hematopoietic stem cells are also CD172a+.
  • the human CD34+CD84- hematopoietic stem cells are also CD117+.
  • the human CD34+CD84- hematopoietic stem cells are also CD133+.
  • a population of cultured hematopoietic cells enriched for the human hematopoietic stem cells contemplated herein, is provided.
  • a population of cultured hematopoietic cells enriched for human CD34+CD84- hematopoietic stem cells is contemplated.
  • a population of cultured hematopoietic cells enriched for human CD34+CD84- CD90+ CD45RA- hematopoietic stem cells is contemplated.
  • a population of cultured hematopoietic cells enriched for human CD34+CD84- and one or more of hematopoietic stem cell markers selected from the group consisting of CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+ is contemplated.
  • a human CD34+CD84- hematopoietic stem cell comprises a gene therapy vector.
  • the CD34+CD84- hematopoietic stem cell further comprises one or more of hematopoietic stem cell markers selected from the group consisting of CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+.
  • a human CD34+CD84- hematopoietic stem cell comprises one or more genome modifications.
  • the CD34+CD84- hematopoietic stem cell further comprises one or more of hematopoietic stem cell markers selected from the group consisting of CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+.
  • a human CD34+CD84- hematopoietic stem cell comprises one or more exogenous polynucleotides.
  • the CD34+CD84- hematopoietic stem cell further comprises one or more of hematopoietic stem cell markers selected from the group consisting of CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+.
  • a composition comprises at least 50%, at least 75%, at least 85%, at least 90%, or at least 95% of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, or one or more human hematopoietic stem cells contemplated herein.
  • a composition comprises at least 50%, at least 75%, at least 85%, at least 90%, or at least 95% human CD34+CD84- hematopoietic stem cells.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier comprises a physiologically acceptable cell culture medium.
  • a pharmaceutical composition comprises a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, or one or more human hematopoietic stem cells contemplated herein; and a pharmaceutically acceptable cell culture medium.
  • a method of treating a subject in need thereof comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • hemoglobinopathy comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • a method of treating sickle cell disease in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • a method of treating a ⁇ -thalassemia in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- - hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding a globin.
  • the retroviral vector or the lentiviral vector is an
  • a method of treating adrenoleukodystrophy or adrenomyelonephropathy in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding an ATP -binding cassette, sub-family D, member 1 (ABCD1) polypeptide.
  • a retroviral vector or a lentiviral vector encoding an ATP -binding cassette, sub-family D, member 1 (ABCD1) polypeptide.
  • the retroviral vector or lentiviral vector comprises a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter or transcriptionally active fragment thereof operably linked to a polynucleotide encoding an ATP-binding cassette, subfamily D, member 1 (ABCD1) polypeptide.
  • MND primer-binding site substituted
  • a method of treating ADA-SCID in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding an adenosine deaminase.
  • the retroviral vector or lentiviral vector comprises an elongation factor 1 alpha promoter or and MND promoter operably linked to a polynucleotide encoding adenosine deaminase.
  • a method of treating X-SCID in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding an interleukin 2 receptor gamma.
  • the retroviral vector or lentiviral vector comprises an elongation factor 1 alpha promoter operably linked to a polynucleotide encoding interleukin 2 receptor gamma.
  • a method of treating Batten's disease in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding a tripeptidyl peptidase 1.
  • the retroviral vector or lentiviral vector comprises an elongation factor 1 alpha promoter or comprises a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl 587rev primer-binding site substituted (MND) promoter operably linked to a polynucleotide encoding tripeptidyl peptidase 1.
  • MND primer-binding site substituted
  • a method of treating MPS I in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding an alpha-L iduronidase.
  • the retroviral vector or lentiviral vector comprises an elongation factor 1 alpha promoter or comprises a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter operably linked to a polynucleotide encoding alpha-L iduronidase.
  • MND primer-binding site substituted
  • a method of treating MPS II in a subject comprises administering an effective amount of a population of human hematopoietic stem cells, a population of cultured human hematopoietic stem cells, one or more human hematopoietic stem cells, or a pharmaceutical composition contemplated herein to the subject.
  • one or more of the human CD34+CD84- hematopoietic stem cells are transduced with a retroviral vector or a lentiviral vector encoding an iduronate 2-sulfatase.
  • the retroviral vector or lentiviral vector comprises an elongation factor 1 alpha promoter or comprises a myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter operably linked to a polynucleotide encoding iduronate 2-sulfatase.
  • MND primer-binding site substituted
  • a method for isolating human CD34+CD84- hematopoietic stem cells from a heterologous cell population comprises contacting a heterologous cell population with one or more binding agents to isolate human CD34+CD84- hematopoietic stem cells from the population.
  • the one or more binding agents are antibodies or antigen binding fragments thereof.
  • the one or more binding agents bind CD34 and CD84 and one or more cell surface markers selected from the group consisting of: CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+.
  • the isolating the human CD34+CD84- hematopoietic stem cells comprises steps of positive and negative selection.
  • the human CD34+CD84- hematopoietic stem cells comprise one or more gene therapy vectors, genome modifications, or exogenous polynucleotides.
  • the heterologous cell population is selected from the group consisting of: bone marrow, mobilized peripheral blood, umbilical cord blood, placenta, or fractions thereof.
  • a method for quantitating human CD34+CD84- hematopoietic stem cells from a heterologous cell population comprises contacting a heterologous cell population with one or more binding agents to identify human CD34+CD84- hematopoietic stem cells from the population, and quantitating the identified cells.
  • the one or more binding agents are antibodies or antigen binding fragments thereof.
  • the one or more binding agents bind CD34 and CD84 and one or more cell surface markers selected from the group consisting of: CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+.
  • the isolating the human CD34+CD84- hematopoietic stem cells comprises steps of positive and negative selection.
  • the human CD34+CD84- hematopoietic stem cells comprise one or more gene therapy vectors, genome modifications, or exogenous polynucleotides.
  • the heterologous cell population is selected from the group consisting of: bone marrow, mobilized peripheral blood, umbilical cord blood, placenta, or fractions thereof.
  • a kit comprises one or more binding agents to isolate or quantitate human CD34+CD84- hematopoietic stem cells from a heterologous cell population.
  • one or more binding agents are antibodies or antigen binding fragments thereof.
  • the one or more binding agents bind CD34 and CD84 and one or more cell surface markers selected from the group consisting of: CD90+, CD45RA-, CD49f+, CD38Lo/, CD164+, CD172a+, CD117+, and CD133+.
  • the human CD34+CD84- hematopoietic stem cells comprise one or more gene therapy vectors, genome modifications, or exogenous polynucleotides.
  • the kit is used for isolating or quantitating human CD34+CD84- hematopoietic stem cells from bone marrow, mobilized peripheral blood, umbilical cord blood, or placenta.
  • Figure 1 shows a viSNE phenotype analysis of hematopoietic cells isolated from bone marrow, mobilized peripheral blood, and cultured hematopoietic cells isolated from mobilized peripheral blood.
  • FIG. 1 shows the results from cell surface marker expression analysis
  • CD34 + cells isolated from bone marrow CD34 + cells isolated from bone marrow.
  • Figure 3 shows the results from a lineage tracing analysis from CD34 + cells isolated from bone marrow and mobilized peripheral blood.
  • HCT hematopoietic cell transplantation
  • HSC hematopoietic stem cell
  • hematopoietic material in order to increase the efficiency of therapeutic applications and minimize adverse effects of HCT.
  • Primary hematopoietic cells, including stem cells, may alter their cell surface marker expression during modification and/or culture; thereby making the stem cells difficult to re-isolate post-manipulation.
  • CD34 + CD38 " fraction CD34 + CD38 " fraction.
  • CD38 expression decreases in all cells following culture.
  • the variation in cell surface marker expression greatly impairs the ability to identify HSCs after culture.
  • the present invention offers solutions to this and other problems
  • hematopoietic stem cell compositions contemplate improved hematopoietic stem cell compositions with increased therapeutic efficacy and methods of making and using the same.
  • the present inventors have identified novel markers that reliably and reproducibly identify hematopoietic stem cells, whether they have been freshly isolated, genetically modified, and/or cultured. Without wishing to be bound by any particular theory, it is contemplated that cell-based therapies are improved by selecting for HSCs after modification and/or culture and prior to administration to a subject.
  • populations of human hematopoietic stem cells are contemplated that express one or more cell surface markers of HSCs and that do not express, or lack expression of, CD84.
  • HSCs lack CD84 expression before and after culture and thus, can be used to distinguish true HSCs from less potent more differentiated hematopoietic progenitor cells.
  • populations of human hematopoietic cells are contemplated that are enriched for HSCs by selecting HSCs that express one or more cell surface HSC markers, e.g,. CD34, and that do not express, or lack expression of, CD84.
  • the HSC has been modified by introducing genetic material into the cell.
  • compositions comprising HSCs that express one or more cell surface HSC markers and that do not express, or lack expression of, CD84.
  • Methods of treating diseases comprising administering a subject in need thereof an effective amount of the HSCs or compositions contemplated herein are also provided in particular preferred embodiments.
  • an element means one element or one or more elements.
  • the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.
  • the term “substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • an “increased” or “enhanced” quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length is a “statistically significant”, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) a reference.
  • a “decrease” or “lower” or “lessen” or “reduce” or “abate” or “ablate” or “inhibit” or “dampen” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length) that is less than a reference.
  • a “decreased” or “reduced” quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length is typically “statistically significant”, and may include an decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) a reference.
  • maintain or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is not significantly different or measurable different from a reference.
  • Cells contemplated in particular embodiments may be autologous/autogeneic ("self) or non-autologous ("non-self,” e.g., allogeneic, syngeneic or xenogeneic).
  • autologous refers to cells from the same subject.
  • Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
  • Syngeneic refers to cells of a different subject that are genetically identical to the cell in comparison.
  • Xenogeneic refers to cells of a different species to the cell in comparison.
  • stem cell refers to a cell which is an undifferentiated cell capable of
  • Stem cells are subclassified according to their developmental potential as totipotent, pluripotent, multipotent and oligo/unipotent.
  • Self-renewal refers a cell with a unique capacity to produce unaltered daughter cells and to generate specialized cell types (potency). Self-renewal can be achieved in two ways. Asymmetric cell division produces one daughter cell that is identical to the parental cell and one daughter cell that is different from the parental cell and is a progenitor or differentiated cell. Symmetric cell division produces two identical daughter cells. "Proliferation” or “expansion” of cells refers to symmetrically dividing cells.
  • progenitor or “progenitor cells” refers to cells have the capacity to self-renew and to differentiate into more mature cells. Many progenitor cells differentiate along a single lineage, but may have quite extensive proliferative capacity. Hematopoietic stem cells (HSCs) give rise to committed hematopoietic progenitor cells (HPCs) that are capable of generating the entire repertoire of mature blood cells over the lifetime of an organism.
  • HSCs Hematopoietic stem cells
  • HPCs committed hematopoietic progenitor cells
  • HSC hematopoietic stem cell
  • myeloid e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages e.g., T-cells, B-cells, NK-cells
  • myeloid e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages e.g., T-cells, B-cells, NK-cells
  • the term "population of cells” refers to a plurality of cells that may be made up of any number and/or combination of homogenous or heterogeneous cell types, as described elsewhere herein.
  • a population of hematopoietic cells may be isolated or obtained from umbilical cord blood, placental blood, bone marrow, or peripheral blood.
  • a population of cells may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% or any intervening percentage of hematopoietic stem cells.
  • ex vivo refers generally to activities that take place outside an organism, such as modification and/or culture done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be vitro " though in certain embodiments, this term can be used interchangeably with ex vivo. In one embodiment, HSCs are modified and/or cultured ex vivo.
  • vivo refers generally to activities that take place inside an organism.
  • genetically engineered or “genetically modified” refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. Genetic modifications may be targeted or non-targeted to a particular site in a cell's genome.
  • gene therapy refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide.
  • gene editing refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell ' s genome, which restores, corrects, disrupts, and/or modifies expression of a gene or gene product.
  • reprogrammed nuclease engineered nuclease
  • nuclease variant refers to a nuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed and/or modified from a parental or naturally occurring nuclease, to bind and cleave a double-stranded DNA target sequence.
  • a “target site” or “target sequence” is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind and/or cleave, provided sufficient conditions for binding and/or cleavage exist.
  • exogenous molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods.
  • exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
  • lipid-mediated transfer i.e., liposomes, including neutral and cationic lipids
  • electroporation direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
  • an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • isolated means material, e.g., a polynucleotide, a polypeptide, a cell, that is substantially or essentially free from components that normally accompany it in its native state.
  • the term “isolated” means material, e.g., a polynucleotide, a polypeptide, a cell, that is substantially or essentially free from components that normally accompany it in its native state.
  • the term “isolated” means material, e.g., a polynucleotide, a polypeptide, a cell, that is substantially or essentially free from components that normally accompany it in its native state.
  • the term “isolated” means material, e.g., a polynucleotide, a polypeptide, a cell, that is substantially or essentially free from components that normally accompany it in its native state.
  • polynucleotide or “nucleic acid” refers to genomic
  • polynucleotide refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)).
  • mRNA messenger RNA
  • gRNA genomic RNA
  • RNA(+) plus strand RNA
  • RNA(-) minus strand RNA
  • polyribonucleotide or “ribonucleic acid” also refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)) and inhibitory RNAs, including but not limited to siRNA, shRNA, piRNA, miRNA or microRNA, and shRNAs embedded in a microRNA backbone (shmir).
  • mRNA messenger RNA
  • RNA genomic RNA
  • RNA(+) plus strand RNA
  • RNA(-) minus strand RNA
  • inhibitory RNAs including but not limited to siRNA, shRNA, piRNA, miRNA or microRNA, and shRNAs embedded in a microRNA backbone (shmir).
  • polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of reference sequence, typically where the variant maintains at least one biological activity of the reference sequence.
  • Exemplary polynucleotides include expression control sequences and polynucleotides encoding a therapeutic polypeptide including, but not limited to, a globin polypeptide, an antisickling globin polypeptide, an adenosine deaminase polypeptide, an interleukin 2 receptor gamma polypeptide, a tripeptidyl peptidase 1 polypeptide, an alpha-L iduronidase polypeptide, an iduronate 2-sulfatase polypeptide, or an ATP-binding cassette, sub-family D (ALD), member 1 (ABCD1) polypeptide, as discussed elsewhere herein are contemplated.
  • a therapeutic polypeptide including, but not limited to, a globin polypeptide, an antisickling globin polypeptide, an adenosine deaminase polypeptide, an interleukin 2 receptor gamma polypeptide, a tripeptidyl peptidas
  • Polynucleotides regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Art sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably.
  • promoters and/or enhancers such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., Lox
  • expression control sequence refers to a polynucleotide sequence that comprises one or more promoters, enhancers, or other transcriptional control elements or combinations thereof that are capable of directing, increasing, regulating, or controlling the transcription or expression of an operatively linked polynucleotide.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer or other expression control sequence) and a second polynucleotide sequence, e.g., a polynucleotide encoding a therapeutic polypeptide, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally- occurring amino acid polymers.
  • polypeptides include, but are not limited to globin polypeptides, suitable for use in the compositions and methods of particular embodiments. Also, see, e.g., US Patents 6,051,402; 7,901,671 ; and
  • polypeptides include but are not limited to a globin polypeptide, an antisickling globin polypeptide, an adenosine deaminase polypeptide, an interleukin 2 receptor gamma polypeptide, a tripeptidyl peptidase 1 polypeptide, an alpha-L iduronidase polypeptide, an iduronate 2-sulfatase polypeptide, or an ATP- binding cassette, sub-family D (ALD), member 1 (ABCD1) polypeptide.
  • ALD sub-family D
  • ABCD1 member 1
  • vector is used herein to refer to a molecule capable transferring or transporting another nucleic acid molecule.
  • Useful vectors include, but are not limited to, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
  • Useful viral vectors include, but are not limited to, replication defective retroviruses and lentiviruses, adenoviruses, adeno- associated viruses, herpes simplex viruses, and vaccinia viruses.
  • viral vector is widely used to refer either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer.
  • the term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. In one embodiment, the viral vector is episomal.
  • retrovirus refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome.
  • retroviruses include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • Harvey murine sarcoma virus HaMuSV
  • murine mammary tumor virus MuMTV
  • GaLV
  • lentivirus refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV based vector backbones i.e., HIV cis-acting sequence elements
  • HIV cis-acting sequence elements are preferred.
  • lentiviral vectors are known in the art, see Naldini et al, (1996a, 1996b, and 1998); Zufferey et al, (1997); Dull et al , 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce a viral vector or transfer plasmid of the present invention.
  • AAV adeno-associated virus
  • rAAV Recombinant AAV
  • ITRs inverted terminal repeats
  • the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein, in its entirety.
  • adenovirus refers to adenoviral-based vectors capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, Elb, and/or E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non- dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity.
  • Adenovirus vectors have been described in Graham et al, 1977, Jones & Shenk, 1978, Graham & Prevec, 1991, Levrero et al., 1991 and Gomez-Foix et al., 1992, for example.
  • the terms "herpes simplex virus” or "HSV” refer to a modified HSV vector deficient in one or more essential or non-essential HSV genes.
  • the mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb.
  • the HSV based viral vector is replication deficient.
  • Most replication deficient HSV vectors contain a deletion to remove one or more intermediate-early, early, or late HSV genes to prevent replication.
  • the HSV vector may be deficient in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and a combination thereof.
  • HSV vector has its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb.
  • HSV -based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583, each of which are incorporated by reference herein in its entirety.
  • a “subject,” as used herein, includes any animal that exhibits a symptom of a monogenic disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.
  • a subject includes any animal that exhibits symptoms of a disease, disorder, or condition of the hematopoietic system.
  • Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog).
  • Non-human primates and, preferably, human patients, are subjects. Typical subjects include animals that exhibit aberrant amounts (lower or higher amounts than a "normal” or "healthy” subject) of one or more physiological activities that can be modulated by gene therapy.
  • the term "patient” refers to a subject that has been diagnosed with a particular disease, disorder, or condition that can be treated with the
  • treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • prevention and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
  • the term “amount” refers to "an amount effective” or “an effective amount” of therapeutic cells to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.
  • a “prophylactically effective amount” refers to an amount of therapeutic cells effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • a “therapeutically effective amount” of therapeutic cells may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic cells are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” includes an amount that is effective to "treat" a subject (e.g., a patient).
  • HSCs human hematopoietic stem cells
  • the HSCs contemplated by the present disclosure express one or more cell surface markers of HSCs and that do not express, or lack expression of, CD84.
  • HSC marker or “marker of HSC” refers to a protein that is expressed in an HSC and/or whose expression distinguishes between an HSC and another hematopoietic and/or non- hematopoietic cell.
  • HSC markers are preferably cell surface expressed proteins, referred to as "cell surface markers.”
  • Cell surface markers are advantageous in particular embodiments, because expression of cell surface proteins may be determined by antibodies that bind the proteins.
  • Cells expressing certain combinations of cell surface markers may be distinguished by positively and negatively selection based on binding to labeled binding agents such as antibodies.
  • cells may be labeled with one or more labeled antibodies that bind to an HSC marker and be positively selected for using flow cytometry or antibody conjugated beads, e.g., antibody conjugated magnetic beads.
  • hematopoietic cells that are not HSCs can be "panned" from a populations of cells based on negative selection by being labeled with one or more labeled antibodies that bind to a marker of differentiated or lineage committed hematopoietic cells.
  • HSCs are selected with a combination of positive and negative selection.
  • a population of cells comprising one or more isolated hematopoietic stem cells that are CD34 + CD84 " hematopoietic stem cells are
  • a population of cells comprising one or more isolated hematopoietic stem cells that are CD34 + CD84 " and comprise one or more of hematopoietic stem cell markers selected from the group consisting of CD90 + ,
  • CD45RA " , CD49f + , CD38 Lo/ , CD164 + , CD172a + , CD117 + , and CD133 + are
  • a population of hematopoietic cells comprises one or more isolated CD34 + CD84 " hematopoietic stem cells. Populations of cells
  • a population of hematopoietic cells comprises one or more isolated CD34 + CD84 " hematopoietic stem cells modified by introducing genetic material into the cell.
  • CD34 + CD84 " hematopoietic stem cells may be modified by introducing one or more exogenous polynucleotides into the cells.
  • the exogenous polynucleotides may comprise mRNA or DNA.
  • a polynucleotide comprising one or more expression control sequences operably linked to a polynucleotide encoding a therapeutic polypeptide is introduced into the cell.
  • an mRNA encoding a a therapeutic polypeptide is introduced into the cell.
  • a population of hematopoietic cells comprises one or more isolated CD34 + CD84 " hematopoietic stem cells modified by introducing a vector, e.g., viral vector into the cell.
  • the viral vector may be a retroviral vector, such as a lentiviral vector, an AAV, a vaccinia viral vector, and adenoviral vector, or an HSV vector comprising one or more exogenous polynucleotides.
  • the exogenous polynucleotides may encode one or more therapeutic polypeptides or one or more expression control sequences operably linked to a polynucleotide encoding a therapeutic polypeptide.
  • the vector comprises one or more expression control sequences operably linked to a polynucleotide encoding a globin, a ⁇ -globin, a ⁇ -globin, and anti-sickling globin, an ATP -binding cassette, sub-family D, member 1 (ABCDl) polypeptide, an adenosine deaminase polypeptide, an interleukin 2 receptor gamma polypeptide, a tripeptidyl peptidase 1 polypeptide, an alpha-L iduronidase polypeptide, or an iduronate 2-sulfatase polypeptide.
  • ABCDl sub-family D, member 1
  • a population of hematopoietic cells comprises one or more isolated CD34 + CD84 " hematopoietic stem cells modified by introducing one or more engineered nucleases that bind and cleave a target site in the cell's genome.
  • engineered nucleases that can be introduced into the cells to effect gene editing include, but are not limited to, homing endonuclease variants, megaTALs, TALENs, zinc finger nucleases, CRISPR/CAS9 nuclease systems, and ARCUS nucleases.
  • the engineered nuclease is introduced into the cells and disrupts gene function and/or expression by creating a double-strand break in the genome that is repaired by non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • a homology repair template and an engineered nuclease are introduced into the cells and disrupts or restores gene function and/or expression by creating a double-strand break in the genome that is repaired by homology directed repair (HDR) and insertion of the homology repair template into the genome at the site of the double-strand break.
  • HDR homology directed repair
  • a population of hematopoietic cells comprises one or more isolated CD34 + CD84 " hematopoietic stem cells cultured ex vivo.
  • a population of hematopoietic cells comprises one or more isolated CD34 + CD84 " hematopoietic stem cells modified by introducing genetic material into the cell and cultured ex vivo.
  • compositions comprising HSCs that express one or more cell surface HSC markers and that do not express, or lack expression of, CD84 are contemplated in various embodiments.
  • compositions comprise one or more isolated CD34 + CD84 " hematopoietic stem cells.
  • compositions comprise one or more isolated CD34 + CD84 " hematopoietic stem cells that comprise one or more hematopoietic stem cell markers selected from the group consisting of CD90 + , CD45RA " , CD49f + , CD38 Lo/ , CD164 + , CD172a + , CD117 + , and CD133 + .
  • Compositions may comprise one or more isolated CD34 + CD84 "
  • compositions contemplated in particular embodiments may comprise genetically modified and/or cultured HSCs and/or HSCs comprising one or more, viral vectors, polypeptides, or polynucleotides, formulated in
  • compositions include, without limitation, cultures contemplated herein, and in particular embodiments, the terms composition and culture may be used synonymously.
  • compositions contemplated herein may comprise a population of hematopoietic cells that has been enriched for CD34 + CD84 " " hematopoietic stem cells formulated in pharmaceutically-acceptable or physiologically-acceptable solutions (e.g. , culture medium) for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
  • pharmaceutically-acceptable or physiologically-acceptable solutions e.g. , culture medium
  • compositions contemplated herein comprise a population of cells comprising a therapeutically-effective amount of CD34 + CD84 " hematopoietic stem cells, as described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents (e.g., pharmaceutically acceptable cell culture medium).
  • a composition comprises a population of hematopoietic cells and a therapeutically-effective amount of CD34 + CD84 " hematopoietic stem cells.
  • the population of hematopoietic cells comprises at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% CD34 + CD84 " hematopoietic stem cells, including any intervening percentages.
  • the population of hematopoietic cells comprises about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% CD34 + CD84 " hematopoietic stem cells, including any intervening percentages.
  • the population of hematopoietic cells comprises at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% CD34 + CD84 " hematopoietic stem cells, including any intervening percentages.
  • compositions contemplated in particular embodiments comprise a population of hematopoietic cells comprising CD34 + CD84 " hematopoietic stem cells that have been cultured for about 6, about 12, about 24, about 36, about 48, about 60, or about 72 hours or more.
  • a composition comprises a population of hematopoietic cells comprising CD34 + CD84 " hematopoietic stem cells that have one of the following ⁇ -globin alleles: ⁇ ⁇ / ⁇ °, ⁇ ⁇ / ⁇ °, ⁇ °/ ⁇ °, ⁇ ⁇ / ⁇ ⁇ , ⁇ ⁇ / ⁇ + , ⁇ ⁇ / ⁇ + , ⁇ °/ ⁇ + , ⁇ + / ⁇ + , ⁇ ⁇ / ⁇ ⁇ , ⁇ ⁇ / ⁇ ⁇ , ⁇ °/ ⁇ ⁇ , ⁇ ⁇ / ⁇ ⁇ , ⁇ + / ⁇ ⁇ or ⁇ 8 / ⁇ ⁇ ⁇
  • compositions contemplated herein comprise a pharmaceutically acceptable carrier and one or more CD34 + CD84 " hematopoietic stem cells produced, genetically modified, cultured, and/or isolated according to methods described herein.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction 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 therapeutic cells are administered.
  • diluent such as cell culture media, 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.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • a composition comprising a carrier is suitable for parenteral administration, e.g. , intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
  • parenteral administration e.g. , intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the transduced cells, use thereof in the pharmaceutical compositions is contemplated.
  • compositions contemplated herein comprise one or more CD34 + CD84 " hematopoietic stem cells and a pharmaceutically acceptable carrier, e.g., pharmaceutically acceptable cell culture medium.
  • compositions contemplated herein comprise one or more genetically modified
  • a composition comprising a cell-based composition contemplated herein can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals.
  • the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition.
  • the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc. , when combined with other components of the composition.
  • the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g. , pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g.
  • a lubricant e.g. , magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.
  • a disintegrant e.g., starch, sodium starch glycolate, etc.
  • a wetting agent e.g. , sodium lauryl sulfate, etc.
  • compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins,
  • hydroxymethylcelluloses polyvinylpyrrolidones and the like.
  • Such carrier solutions also can contain buffers, diluents and other suitable additives.
  • buffer refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
  • buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
  • PBS Dulbecco's phosphate buffered saline
  • D5W 5% dextrose in water
  • normal/physiologic saline (0.9% NaCl).
  • the pharmaceutically acceptable carriers and/or diluents may be present in amounts sufficient to maintain a pH of the therapeutic composition of about 7.
  • the therapeutic composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In still another embodiment, the therapeutic composition has a pH of about 7.4.
  • compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium.
  • the compositions may be a suspension.
  • the term "suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
  • compositions contemplated herein are formulated in a suspension, where the hematopoietic stem cells are dispersed within an acceptable liquid medium or solution, e.g., saline or serum-free medium, in an intravenous (IV) bag or the like.
  • acceptable liquid medium or solution e.g., saline or serum-free medium
  • IV intravenous
  • Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium.
  • a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of hematopoietic stem cells.
  • the composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
  • compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects.
  • the pharmaceutically acceptable cell culture medium is a serum free medium.
  • Serum-free medium has several advantages over serum containing medium, including a simplified and better defined composition, a reduced degree of
  • the serum-free medium is animal-free, and may optionally be protein-free.
  • the medium may contain biopharmaceutically acceptable recombinant proteins.
  • Animal-free medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal-free medium and the nutrients are obtained from synthetic, plant or microbial sources.
  • Protein-free medium in contrast, is defined as substantially free of protein.
  • serum-free media used in particular compositions includes, but is not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.
  • compositions comprising hematopoietic stem and/or progenitor cells are formulated in PlasmaLyte.
  • compositions comprising hematopoietic stem and/or progenitor cells are formulated in a cryopreservation medium.
  • cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw.
  • cryopreservation media used in particular compositions includes, but is not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
  • compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS 10.
  • the composition is substantially free of mycoplasma, endotoxin, and microbial contamination.
  • substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
  • compositions comprising hematopoietic stem or progenitor cells transduced with a retroviral vector contemplated herein contains about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL, 3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.
  • compositions comprising one or more CD34 + CD84 " hematopoietic stem cells are useful for ex vivo and in vivo cell-based therapies.
  • compositions may comprise a purified or isolated population of CD34 + CD84 " cells in culture, i.e., a cell culture composition.
  • a cell culture composition may comprise a population of cells comprising one or more CD34 + CD84 " and a suitable cell culture medium.
  • Cell culture compositions contemplated in particular embodiments may be formulated for administration to a subject.
  • Hematopoietic stem cells contemplated in particular embodiments are suitable for administration to a subject in need thereof.
  • a subject in need thereof a subject in need thereof.
  • CD34 + CD84 CD90 + CD45RA- HSCs is administered to a subject in need thereof to provide curative, preventative, or ameliorative benefits to a subject diagnosed with or that is suspected of having monogenic disease, disorder, or condition or a disease, disorder, or condition that is amenable to hematopoietic stem cell therapy.
  • CD34 + CD84 CD90 + CD45RA- HSCs are genetically modified to express a therapeutic polypeptide and administered to an individual in need of therapy for a lysosomal storage disorder, an adrenoleukodystrophy or
  • CD34 + CD84 CD90 + CD45RA- HSCs are genetically modified to express a therapeutic polypeptide and administered to an individual to treat, prevent, and/or ameliorate a monogenic disease, disorder, or condition or a disease, disorder, or condition of the hematopoietic system in a subject, e.g., a
  • Illustrative examples of monogenic disorders and their corresponding therapeutic gene(s)/polypeptides that are suitable for use in particular embodiments contemplated herein include, but are not limited to: X-SCID (IL2Ry), ADA-SCID (adenosine deaminase), Batten's disease (tripeptidyl peptidase 1),
  • Mucopolysaccharidosis type 1 - Hurler Syndrome alpha-L iduronidase
  • Mucopolysaccharidosis type 2 - Hunter Syndrome (iduronate 2-sulfatase)
  • Mucopolysaccharidosis type 3 Sanfilippo Syndrome (N-sulfoglucosamine sulfohydrolase), Mucopolysaccharidosis type 4A - Morquio A Syndrome
  • hemoglobinopathy refers to a diverse group of inherited blood disorders that involve the presence of abnormal hemoglobin molecules resulting from alterations in the structure and/or synthesis of hemoglobin.
  • the most common hemoglobinopathies include sickle cell disease, ⁇ -thalassemia, and a- thalassemia.
  • genetically modified CD34 + CD84 CD90 + CD45RA- HSCs are used to treat, prevent, or ameliorate a hemoglobinopathy is selected from the group consisting of: hemoglobin C disease, hemoglobin E disease, sickle cell anemia, sickle cell disease (SCD), thalassemia, ⁇ -thalassemia, thalassemia major, thalassemia intermedia, ⁇ -thalassemia, hemoglobin Bart syndrome and hemoglobin H disease.
  • the genetically modified CD34 + CD84 CD90 + CD45RA- HSCs have a ⁇ -globin genotype selected from the group consisting of: ⁇ ⁇ / ⁇ °, ⁇ °/ ⁇ °, ⁇ °/ ⁇ °, ⁇ ⁇ / ⁇ ⁇ , ⁇ °/ ⁇ + ⁇ ⁇ / ⁇ + , ⁇ °/ ⁇ + , ⁇ + / ⁇ + , ⁇ °/ ⁇ ⁇ , ⁇ ⁇ / ⁇ ⁇ , ⁇ °/ ⁇ ⁇ , ⁇ ⁇ / ⁇ ⁇ , ⁇ + / ⁇ ⁇ or ⁇ 8 / ⁇ ⁇
  • a dose of CD34 + CD84 CD90 + CD45RA- HSCs is delivered to a subject intravenously.
  • a population of CD34 + CD84 " CD90 + CD45RA " HSCs is transduced with a vector and the transduced cells are administered to an individual in need of therapy for an adrenoleukodystrophy or an adrenomyeloneuropathy.
  • a population of CD34 + CD84 CD90 + CD45RA- HSCs is transduced with a vector and the transduced cells are administered to an individual in need of therapy for ADA-SCID, X-SCID, Batten's Disease, MPSI, or MPSII.
  • a method for isolating and/or quantitating human CD34 + CD84 " hematopoietic stem cells from a heterologous cell population may comprise contacting a heterologous cell population, including but not limited to bone marrow, mobilized peripheral blood, umbilical cord blood, and placenta, with one or more binding agents for a time sufficient to isolate human CD34 + CD84 " hematopoietic stem cells from the population.
  • any suitable binding agent may be used to bind and isolate the CD34 + CD84 " hematopoietic stem cells from the population.
  • the binding agents used to bind and isolate the CD34 + CD84 " hematopoietic stem cells from the population comprise antibodies or antigen binding fragments thereof.
  • the binding agents may be directed against CD34 and CD84 and one or more cell surface markers selected from the group consisting of: CD90 + , CD45RA " , CD49f + , CD38 Lo/ , CD164 + , CD172a + , CD117 + and CD133 + . It would be understood that both positive and negative antibody-based selection methods may be employed.
  • a CD34 antibody may be used to positively select for hematopoietic stem cells expressing this marker (CD34 + ) and a CD84 antibody may be used to negatively select undesired cells that express CD84; the resulting population would then comprise CD34 + CD84 " hematopoietic stem cells.
  • the isolated cells may then be counted.
  • the kit comprise one or more binding agents and instructions for contacting a heterologous cell population, including but not limited to bone marrow, mobilized peripheral blood, umbilical cord blood, and placenta, with the one or more binding agents for a time sufficient to isolate human CD34 + CD84 " hematopoietic stem cells from the population.
  • any suitable binding agent may be used to bind and isolate the CD34 + CD84 " hematopoietic stem cells from the population.
  • the binding agents used to bind and isolate the binding agents used to bind and isolate the binding agents
  • CD34 + CD84 " hematopoietic stem cells from the population comprise antibodies or antigen binding fragments thereof.
  • the binding agents may be directed against CD34 and CD84 and one or more cell surface markers selected from the group consisting of: CD90 + , CD45RA " , CD49f + , CD38 Lo/ , CD164 + , CD172a + , CD117 + and CD133 + . It would be understood that both positive and negative antibody-based selection methods may be employed.
  • a CD34 antibody may be used to positively select for hematopoietic stem cells expressing this marker (CD34 + ) and a CD84 antibody may be used to negatively select undesired cells that express CD84; the resulting population would then comprise CD34 + CD84 " hematopoietic stem cells.
  • BM bone marrow
  • mPB peripheral blood
  • MNCs bone marrow mononuclear cells
  • CD34 + cells were then isolated from the BM MNCs using the CD34 MicroBead Kit and MACS cell separation columns (Miltenyi Biotec).
  • mPB was collected by apheresis, washed to remove platelets and incubated with CliniMACS CD34 reagent. Labeled CD34 + cells were then enriched using the
  • CliniMACS Plus instrument (Miltenyi Biotec). Enriched cells were aliquoted and cryopreserved in CryoStor CS10.
  • mPB CD34 + cells were thawed and cultured in SCGM (CellGenix) supplemented with 100 ng/ml Flt3, Scf, and TPO (CellGenix) for 72 hrs at 37°C and 5% C02.
  • CyTOF For CyTOF, cells were stained for BMI-1, CD117, CD123, CD2, CD34, CD38, CD45, CD71, CD90, CEBPa, FoxOl, FoxPl, HLA-DR, PBXl, SATBl, TCRab, TdT, DNA (iridium), and viability (CisPlatin), using lanthani de-conjugated antibodies and Cell-ID reagents (Fluidigm, Inc). Mass cytometry data was acquired using CyTOF 2 instrument (Fluidigm), normalized, and DNA+ viable CD34 + single cells were analyzed using ViSNE algorithm (Cytobank).
  • BMI-1 BMI-1, CD117, CD123, CD2, CD38, CD71, CD90, CEBPa, FoxOl, FoxPl, HLA-DR, PBXl, SATBl, TCRab, and TdT were used for ViSNE analysis.
  • a phenotypic tSNE map of single CD34 + cells from human bone marrow (Figure 1; BM; left panels) and mobilized peripheral blood (Figure 1; mPB; middle panels) show similar but distinct distribution of cell types.
  • CD34 + cells substantially alter their phenotypes after culture ( Figure 1; cultured CD34 + from mPB; right panels; dark arrows).
  • CD38 expression is downregulated in culture and consequently most cultured cells are CD38 negative ( Figure 1 ; cultured CD34 + from mPB; right center panel; white arrow), making it impossible the detection of hematopoietic stem cells post culture.
  • Enriched cells were aliquoted and cryopreserved in CryoStor CS10. 5 million cells were stained with CD7-, CD33, CD34, CD38, and 7-AAD and aliquoted into
  • CD34 + cells isolated from bone marrow and mobilized peripheral blood were thawed, resuspended in SCGM, and labeled with Cell-Trace CFSE
  • LT-HSCs long-term hematopoietic stem cells
  • CD34 + cells were stained for Fluorescein (CFSE), CD34, CD38, CD84, CD90, CD117, CD164, and CD172a, DNA (iridium), and viability (CisPlatin), using lanthani de-conjugated antibodies and Cell-ID reagents (Fluidigm, Inc). Mass cytometry data was acquired using CyTOF 2 instrument (Fluidigm), normalized, and DNA+ viable CD34 + single cells were analyzed using Cytobank. Figure 3.
  • CD84 is a candidate stem cell marker when used in combination with other hematopoietic stem cell markers, serving as a surrogate for CD38 which expression is downregulated in vitro ( Figure 3, arrows).

Abstract

Cette invention concerne des compositions de cellules souches hématopoïétiques améliorées et leurs procédés d'utilisation.
PCT/US2018/049355 2017-09-05 2018-09-04 Composition de cellules souches hématopoïétiques WO2019050841A1 (fr)

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US20160151422A1 (en) * 2006-05-17 2016-06-02 Cognate Bioservices, Inc. Isolation and purification of hematopoietic stem cells from post-liposuction lipoaspirates
US20160324982A1 (en) * 2015-04-06 2016-11-10 President And Fellows Of Harvard College Compositions and methods for non-myeloablative conditioning
WO2016210292A1 (fr) * 2015-06-25 2016-12-29 Children's Medical Center Corporation Procédés et compositions se rapportant à l'expansion, l'enrichissement et la conservation de cellules souches hématopoïétiques
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Publication number Priority date Publication date Assignee Title
US5681559A (en) * 1993-08-25 1997-10-28 Systemix, Inc. Method for producing a highly enriched population of hematopoietic stem cells
US20050158857A1 (en) * 2003-09-26 2005-07-21 The Regents Of The University Of Michigan Hematopoietic stem cell identification and isolation
US20160151422A1 (en) * 2006-05-17 2016-06-02 Cognate Bioservices, Inc. Isolation and purification of hematopoietic stem cells from post-liposuction lipoaspirates
US20160324982A1 (en) * 2015-04-06 2016-11-10 President And Fellows Of Harvard College Compositions and methods for non-myeloablative conditioning
WO2016210292A1 (fr) * 2015-06-25 2016-12-29 Children's Medical Center Corporation Procédés et compositions se rapportant à l'expansion, l'enrichissement et la conservation de cellules souches hématopoïétiques
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