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WO2020185856A1 - Méthodes pour accroître la production de plaquettes - Google Patents

Méthodes pour accroître la production de plaquettes Download PDF

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Publication number
WO2020185856A1
WO2020185856A1 PCT/US2020/022023 US2020022023W WO2020185856A1 WO 2020185856 A1 WO2020185856 A1 WO 2020185856A1 US 2020022023 W US2020022023 W US 2020022023W WO 2020185856 A1 WO2020185856 A1 WO 2020185856A1
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Prior art keywords
vincristine
megakaryocyte
vinblastine
cell
aurora kinase
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Arunoday K. BHAN
George Q. Daley
Thorsten M. Schlaeger
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Boston Childrens Hospital
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Boston Childrens Hospital
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    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • 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
    • A61K35/14—Blood; Artificial blood
    • 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
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

  • the field of the invention relates to methods and compositions for increasing platelet production from megakaryocytes.
  • Platelets are tiny blood cells that play an integral role in blood clotting.
  • platelets which are also called“thrombocytes,” are involved in inflammation, blood vessel growth, and tumor metastasis. The entire platelet population is replaced every 10 days, thus requiring a high amount of ongoing platelet production.
  • Platelet supplementation is necessary in a variety of clinical settings including (i) in the treatment of cancer, (ii) surgery, (iii) serious injuries or trauma, and (iv) in the treatment of blood disorders and transplants.
  • a megakaryocyte e.g., an in vitro cultured megakaryocyte or immortalized megakaryocyte.
  • a method for increasing platelet production from a megakaryocyte comprising: contacting a megakaryocyte (MK) or population thereof with an effective amount of a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or an Aurora kinase inhibitor (e.g., CCT137690).
  • a vinca alkaloid such as vincristine, vinblastine, vinorelbine, or vindesine
  • an Aurora kinase inhibitor e.g., CCT137690
  • the Aurora kinase inhibitor is CCT137690, ZM447439, Hesperadin, VX-680, barasertib, alisertib, danusertib, AT9283, PF- 03914735, or AMG 900, among others.
  • vincristine and/or vinblastine is used in combination with at least one Aurora kinase inhibitor.
  • the method comprises contacting with a combination of agents (e.g., at least two, 3, 4, 5, or 6 different agents).
  • a combination of agents specifically includes the following: CCT137690 & vincristine, CCT137690 & vinblastine, vinblastine & vincristine, vinblastine & vincristine & CCT137690, vincristine and/or vinblastine & barasertib, vincristine and/or vinblastine & PF03814735, vincristine and/or vinblastine & ZM447434, and the like.
  • the effect of the combination of agents is additive.
  • the combination of agents produces a synergistic effect.
  • a combination comprising an Aurora kinase inhibitor, such as CCT137690, with vincristine can produce a synergistic effect on platelet production.
  • the different agents can be contacted with the megakaryocyte or population thereof at the same time, i.e., simultaneously, or the different agents can be contacted with the megakaryocyte or population thereof at different times, i.e., sequentially.
  • two or more different agents are contacted with the megakaryocyte or population thereof at the same time.
  • the agents e.g., vinblastine and/or vincristine, and the Aurora kinase inhibitor is contacted with the megakaryocyte or population thereof at different times.
  • the megakaryocyte or population thereof is contacted with the Aurora kinase inhibitor for a period of time before the megakaryocyte or population thereof is contacted with vinblastine and/or vincristine.
  • the time between contacting with the Aurora kinase inhibitor and vinblastine and/or vincristine is from a few minutes to days.
  • the time between contacting with the Aurora kinase inhibitor and vinblastine and/or vincristine can be 30 minutes, 60 minutes, 2 hours, 5 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days or more. In some embodiments of this aspect and all other aspects provided herein, the time between contacting with the Aurora kinase inhibitor and vinblastine and/or vincristine is about 3 days.
  • the combination of the Aurora kinase inhibitor e.g., CCT137690
  • vincristine synergist ically increases platelet production.
  • the combination of the Aurora kinase inhibitor e.g., CCT137690
  • vinblastine synergist ically increases platelet production.
  • control wells e.g., wells containing imMKCLs that were not exposed to CCT137690 and/or vincristine
  • agent e.g., CCT137690 or vincristine alone
  • combination of agents e.g. CCT137690 and vincristine treated imMKCL wells
  • the method is performed in vitro.
  • platelet production is increased by at least 10% compared to an untreated control.
  • the untreated control comprises a substantially similar megakaryocyte or population thereof that is not contacted with a vinca alkaloid, such as vincristine or vinblastine, or an Aurora kinase inhibitor.
  • the increase in platelet production is an increase in platelet number.
  • the increase in platelet number is an increase in the number of platelets per megakaryocyte (plate lets/MK).
  • the population of megakaryocytes is a homogenous population.
  • the effective amount of vincristine, vinblastine and/or the aurora kinase is between O.OImM - IOOmM, inclusive.
  • the effective amount of vincristine, vinblastine and/or the aurora kinase is between 0.1mM-5mM.
  • the effective amount of vincristine, vinblastine and/or the aurora kinase is between 0.75mM- 1.50mM.
  • the Aurora kinase inhibitor comprises CCT137690.
  • CCT137690 is a potent inhibitor of Aurora kinases with IC50 values of 0.015, 0.019 and 0.025 mM for Aurora A, Aurora C and Aurora B, respectively.
  • CCT137690 possesses antiproliferative activity in various human tumor cell lines.
  • the megakaryocyte or population thereof is grown on a substrate.
  • the megakaryocyte or population thereof is grown, e.g., on a substrate, for a period time before contacting with vincristine, vinblastine, vinorelbine, or vindesine, or the Aurora kinase inhibitor.
  • the megakaryocyte or population thereof can be grown for a few minutes to days before contacting with vincristine, vinblastine, vinorelbine, or vindesine, or the Aurora kinase inhibitor.
  • the megakaryocyte or population thereof can be grown for 30 minutes, 60 minutes, 2 hours, 5 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days or more before contacting with vincristine, vinblastine, vinorelbine, or vindesine, or the Aurora kinase inhibitor.
  • the megakaryocyte or population thereof can be grown for about 3 days before contacting with vincristine, vinblastine, vinorelbine, or vindesine, or the Aurora kinase inhibitor
  • the substrate comprises laminin, fibronectin, Poly-L-Lysine, or methylcellulose.
  • the number of platelets is counted using FACS or a burst/spreading assay.
  • the megakaryocyte or population thereof is contacted with an effective amount of both vincristine and vinblastine, and optionally an effective amount of an Aurora kinase inhibitor (e.g., CCT137690).
  • an Aurora kinase inhibitor e.g., CCT137690.
  • the megakaryocyte cell or population thereof is an immortalized megakaryocyte cell or population thereof.
  • the vincristine, vinblastine and/or Aurora kinase is contacted with the megakaryocyte cell(s) after induction of terminal differentiation.
  • the vincristine, vinblastine, and/or Aurora kinase inhibitor is contacted with the megakaryocyte cell(s) at least one day after induction of terminal differentiation.
  • the vincristine, vinblastine and/or Aurora kinase inhibitor is contacted with the megakaryocyte cell(s) from day 3 after induction of terminal differentiation.
  • the vincristine, vinblastine and/or Aurora kinase inhibitor is contacted with the megakaryocyte cell(s) until functional platelets are generated.
  • the vincristine, vinblastine, and/or Aurora kinase inhibitor is contacted with the megakaryocyte cell(s) until a maximal output of functional platelets are generated.
  • kits for increasing platelet production comprising: vincristine, vinblastine, and/or an Aurora kinase inhibitor, instructions for use thereof, and at least one of the following: (i) laminin substrate, (ii) megakaryocyte cells, or (iii) positive control comprising human platelets or platelet lysates in a known amount.
  • the megakaryocyte cells are immortalized megakaryocyte cells.
  • the megakaryocyte or population thereof is grown on a baffled surface.
  • the megakaryocyte or population thereof is grown on a flat surface.
  • FIG. 1 depicts an exemplary workflow for a high-throughput CV7000 imaging screen to detect platelet generation and pro-platelet phenotype.
  • FIG. 2 depicts Fiji based identification and counting of platelet sized particles and megakaryocyte sized imMKCLs from the captured images. Depiction of normal maturation kinetics of megakaryocytes via comparison of Day6 megakaryocyte membrane content vs DNA content, Day6 megakaryocyte circularity vs DNA content, Day6 megakaryocyte Area vs DNA content.
  • FIG. 3 depicts Fiji based counting of plate lets/imMKCLs to identify“hit” compounds. Also depicts screening performed to identify pro-platelet phenotype in various drug/compound treated wells.
  • FIGs. 4A-4B depicts exemplary data of the “hit” compounds vincristine and vinblastine (FIG. 4A) and identification of“hit” compound-treated wells that produce a significant pro-platelet phenotype from the imMKCLs.
  • Initial analyses identify vincristine and vinblastine as “hit” compounds (FIG. 4B).
  • FIGs. 5A-5B depicts exemplary methods and data relating to the validation of “hit” compounds. These data show that vincristine and vinblastine treatment results in high platelet yields (FIG. 5A). Also depicted is an exemplary scheme used to validate“hit” compounds in clone 38 imMKCLs in static conditions. FACS based data shows that Vincristine and vinblastine treatment promotes platelet yields by ⁇ 3-4 fold. Vincristine and vinblastine also promote extensive pro-platelet phenotype from the imMKCLs as compared to the corresponding controls (FIG. 5B).
  • FIG. 6 shows exemplary data relating to the effect of vincristine and vinblastine on platelet production in static conditions. Platelet count was performed 72h post exposure of clone 38 imMKCLs to vincristine and vinblastine.
  • FIGs. 7A-7B shows exemplary data relating to the effects of vincristine and vinblastine on platelet production in shaking conditions. Platelet count was performed 72h post exposure of clone 38 imMKCLs to vincristine and/or vinblastine. This figure also shows the gating strategy used to identify and segregate platelets from contaminating imMKCLs in the culture, and identify live, non-apoptotic, CD41a+ CD42b+ platelets (FIG. 7A). Also depicted is exemplary platelet output in the form of platelet/imMKCL ratio from variously passaged clone 38 imMKCLs with and without incubation of vincristine or vinblastine. Vincristine effects 2-fold increase in platelet output as compared to the control from both flat and baffled surface flasks (FIG. 7B).
  • FIG. 8 shows exemplary data relating to vincristine and vinblastine validation, which were identified using the high-throughput imaging library screen.
  • FIG. 9 shows exemplary data relating to the temporal treatment of vincristine on platelet production in shaking conditions. Platelet count was performed post-treatment (C-38).
  • FIG. 10 depicts an exemplary scheme used to identify compounds in clone 38 imMKCLs that promote endoreplication/endomitosis.
  • FACS based data shows that CCT137690, Barasertib, PF03814735 promote endoreplication/endomitosis.
  • Figure shows concentration titration as well as various biological replicates depicting that CCT137690 promotes higher endomitosis as compared to other compounds.
  • FIG. 11 shows the gating strategy used to identify and segregate singlet imMKCLs on D3 in the absence of DOX from the culture.
  • CD41a+CD42b+ megakaryocyte fraction was identified and measurement of ploidy in this CD41a+CD42b+ fraction was performed.
  • FIG. 12 shows an exemplary combinatorial and sequential treatment plan of imMKCLs with CCT137690 and vincristine to develop a synergistic or additive effect, as compared to the flat surface imMKCL (untreated) controls, the Baffled surface imMKCL cultures treated with Vincristine and CCT137690 possess ⁇ 14-16-fold higher number of platelets
  • FIG. 13 shows an exemplary scheme of“scale up” using the optimized CCT137690 and vincristine treatment plan and PBS biotech bioreactor.
  • Treatment of 10 L 7 imMKCLs in lOOmL of differentiation media with the optimized CCT137690 and vincristine treatment plan enables generation of 1.2-1.5c10 L 9 platelets.
  • FIG. 14 shows TEM based ultrastructure/organelle comparison of CCT137690 and vincristine treated platelets and fresh donor platelets
  • FIG. 15 shows FACS based in vitro activation analyses and comparison of CCT137690 and vincristine treated imMKCL-platelets and fresh donor platelets.
  • FIG. 16 shows LTA based aggregation analyses and comparison of CCT137690 and vincristine treated imMKCL-platelets and fresh donor platelets.
  • FIG. 17 shows tail vein bleeding time analyses in thrombocytopenic mice (generated via busulphan treatment). This exemplary assay shows the tail vein bleeding times post infusion of CCT137690 and Vincristine treated imMKCL-platelets and fresh donor platelets.
  • FIG. 18 Figure shows thrombus incorporation analyses and simultaneous comparison of CCT137690 and Vincristine treated imMKCL-platelets and fresh donor platelets.
  • compositions and methods described herein are related, in part, to the discovery that vincristine, vinblastine, and/or Aurora kinase inhibitor can induce platelet production. That is, vincristine, vinblastine, an Aurora kinase inhibitor, or any combination thereof can increase the number of platelets produced by a megakaryocyte or population thereof, as compared to the number of platelets produced by a substantially similar megakaryocyte that is not treated with vincristine, vinblastine, and/or an Aurora kinase inhibitor.
  • the term in v/YO-differentiated megakaryocytes refers to megakaryocyte cells that are generated in culture, typically via step-wise differentiation from a precursor cell such as a human embryonic stem cell, an induced pluripotent stem cell, an early mesoderm cell, a lateral plate mesoderm cell or a cardiac progenitor cell.
  • the term“in v/Yro-differentiated megakaryocytes” excludes adult human megakaryocytes isolated from a subject (e.g., primary megakaryocytes).
  • the in v/Yro-differentiated megakaryocytes are synchronized in culture.
  • the term“immortalized megakaryocytes” refers to megakaryocytes that can be expanded or passaged in an in vitro culture.
  • markers are used to describe a characteristic and/or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interest and can vary with specific cells. Markers are characteristics, whether morphological, structural, functional or biochemical (enzymatic) characteristics of the cell of a particular cell type, or molecules expressed by the cell type. In one aspect, such markers are proteins. Such proteins can possess an epitope for antibodies or other binding molecules available in the art. However, a marker can consist of any molecule found in or on a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids.
  • morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio.
  • functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages.
  • Markers can be detected by any method available to one of skill in the art. Markers can also be the absence of a morphological characteristic or absence of proteins, lipids etc. Markers can be a combination of a panel of unique characteristics of the presence and/or absence of polypeptides and other morphological or structural characteristics. In one embodiment, the marker is a cell surface marker.
  • a stem cell as the term is defined herein, can differentiate to lineage-restricted precursor cells (such as a human hematopoietic progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as a tissue specific precursor, such as a megakaryocyte progenitor cell), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • lineage-restricted precursor cells such as a human hematopoietic progenitor cell
  • a tissue specific precursor such as a megakaryocyte progenitor cell
  • end-stage differentiated cell which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • the terms“dedifferentiation” or“reprogramming” or“retrodifferentiation” refer to the process that generates a cell that re-expresses a more stem cell phenotype or a less differentiated phenotype than the cell from which it is derived.
  • a multipotent cell can be dedifferentiated to a pluripotent cell. That is, dedifferentiation shifts a cell backward along the differentiation spectrum of totipotent cells to fully differentiated cells.
  • reversal of the differentiation phenotype of a cell requires artificial manipulation of the cell, for example, by expressing stem cell-specific mRNA and/or proteins. Reprogramming is not typically observed under native conditions in vivo or in vitro.
  • the term“somatic cell” refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from proliferation of such a cell in vitro.
  • a somatic cell refers to any cells forming the body of an organism, as opposed to germline cells. Every cell type in the mammalian body— apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells— is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all substantially made up of somatic cells organism other than an embryo or a fetus or results from proliferation of such a cell in vitro.
  • substantially pure refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to the cells making up a total cell population.
  • the terms "substantially pure” or “homogeneous,” with regard to a population of megakaryocytes or platelets, refers to a population of cells that contain fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are not megakaryocytes or platelets, respectively.
  • selection refers to isolating different cell types into one or more populations and collecting the isolated population as a target cell population which is enriched, for example, in a specific target cell. Selection can be performed using positive selection, whereby a target enriched cell population is retained, or negative selection, whereby non-target cell types are discarded (thereby enriching for desired target cell types in the remaining cell population).
  • positive selection refers to selection of a desired cell type by retaining the cells of interest.
  • positive selection involves the use of an agent to assist in retaining the cells of interest, e.g., use of a positive selection agent such as an antibody which has specific binding affinity for a surface antigen on the desired or target cell.
  • positive selection can occur in the absence of a positive selection agent, e.g., in a“touch-free” or closed system, for example, where positive selection of a target cell type is based on any of cell size, density and/or morphology of the target cell type.
  • negative selection refers to selection of undesired or non-target cells for depletion or discarding, thereby retaining (and thus enriching) the desired target cell type.
  • negative selection involves the use of an agent to assist in selecting undesirable cells for discarding, e.g., use of a negative selection agent such as a monoclonal antibody which has specific binding affinity for a surface antigen on unwanted or non-target cells.
  • negative selection does not involve a negative selection agent.
  • negative selection can occur in the absence of a negative selection agent, e.g., in a“touch-free” or closed system, for example, where negative selection of an undesired (non-target) cell type to be discarded is based on any of cell size, density and/or morphology of the undesired (non-target) cell type.
  • “decrease”,“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount.
  • “reduce,”“reduction” or“decrease” or“inhibit” typically means a decrease by at least 10% as compared to a reference level and can include, for example, a decrease by at least about
  • “reduction” or“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • the terms“increased” ‘increase” or“enhance” or“activate” are all used herein to generally mean an increase of a property, level, or other parameter by a statically significant amount; for the avoidance of any doubt, the terms“increased”,“increase” or“enhance” or“activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase
  • vinca alkaloid refers to a set of compounds having anti-mitotic and anti-microtubule effects and typically used as chemotherapy agents in the treatment of cancer.
  • the original vinca alkaloids were first derived from a plant of the Vinca genus (e.g., Vinca rosea) but newer compounds are synthetic derivatives of the plant compounds.
  • Exemplary vinca alkaloids include, but are not limited to, vinblastine, vincristine, vindesine, vinorelbine, vincaminol,ieridine, vinbumine, vinpocetine, minovincine, methoxyminovincine, minovincinine, vincadafformine, desoxyvincaminol and vincamajine.
  • Aurora kinase inhibitor refers to a compound(s) that inhibit Aurora kinase, thus affecting downstream cellular processes such as cellular mitosis, phosphorylation of histone H3, etc.
  • exemplary Aurora kinase inhibitors include, but are not limited to, CCT137690, ZM447439, Hesperadin, VX-680, barasertib, alisertib, danusertib, AT9283, PF-03914735, or AMG 900, among others.
  • the Aurora kinase inhibitor is an Aurora kinase-A inhibitor, an Aurora kinase -B inhibitor or a pan-Aurora kinase inhibitor.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • Platelets are small (e.g., 2-3 um in diameter), irregularly shaped clear cell fragments that are formed from fragmentation of precursor megakaryocytes.
  • Megakaryocytes are derived from hematopoietic stem cell precursor cells in the bone marrow in response to thrombopoietin (TPO). TPO induces differentiation of progenitor cells in the bone marrow towards a final megakaryocyte phenotype.
  • TPO thrombopoietin
  • megakaryocytes develop through the following lineage: CFU-ME (pluripotential hematopoietic stem cell or hemocytoblast) megakaryoblast promegakaryocyte megakaryocyte.
  • CFU-ME pluripotential hematopoietic stem cell or hemocytoblast
  • megakaryoblast promegakaryocyte megakaryocyte.
  • the cell eventually reaches megakaryoblast stage and loses its ability to divide. However, it is still able to replicate its DNA and continue development
  • a megakaryocyte for use with the methods and compositions described herein can be an immortalized megakaryocyte or population thereof.
  • the immortalized megakaryocyte or population thereof is/are conditionally immortalized, wherein expression of the gene(s) necessary for the immortalized phenotype are controlled using an inducible promoter (e.g., a doxy cy cline controlled promoter).
  • stem cells are undifferentiated cells in early stage of development and are capable of giving rise to more cells of the same type or differentiating into a diverse range of cell lineages.
  • the main different types of stem cells are human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC), and adult hematopoietic stem cells (HSC).
  • the methods and compositions described herein can utilize in vitro derived megakaryocytes to produce platelets in vitro.
  • the following describes various stem cells that can be used to prepare a megakaryocyte of a population thereof for the purpose of megakaryopoiesis.
  • Stem cells are cells that retain the ability to renew themselves through mitotic cell division and can differentiate into more specialized cell types.
  • Three broad types of mammalian stem cells include: embryonic stem (ES) cells that are found in blastocysts, induced pluripotent stem cells (iPSCs) that are reprogrammed from somatic cells, and adult stem cells that are found in adult tissues.
  • ES embryonic stem
  • iPSCs induced pluripotent stem cells
  • Other sources of pluripotent stem cells can include amnion-derived or placental-derived stem cells.
  • stem cells can differentiate into all of the specialized embryonic tissues.
  • stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
  • Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.
  • Megakaryocytes useful in the methods and compositions described herein can be differentiated from both embryonic stem cells and induced pluripotent stem cells, among others.
  • the embryonic stem cells or induced pluripotent stem cells are human cells.
  • the compositions and methods provided herein do not encompass generation or use of human megakaryocytes or platelets made from cells taken from a viable human embryo.
  • Embryonic stem cells and methods for their retrieval are well known in the art and are described, for example, in Trounson A O (Reprod Fertil Dev (2001) 13: 523), Roach M L (Methods Mol Biol (2002) 185: 1), and Smith A G (Annu Rev Cell Dev Biol (2001) 17:435).
  • the term "embryonic stem cell” is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see e.g., US Patent Nos.
  • An embryonic stem comprises characteristics including, without limitation, an embryonic stem cell specific gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
  • Embryonic stem cells are considered to be undifferentiated when they have not committed to a specific differentiation lineage. Such cells display morphological characteristics that distinguish them from differentiated cells of embryo or adult origin. Undifferentiated embryonic stem (ES) cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli.
  • the human megakaryocytes and/or platelets described herein are derived from embryonic stem cells. In some embodiments, the human megakaryocytes and/or platelets described herein are not derived from embryonic stem cells or any other cells of embryonic origin.
  • Cells derived from embryonic sources can include embryonic stem cells or stem cell lines obtained from a stem cell bank or other recognized depository institution.
  • Other means of producing stem cell lines include methods comprising the use of a blastomere cell from an early stage embryo prior to formation of the blastocyst (at around the 8-cell stage). Such techniques correspond to the pre implantation genetic diagnosis technique routinely practiced in assisted reproduction clinics. The single blastomere cell is co-cultured with established ES-cell lines and then separated from them to form fully competent ES cell lines.
  • Adult Stem Cells are stem cells derived from tissues (e.g., bone marrow) of a post-natal or post-neonatal organism or from an adult organism.
  • An adult stem cell e.g., hematopoietic stem cell
  • An adult stem cell is structurally distinct from an embryonic stem cell not only in markers it does or does not express relative to an embryonic stem cell, but also by the presence of epigenetic differences, e.g. differences in DNA methylation patterns.
  • the human megakaryocytes and/or platelets described herein are derived from an adult stem cell; in one embodiment, they are derived in vitro from a hematopoietic stem cell (i.e., megakaryocytes do not need to be isolated directly from the bone marrow of an adult).
  • iPSCs Induced Pluripotent Stem Cells
  • the methods and compositions described herein utilize megakaryocytes that are differentiated in vitro from induced pluripotent stem cells for platelet production.
  • An advantage of using iPSCs to generate a megakaryocyte or population thereof for the methods described herein is that the cells can be derived from the same subject to which the resulting platelets are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then re- differentiated into a human megakaryocyte for the production of autologous platelets.
  • the megakaryocytes and/or platelets are derived from non-autologous sources.
  • the use of iPSCs negates the need for cells obtained from an embryonic source.
  • the stem cells used to generate megakaryocytes and/or platelets for use in the compositions and methods described herein are not embryonic stem cells.
  • Reprogramming is a process of driving the differentiation of a cell backwards to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. However, simply culturing such cells included in the term differentiated cells does not render these cells non-differentiated cells (e.g., undifferentiated cells) or pluripotent cells. The transition of a differentiated cell to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character when differentiated cells are placed in culture. Reprogrammed cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.
  • the cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming.
  • reprogramming encompasses complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state.
  • reprogramming encompasses complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming.
  • reprogramming of a differentiated cell causes the differentiated cell to assume an undifferentiated state with the capacity for self-renewal and differentiation to cells of all three germ cell lineages.
  • the resulting cells are referred to as“reprogrammed cells,” or“induced pluripotent stem cells (iPSCs or iPS cells).”
  • iPS cells can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming.
  • pluripotent stem cells from somatic cells e.g., any cell of the body with the exclusion of a germ line cell; fibroblasts etc.
  • any method that re-programs a somatic cell to the pluripotent phenotype would be appropriate for use in the methods described herein.
  • the efficiency of reprogramming i.e., the number of reprogrammed cells derived from a population of starting cells can be enhanced by the addition of various small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3: 132-135.
  • agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX- 01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.
  • isolated clones can be tested for the expression of a stem cell marker.
  • a stem cell marker can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl.
  • a cell that expresses Oct4 or Nanog is identified as pluripotent.
  • Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, detection does not involve only RT-PCR, but also includes detection of protein markers. Intracellular markers may be best identified via RT-PCR, while cell surface markers are readily identified, e.g., by immunocytochemistry.
  • the pluripotent stem cell character of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate to cells of each of the three germ layers.
  • teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones.
  • the cells are introduced to nude mice and histology and/or immunohistochemistry is performed on a tumor arising from the cells.
  • the growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
  • Reprogrammed somatic cells as disclosed herein can express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA- 1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; b-111-tubulin; a-smooth muscle actin (a-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Daxl; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Natl); (ES cell associated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; EC ATI 5- 2; Fthl 17; Sall4; undifferentiated embryonic cell transcription factor (Utfl); Rex
  • markers can include Dnmt3L; Soxl5; Stat3; Grb2; b-catenin, and Bmil.
  • Such cells can also be characterized by the down-regulation of markers characteristic of the somatic cell from which the induced pluripotent stem cell is derived.
  • v/Yro-differentiation of megakaryocytes produces an end-result of a cell having the phenotypic and morphological features of a megakaryocyte but that the differentiation steps of in v/Yro-differentiation need not be the same as the differentiation that occurs naturally in the embryo. That is, during differentiation to a megakaryocyte, it is specifically contemplated herein that the step-wise differentiation approach utilized to produce such cells need not proceed through every progenitor cell type that has been identified during embryogenesis and can essentially“skip” over certain stages of development that occur during embryogenesis.
  • megakaryocytes undergo a process called endomitosis, where they double their nuclear and cellular contents without cell division.
  • endomitosis the megakaryocyte grows to enormous size and can have more than 64 times the normal nuclear contents.
  • the increase of nuclear contents, or polyploidy plays a fundamental role in the platelet formation by allowing the cell to produce the large amounts of proteins and organelles necessary for platelet formation and function.
  • mature megakaryocytes also have vast quantities of extra cell membrane with which to make platelets. Inducing polyploidization or maturation of megakaryocytes in vitro can be achieved using the following reagents alone or in different combinations.
  • Overexpression of Ectopic genes It is desirable to generate self-renewing megakaryocytes progenitor cells that can be quickly matured in order to initiate platelet production.
  • self-renewing megakaryocyte progenitor cells are immortalized cells.
  • the control of the maturation of megakaryocytes from self-renewing cells is achieved by overexpression of one or more megakaryocyte maturation markers, the expression of which is under the control of an inducible promoter.
  • a doxycycline (dox) -inducible promoter can be used to regulate expression of a megakaryocyte maturation marker such that in the presence of doxy cy cline the maturation marker is expressed (or vice versa).
  • One system for maturing megakaryocytes in vitro uses a lentivirus to express the transcription factors MYC and BMI1, and one or more pro-survival proteins (e.g., Bcl-xL or BCL2L1) under the control of a dox-inducible promoter.
  • a dox-inducible promoter e.g., Bcl-xL or BCL2L1
  • a second system employs controlled expression of GATA1 by a dox-inducible promoter.
  • the megakaryocyte progenitor cells Under conditions where GATA1 expression is repressed, the megakaryocyte progenitor cells self-renew and when GATA1 expression is induced the cells mature into megakaryocytes that release platelets.
  • Other genes that can be targeted to modulate maturation of megakaryocytes include, but are not limited to, gpl30/interleukin-6-dependent genes, Notch, stromal cell-derived factor 1 (SDFl)/fibroblast growth factor 4-dependent signaling pathways and interleukin 1 -alpha signaling pathways. It is specifically contemplated herein that vincristine and/or vinblastine can be used in combination with any of these systems, or alternatively in isolation, to increase the efficiency of platelet production.
  • Rho Rock inhibitors The final steps of megakaryocyte cell division require regulation of actin and myosin to form the cleavage furrow and contractile ring. The inhibition of actin and myosin during cytokinesis allows megakaryocytes to replicate DNA material without undergoing cell division.
  • the Rho/Rock pathway signals through myosin light chain (MLC) and filamin and activates both stress fibers and lamellipodia formation.
  • Y27632 inhibits the Rho/Rock pathway and consequently inhibits myosin activation and the contractile ring formation, presumably allowing the megakaryocyte to undergo polyploidization.
  • Rho/Rock inhibitors include, but are not limited to, Y27632, thiazovivin, GSK429286A, fasudil HC1, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, and H-1152P.
  • NIC Nicotinamide Decreases in p53 activity are responsible for accelerated DNA synthesis, higher ploidy and delayed apoptosis. NIC increases p53 activity and thus increases endomitosis and megakaryocyte polyploidization.
  • Src inhibitors The inhibition of Src family kinases increases megakaryocyte polyploidization through the Lyn Fyn pathway and inhibition of actin polymerization.
  • Exemplary Src inhibitors include, but are not limited to, saracatinib (AZD0530), bosutinib (SKI-606), danusertib (PHA- 739358), NVP-BHG712, quercetin (sophoretin), PCI-32765, KX2-391, AP23846, and PP2.
  • Aurora-B inhibitors are responsible for controlling microtubule formation and subsequent chromosome separation during mitosis. Its inhibition increases microtubule destruction through stathmin and mitotic centromere-associated kinesin (MCAK) action.
  • MCAK mitotic centromere-associated kinesin
  • Aurora-B kinase inhibitors include, but are not limited to, AMG 900, AT9283, Aurora A Inhibitor I, AZD1152, AZD1152-HQPA (barasertib), CCT129202, CYC 116, danusertib (PHA-739358), ENMD-2076, GSK1070916, hesperadin, JNJ-7706621, KW-2449, MLN8054, MLN8237 (alisertib), PF-03814735, PHA-680632, SNS-314, TAK-901, VX-680 (MK-0457, tozasertib), and ZM-447439.
  • MLCK Myosin light chain kinase
  • MLC Myosin light chain kinase
  • MLCK inhibitors include, but are not limited to, A3 HC1, Go 7874 HC1, InSolutionTM K-252a (Nocardiopsis sp.), K-252a (Nocardiopsis sp.), K-252b (Nocardiopsis sp.), ML-7 HC1, ML-9 HC1, MLCK inhibitor peptide 18, piceatannol, and staurosporine ( Streptomyces sp).
  • PMA Protein kinase C
  • Blebbistatin inhibits myosin II and consequently the last steps of cytokinesis and cell division, thus allowing the cell to undergo polyploidization and increase the nuclear material.
  • Other stimulators of megakaryocyte maturation include the transcription factor‘all trans retinoic acid,’ and the histone deacetylase inhibitor valproic acid.
  • Racl activation, CDC42 activation, hirudin and c-Myc inhibition also increase proplatelet formation.
  • some physical stimuli can induce megakaryocyte maturation and polyploidization, such as an increased oxygen concentration during culture, culturing at a temperature higher than conventional culturing temperature (e.g., 39°C).
  • the successful maturation of megakaryocytes from pluripotent stem cells can be determined by morphology (e.g., presence of DNA polyploidy (e.g., up to 128N), increase in cell size (up to a diameter of 60 um), increased granularity, and the presence of intracellular organelles reminiscent of pro-platelets), or by the presence of cell surface markers (e.g., CD41, CXCR4, or the TPO receptor, among others).
  • morphology e.g., presence of DNA polyploidy (e.g., up to 128N), increase in cell size (up to a diameter of 60 um), increased granularity, and the presence of intracellular organelles reminiscent of pro-platelets), or by the presence of cell surface markers (e.g., CD41, CXCR4, or the TPO receptor, among others).
  • Cell surface markers are useful with the methods and compositions described herein to identify the differentiation or dedifferentiation state of a cell. Both cell surface markers and intracellular markers can be detected, for example, using an antibody for binding, e.g., cell surface markers or by PCR for intracellular markers. In some embodiments, antibodies or similar agents specific for a given marker, or set of markers, can be used to separate and isolate the desired cells using fluorescent activated cell sorting (FACS), panning methods, magnetic particle selection, particle sorter selection and other methods known to persons skilled in the art, including density separation (Xu et al. (2002) Circ. Res. 91:501; U.S.S.N. 20030022367) and separation based on other physical properties (Doevendans et al. (2000) J. Mol. Cell. Cardiol. 32:839-851).
  • FACS fluorescent activated cell sorting
  • negative selection can be performed, including selecting and removing cells with undesired markers or characteristics, for example fibroblast markers, epithelial cell markers etc.
  • Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells.
  • Exemplary ES cell markers include stage -specific embryonic antigen (SSEA)- 3, SSEA-4, TRA-I-60, TRA-1-81, alkaline phosphatase or those described in e.g., U.S.S.N. 2003/0224411; or Bhattacharya (2004) Blood 103(8):2956-64, each herein incorporated by reference in their entirety.
  • Exemplary markers expressed on cardiac progenitor cells include, but are not limited to, TMEM88, GATA4, ISL1, MYL4, and NKX2-5.
  • the methods described herein relate to increasing the amount of platelets produced in vitro from megakaryocytes or the efficiency of platelet production from cultured megakaryocytes by contacting a megakaryocyte cell or population thereof with an effective amount of vincristine, vinblastine, and/or an Aurora kinase inhibitor.
  • An “effective amount” refers to an amount of vincristine, vinblastine, and/or an Aurora kinase inhibitor that increases the number of platelets produced from a given megakaryocyte (e.g., platelet/megakaryocyte) by at least 10% as compared to a megakaryocyte grown under substantially identical conditions but in the absence of vincristine, vinblastine, and/or an Aurora kinase inhibitor.
  • an effective amount of vincristine, vinblastine, and/or an Aurora kinase inhibitor can increase the number of platelets produced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10- fold, at least 25 -fold, at least 50-fold, at least 100-fold or more as compared to a megakaryocyte not treated with vincristine, vinblastine, and/or an Aurora kinase inhibitor.
  • the effective amount of vincristine, vinblastine, and/or the Aurora kinase inhibitor is within the range of 4nM - 100 mM, inclusive.
  • the range of vincristine, vinblastine, and/or the Aurora kinase inhibitor for use with the methods and compositions described herein is: 0.04mM - 50 mM, for example, 0.04-40 mM, 0.04-30 mM, 0.04-20 mM, 0.04- 10 mM, 0.04-5 mM, 0.04-4 mM, 0.004-3 mM, 0.04-2 mM, 0.04-1 mM, 0.04-0.01 mM, 0.04-0-0.75 mM, 0.1-40 mM, 0.5-40 mM, 1-40 mM, 5-40 mM, 10-40mM, 20-40 mM, 30-40 mM, 0.1-5 mM, 0.1-4 mM, 0.1-3 mM, 0.1-2 m
  • the vincristine, vinblastine, and/or an Aurora kinase inhibitor is contacted with the megakaryocyte cells at day 0 of a platelet biogenesis protocol, such as the protocol provided herein in the working Examples.
  • the vincristine, vinblastine, and/or Aurora kinase inhibitor is contacted with the megakaryocyte cells at day 1, 2, 3, 4, 5, or 6 of a platelet biogenesis protocol.
  • the vincristine, vinblastine, and/or Aurora kinase inhibitor is contacted with the megakaryocyte cells from day 3 to the end of the platelet biogenesis protocol (e.g., from days 3-6, or days 3-5, or days 3-7).
  • the vincristine, vinblastine, and/or Aurora kinase inhibitor is contacted with the megakaryocyte cells for at least 6h, at least 12h, at least 24h, at least 2 days, at least 3 days, at least 4 days, at least 5 days or more (e.g., until the end of the platelet biogenesis protocol).
  • the Aurora kinase inhibitor is an Aurora B kinase inhibitor.
  • Aurora-B kinase inhibitors include, but are not limited to, AMG 900, AT9283, Aurora A Inhibitor I, AZD1152, AZD1152-HQPA (barasertib), CCT129202, CYC116, danusertib (PHA-739358), ENMD- 2076, GSK1070916, hesperadin, JNJ-7706621, KW-2449, MLN8054, MLN8237 (alisertib), PF- 03814735, PHA-680632, SNS-314, TAK-901, VX-680 (MK-0457, tozasertib), and ZM-447439.
  • formulations of vincristine, vinblastine, and/or Aurora kinase inhibitors are preferably non-toxic to cultured cells and do not substantially alter cell viability, cell growth rate etc.
  • Any cell culture medium and/or supplements can be used to optimize the growth and maintenance of self-renewing megakaryocyte progenitor cells or immortalized megakaryocytes for the purpose of generating platelets in vitro. Such methods of optimization are known to those of skill in the art or can be determined using techniques known in the art. It is also contemplated herein that the cells can be cultured on a desired substrate, such as laminin, fibronectin, poly-lysine or methy cellulose.
  • the number of platelets produced under a given set of conditions can be determined using FACS or a megakaryocyte bursting assay as described herein in the Examples section.
  • FACS counting can be performed on the basis of size; that is FACS can be used to sort platelets from megakaryocytes based on size.
  • suitable cell surface markers useful for counting platelets include, but are not limited to, CD41, CD61 and/or CD62.
  • a composition i.e., a synergistic composition, comprising vincristine and/or vinblastine in combination with an Aurora kinase inhibitor.
  • a composition comprising an effective amount of vincristine and/or vinblastine and an effective amount of an Aurora kinase inhibitor.
  • the composition comprises vincristine and an Aurora kinase inhibitor.
  • vincristine and an Aurora kinase inhibitor selected from the group consisting of CCT137690, ZM447439, Hesperadin, VX-680, barasertib, alisertib, danusertib, AT9283, PF- 03914735, or AMG 900.
  • the composition comprises vincristine and the Aurora kinase inhibitor CCT137690.
  • the composition comprises vinblastine and an Aurora kinase inhibitor.
  • vinblastine and an Aurora kinase inhibitor selected from the group consisting of CCT137690, ZM447439, Hesperadin, VX-680, barasertib, alisertib, danusertib, AT9283, PF- 03914735, or AMG 900.
  • the composition comprises vinblastine and the Aurora kinase inhibitor CCT137690.
  • the composition further comprises megakaryocyte cell(s). It is noted that the megakaryocytes can be cultured megakaryocytes, e.g., immortalized and/or matured megakaryocytes. In some other embodiments, the composition further comprises plate let(s). In still other embodiments, the composition further comprises both megakaryocyte cell(s) and platelet(s).
  • the methods and compositions for increasing the number and/or efficiency of platelet production using vincristine and/or vinblastine can be performed in a large-scale manner, for example, in a bioreactor.
  • the specific methods for increasing the scale of platelet production are not critical to the invention and as such any method known in the art can be used in combination with the methods described herein.
  • kits for enhancing platelet production from cultured megakaryocytes are described herein. Described herein are kit components that can be included in one or more of the kits described herein.
  • the kit comprises an effective amount of vincristine, vinblastine, and/or Aurora kinase inhibitor formulated for use with cultured megakaryocytes (e.g., immortalized and/or matured megakaryocytes).
  • cultured megakaryocytes e.g., immortalized and/or matured megakaryocytes.
  • vincristine, vinblastine, and/or Aurora kinase inhibitor can be supplied in a lyophilized form or a concentrated form that can diluted prior to use with cultured cells.
  • Preferred formulations include those that are non-toxic to the cells and/or does not affect growth rate or viability etc.
  • vincristine, vinblastine, and/or Aurora kinase inhibitor can be supplied in aliquots or in unit doses.
  • the kit further comprises a vector comprising a nucleic acid encoding a gene to mature megakaryocytes under the control of an inducible promoter for maturing megakaryocytes in vitro.
  • kits includes the components described herein, e.g., a composition comprising vincristine and/or vinblastine, a composition(s) that includes a vector comprising e.g., a megakaryocyte maturation marker (e.g., GATA-1, CD42b etc.) under the control of an inducible promoter (e.g., doxycycline promoter), a doxycycline composition, and one or more maturation compounds as described throughout the specification.
  • kits can optionally include one or more agents that permit the detection of residual stem cells or a set thereof.
  • the kit optionally comprises informational material.
  • kits provided herein comprise an aliquot of immortalized megakaryocytes and optionally a preferred medium for growth thereof.
  • the kit can also contain a substrate for coating culture dishes, such as laminin, fibronectin, Poly-L-Lysine, or methylcellulose.
  • the compositions in the kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit.
  • a vincristine, vinblastine, and/or Aurora kinase inhibitor composition can be supplied in more than one container, e.g., it can be supplied in a container having sufficient reagent for a predetermined number of differentiation reactions, e.g., 1, 2, 3 or greater.
  • One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the components described herein are substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the megakaryocytes and/or platelets, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to methods for using or administering the components of the kit.
  • the kit can include a component for the detection of a marker for human cells, ES cells, iPS cells, hematopoietic progenitor cells, immature megakaryocytes, mature megakaryocytes, platelets etc.
  • the kit can include one or more antibodies that bind a cell marker, or primers for an RT-PCR or PCR reaction, e.g., a semi -quantitative or quantitative RT-PCR or PCR reaction.
  • Such components can be used to assess the activation of megakaryocyte-specific markers or the loss of ES cell, iPSC, or adult stem cell markers.
  • the detection reagent is an antibody, it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the antibody or other detection reagent can be linked to a label, e.g., a radiological, fluorescent (e.g., GFP) or colorimetric label for use in detection.
  • a label e.g., a radiological, fluorescent (e.g., GFP) or colorimetric label for use in detection.
  • the detection reagent is a primer, it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box.
  • the enclosure can be configured so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.
  • the present invention may be as defined in any one of the following numbered paragraphs.
  • a method for increasing platelet production from a megakaryocyte comprising: contacting a megakaryocyte (MK) or population thereof with an effective amount of vincristine, vinblastine, an Aurora kinase inhibitor, or a combination thereof.
  • MK megakaryocyte
  • the substrate comprises laminin, fibronectin, Poly-L-Lysine, or methylcellulose.
  • Aurora kinase inhibitor is contacted with the megakaryocyte cell(s) until functional platelets are generated.
  • a kit for increasing platelet production comprising: vincristine, vinblastine, and/or Aurora kinase inhibitor, instructions for use thereof, and at least one of the following: (i) Laminin substrate, (ii) Megakaryocyte cells, or (iii) a positive control comprising human platelet lysates in a known amount.
  • EXAMPLE 1 Vincristine and vinblastine stimulate megakaryocyte maturation and platelet production
  • hiPSCs human pluripotent stem cells
  • hiPSCs human pluripotent stem cells
  • PLT platelet
  • This assay was used to screen approximately 4,000 chemical genetics compounds, each at four different concentrations, and image processing and analysis scripts were developed to quantify the maturation of imMKCLs and the production of pro- PLTs and PLTs. About 300,000 images from over 20,000 wells were acquired, processed and analyzed using FIJI scripts, and the resulting data evaluated using MATLAB scripts.
  • This primary screen identified ⁇ 30 compounds (Hit compounds) that substantially promoted a pro-PLT phenotype or the output of PLT-like particles.
  • a secondary validation screen was performed on the hit compounds at 11 different doses ranging from 4hM-100mM. The 15 compounds that elicited a dose dependent increase of PLT release were selected for further evaluation.
  • EXAMPLE 2 Novel High-throughput live-cell imaging assays to identify and validate agents that promote megakaryocyte maturation and trigger platelet release
  • EXAMPLE 3 Exemplary protocol for generation of synergistic effects of CCT137690 and Vincristine
  • the cells are diluted to a concentration of 100,000cells/mL in CCT137690 (Aurora Kinase inhibitor) (working concentration of 90nM) SeV2 differentiation medium. Transfer the differentiation culture to orbital shaker Erlenmeyer flask or PBS biotech Bioreactor vessel.

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Abstract

L'invention concerne des méthodes et des compositions pour améliorer la production de plaquettes à partir d'une cellule mégacaryocytaire ou d'une population de celle-ci. La présente invention concerne, en partie, des méthodes et des compositions comprenant l'utilisation de vincristine et/ou de vinblastine pour améliorer la production de plaquettes à partir d'un mégacaryocyte (par exemple, un mégacaryocyte cultivé in vitro ou un mégacaryocyte immortalisé). Dans un mode de réalisation de cet aspect et de tous les autres aspects décrits ici, la vincristine et/ou la vinblastine sont utilisées en combinaison avec au moins un inhibiteur de la kinase Aurora.
PCT/US2020/022023 2019-03-11 2020-03-11 Méthodes pour accroître la production de plaquettes Ceased WO2020185856A1 (fr)

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