RELATED APPLICATIONS
This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 62/692,277, filed on Jun. 29, 2018, and International Patent Application No. PCT/US2019/040021, filed on Jun. 29, 2019, which applications are incorporated herein in their entireties by reference.
FIELD
This disclosure relates to compositions of, methods for producing and method of using modified megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets and platelets for drug delivery.
BACKGROUND
Platelets are blood cells responsible for clot formation and blood vessel repair at sites of active bleeding. Physiologically, platelets are produced in the bone marrow by parent cells called megakaryocytes (MKs), which comprise <0.1% of cells in the bone marrow. Mature MK sit outside sinusoidal blood vessels in the bone marrow and extend long structures called proplatelets into the circulation. Proplatelets function as the assembly lines for platelet production, and sequentially release platelets from their ends.
Platelets are currently derived entirely from human volunteer donors, and shortages are common. Wide functional variability between donor platelets limit transfusion effectiveness. Mounting platelet demand exceeds current supply by ˜20%, and limited platelet unit inventory is rapidly depleted in emergencies.
Efficient delivery of therapeutic drugs to target sites is desirable to maximize therapeutic efficacy and minimize side effects. Although various nanoparticle-based approaches have been advanced to improve the tissue-targeted delivery of small molecules due to their enhanced permeability and retention (EPR) effects, nanoparticle approaches have not been successful in packaging protein-based therapeutics such as coagulation factors or Ab drugs due to their larger size. Further, less than −1% of injected nanoparticles accumulate in most targeted sites, and adverse immune responses against some components (e.g. PEG) of nano-formulations can compromise the efficacy upon repeated injections (Wilhelm Sea. Analysis of nanoparticle delivery to tumors. Nat Rev Mater. 2016; 1:16014). New methods that leverage existing physiological processes to further enhance the tissue-targeted drug delivery, particularly for protein-based therapeutics, are urgently needed.
Methods are needed for on-demand platelet production of well-defined platelet units to meet current and projected platelet need, as well as for production of new vehicles for drug delivery.
SUMMARY
In some aspects, the present disclosure is directed to compositions and methods of use of iPSCs-derived preMKs, MKs, proplatelets, preplatelets and platelets for drug delivery.
In some aspects, there is provided a method for producing induced pluripotent stem cell (iPSC)-derived cells comprising a therapeutic agent, the method comprising: differentiating the pluripotent cells in a first culture medium into hemogenic endothelial cells; differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors; differentiating the megakaryocytic progenitors into mature megakaryocytes; differentiating the mature megakaryocytes under conditions sufficient to produce a platelet, wherein one the platelet, megakaryocyte, or megakaryocytic progenitor or combinations thereof comprises a therapeutic agent.
In some embodiments, the megakaryocytes are CD42b+, CD61+, and DNA+. In some embodiments, the platelets may be one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P+ in an activated state. The platelets may not express GPVI.
The platelet, megakaryocyte, or megakaryocytic progenitors may be loaded with the therapeutic agent, for example, by receptor-mediated loading, passive loading, or covalent conjugation. The passive loading may include incubating the therapeutic agent with a cellular suspension comprising the platelet, megakaryocyte, or megakaryocytic progenitor. The covalent conjugation may comprises thiolation of membrane proteins and sulfhydryl-reactive crosslinkers, alkyne reactive azides, high affinity binders, and antibody docking to membrane bound epitopes. The covalent conjugation may comprise reacting amines present in the therapeutic agent with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
The therapeutic agent may be a chemokine, a cytokine, a growth factor, a polypeptide, anti-angiogenic agent, a polynucleotide, or a small molecule. The anti-angiogenic agent may be doxorubicin, vincristine, irinotecan, or pacl*taxel. The may be atezolizumab, ipilimumab, bevacizumab, cetuximab, or trastuzumab. The small molecule may be aripiprazole, esomeprazole, or rosuvastatin. The growth factor may be a platelet derived growth factor isoform (PDGF-AA, -AB and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), bone morphogenetic protein 2, -4 or -6 (BMP-2, -4, -6), von Willebrand Factor, keratinocyte growth factor, FVII, FVIII, FIX, epidermal growth factor, or hair growth factor. The cytokine may be Interleukin 1-beta, Interleukin 2, or Interleukin 12.
In some aspects, there is provided a IPSC-derived cell, such as preMKs, MKs, proplatelets, preplatelets or platelets, comprising a therapeutic agent. In some embodiments, such cells may be loaded or conjugated with the therapeutic agent. In some embodiments, such cells may be genetically engineered to express the therapeutic agent. There is also provided a composition comprising a a IPSCc-derived cell, such as preMKs, MKs, proplatelets, preplatelets or platelets, comprising a therapeutic agent. In some embodiments, there is a method of treating a subject comprising administering a therapeutically effective amount of a composition comprising a a IPSCc-derived cell, such as preMKs, MKs, proplatelets, preplatelets or platelets, comprising a therapeutic agent.
One aspect of the present disclosure provides a composition that includes a population of platelets derived from induced pluripotent stem cells (iPSCs), wherein the platelets exhibit increased thrombin generation relative to a population of donor derived platelets having a similar cell density.
In some embodiments, the thrombin generation in the platelets derived from iPSCs is greater than two- to three-fold the thrombin generation in the population of donor derived platelets. In some embodiments, the thrombin generation results in a maximum concentration of thrombin of between about 250 nM and about 850 nM. In some embodiments, the thrombin generation results in between about a 450 and about 600 nM maximum concentration of thrombin. In some embodiments, a lag time between a stimulus and achieving a maximum concentration of thrombin in the population of platelets derived from iPSCs is reduced compared to the lag time for a population of donor derived platelets. the lag time of achieving a maximum thrombin concentration in the population of platelets derived from induced pluripotent stem cells is between about 7 and 15 minutes, and wherein the population comprises between about 0.5×106 platelets/mL and 3×106 platelets/mL, respectively.
In some embodiments, the population of platelets derived from iPSCs comprises platelets that do not express glycoprotein VI. In some embodiments, the population of platelets derived from iPSCs comprises platelets having a biomarker profile selected from the group consisting of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P− biomarker profile, a CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P+ biomarker profile, and a CD61+, CD62P+ biomarker profile. In some embodiments, greater than 70% of the platelets derived from iPSC express CD61+, less than 10% of the platelets derived from iPSC express CD42b, less than 5% of the platelets derived from iPSCs express CD36, less than 35% of the platelets derived from iPSCs express calcein, or a combination thereof. In some embodiments, the population of platelets derived from iPSC has altered cell signaling compared to a donor derived platelet or megakaryocyte. In some embodiments, the altered cell signaling comprises reduced CD62 activation after exposure to TRAP-6 or thrombin or wherein less than 15% of the platelets derived from iPSC comprise CD62p. In some embodiments, the composition further comprises thrombogenic microparticles such that the composition has a peak size of less than approximately 2 μm. In some embodiments, the thrombogenic microparticles range in size between 40 nm and 100 nm in diameter. In some embodiments, the thrombogenic microparticles form greater than 50% of the composition.
The present disclosure will be described in the detailed description which follows, in reference to the noted plurality of drawings by way of non-limiting examples of exemplary embodiments, in which like reference numerals represent similar parts throughout the several views of the drawings, and wherein:
While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.
DETAILED DESCRIPTION Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
The terms “agent,” “therapeutic agent,” “therapeutic composition,” “drug,” or “therapeutic” can be used interchangeably and are meant to include any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
By “alteration” or “change” is meant an increase or decrease. An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.
By “biologic sample” is meant any tissue, cell, fluid, or other material derived from an organism.
By “capture reagent” is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
By “cellular composition” is meant any composition comprising one or more isolated cells.
By “cell survival” is meant cell viability.
As used herein, “clinical grade” is meant to refer to a cell or cell line derived or obtained using current Good Manufacturing Practice (GMP), which permits its clinical use in humans. GMP is a quality assurance system used in the pharmaceutical industry to ensure that the end product meets preset specifications. GMP covers both manufacturing and testing of the final product. It requires traceability of raw materials and also that production follows validated standard operating procedures (SOPs).
By “detectable levels” is meant that the amount of an analyte is sufficient for detection using methods routinely used to carry out such an analysis.
“Detect” refers to identifying the presence, absence or amount of the object to be detected.
The term “covalent conjugation” refers to using a chemical linker that reacts with specific chemical groups on the molecule to be conjugated. In some embodiments, covalent conjugation of a therapeutic composition to another molecule or compound is achieved by reacting amines on the therapeutic composition with the linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include any disease or injury that results in a reduction in cell number or biological function, including ischemic injury, such as stroke, myocardial infarction, or any other ischemic event that causes tissue damage, peripheral vascular disease, wounds, burns, fractures, blunt trauma, arthritis, and inflammatory diseases.
By “effective amount” is meant the amount of an agent required to produce an intended effect.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and hom*ogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the present disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the present disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the present disclosure. An isolated polypeptide of the present disclosure may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
The term “hemogenic endothelial cell” as used herein refers to cells capable of differentiating to give rise to hematopoietic cell types or endothelial cell types, and which may optionally be derived from pluripotent stem cells. Hemogenic endothelial cells are normally adherent to extracellular matrix protein and/or to other hemogenic endothelial cells, and can be characterized by the expression of the markers CD31 and CD34.
By “marker” is meant any protein or other epitope having an alteration in expression level or activity that is associated with a characteristic or condition.
The term “megakaryocyte” (MK) as used herein refers to a large (e.g., diameter >10 m), polyploid hematopoietic cell with the propensity to generate proplatelets and/or platelets. One morphological characteristic of mature megakaryocytes is the development of a large, multi-lobed nucleus. Mature megakaryocytes can stop proliferating, but continue to increase their DNA content through endomitosis, with a parallel increase in cell size.
The term “megakaryocytic progenitor” (preMK), as used herein, refers to a mononuclear hematopoietic cell that is committed to the megakaryocyte lineage and is a precursor to mature megakaryocytes. Megakaryocytic progenitors are normally found in (but not limited to) bone marrow and other hematopoietic locations, but can also be generated from pluripotent stem cells, such as by further differentiation of hemogenic endothelial cells that were themselves derived from pluripotent stem cells.
The term “ricroparticle” refers to a very small (<1 micron) phospholipid vesicle shed from a megakaryocyte or other cell. Microparticles may contain genetic material such as RNA, and express the extracellular markers of their parental cells. Megakaryocyte- and platelet-derived microparticles may have a role in multiple pathways, including hemostasis and inflammation.
By “passive drug loading” is meant the uptake of a therapeutic composition by a cell (e.g., a platelet or progenitor thereof) without conjugation or mechanical or chemical disruption or modification of the cell. For example, liposomal delivery systems can be used for passive drug loading.
The term “platelet” refers to a cell with a diameter of 1-3 microns with no nucleus but does contain RNA. Internally, it contains alpha and dense granules, which contain such factors as P-selectin and serotonin, respectively. Platelets also have an open canalicular system, which refer to channels that are a pathway for the transport of extracellular material into the cell and the release of material from granules to the extracellular environment. They primarily function in the regulation of hemostasis by participating in blood clotting but also have been shown to have a role in inflammation.
The term “preplatelet” refers to a cell with a diameter of 3-10 microns with no nucleus but with RNA. Preplatelets are otherwise morphologically and ultra-structurally similar to platelets and constitute an intermediate cell stage produced by megakaryocytes that break apart through cytoskeletal rearrangement to form individual platelets.
The term “proplatelet” refers to cytosolic extensions from megakaryocytes or just released from megakaryocytes. Proplatelets break apart through cytoskeletal rearrangement to form individual preplatelets and platelets.
The term “pluripotent stem cell” includes embryonic stem cells, embryo-derived stem cells, and induced pluripotent stem cells and other stem cells having the capacity to form cells from all three germ layers of the body, regardless of the method by which the pluripotent stem cells are derived. Pluripotent stem cells are defined functionally as stem cells that can have one or more of the following characteristics: (a) be capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) be capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); or (c) express one or more markers of embryonic stem cells (e.g., express Oct 4, alkaline phosphatase. SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, Nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.).
The term “induced pluripotent stem cells” (iPS cells or iPSCs) refers to a type of pluripotent stem cell generated by reprogramming a somatic cell by expressing a combination of reprogramming factors. The iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. Factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct 4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4. In other embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, a combination of Oct 4, Sox2, Nanog, and Lin28. In certain embodiments, at least two, three, or four reprogramming factors are expressed in a somatic cell to reprogram the somatic cell.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reducing cell death” is meant reducing the propensity or probability that a cell will die. Cell death can be apoptotic, necrotic, or by any other means.
By “reduced level” is meant that the amount of an analyte in a sample is lower than the amount of the analyte in a corresponding control sample.
By “reference” is meant a standard or control condition.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the present disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the present disclosure.
The term “subject” or “patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
By, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
The present disclosure is directed to compositions and methods for producing megakaryocytic progenitors (preMKs), megakaryocytes (MKs), proplatelets, preplatelets or platelets from stem cells, such as, pluripotent stem cells, for example, clinical-grade human induced pluripotent stem cells. The methods enable the continued production of preMKs, MKs, proplatelets, preplatelets or platelets from hemogenic endothelial cells. The preMKs, MKs, proplatelets, preplatelets and platelets derived by the instant methods can be distinguished by one or more of the following: their size range, ploidy profile, biomarker expression, gene expression, granule composition, and growth factor, cytokine and chemokine composition or combinations thereof. The present disclosure is further directed to compositions and methods of use of such preMKs, MKs, proplatelets, preplatelets and platelets for drug delivery.
Unique to megakaryocytes and platelets are the presence of secretory granules wherein multiple proteins promoting clot formation (clotting factors) and tissue repair (cytokine, chemokine, and growth factors) are naturally sequestered. Megakaryocyte and platelet granule exocytosis plays a critical role in thrombosis, immune-system modulation, and tissue regeneration. Upon contact-activation at sites of bone marrow damage or vascular injury, megakaryocytes and platelets can selectively release the contents of their secretory granules to trigger a localized therapeutic response. Platelets will also naturally accumulate at sites of cancer, wherein they selectively adhere to tumors (wounds that never heal), hiding them from the immune system and contributing pro-angiogenic factors such as VEGF, and anti-inflammatory cytokines such as TGF-β through their granules that contribute to angiogenesis and tumor metastasis.
Megakaryocytes and platelets can be loaded or genetically engineered to express molecules (for example, within their granules) to produce ‘designer cells’ that can specifically be applied for expression of coagulation factors, cytokines, chemokines, growth factors, and drugs. These modified cells can be manipulated to be more or less sensitive and responsive to agonists and improve or inhibit clotting time even under conditions that normally cause cell dysfunction or impair coagulation. Likewise, molecules can be directly conjugated on their surface or packaged into secretory granules, which can be leveraged to improve cell specificity to target tissue and carry molecules to therapeutic targets to improve their specificity. In some embodiments, nanoparticles coated with platelet membranes may be used instead of whole platelets.
The present disclosure provides methods and systems for manufacturing of a large number of megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets under cGMP conditions for clinical use and expressing and/or loading of drugs in these cell types for targeting to therapeutic target and selective release.
Aspects of the present disclosure relate to a scalable, cGMP-compliant stem cell-based process that enables the rapid generation of functional megakaryocytes and platelets.
In some aspects of the present disclosure the cGMP-compliant human PSC line can be engineered to conditionally express specific drugs at the megakaryocyte (immediate cellular progenitor) level or the platelet level. For example, megakaryocytes and platelets generated according to the process of the present disclosure can be directed to package drugs into secretory granules as part of normal megakaryocytes or platelet production. Resulting “designer” modified megakaryocytes and platelets can consequently contain the desired drugs, which can be delivered through normal circulation to the targeted sites of injury or disease, avoiding the direct systemic exposure from intravascular transfusion of these factors to the body, and reducing non-specific risks of microaggregate/clot formation and immunogenicity.
Aspects of the present disclosure are directed to compositions comprising the presently disclosed megakaryocytic progenitor, megakaryocytes, proplatelets, preplatelets or platelets derived from iPSCs as a vehicle for drug delivery.
According to some aspects of the present disclosure, methods for producing the instant megakaryocytic progenitor, megakaryocytes, proplatelets, preplatelets or platelets from pluripotent stem cells (such as, for example, clinical-grade hiPSCs) are disclosed.
These methods enable the continued production of megakaryocytic progenitors from hemogenic endothelial cells for extended time frames up to 1 week or more can be subsequently differentiated into mature megakaryocytes. These iPSC-derived megakaryocytes can be distinguished by their size range, ploidy profile, biomarker expression, and growth factor, cytokine and chemokine composition or combinations thereof.
In some embodiments, MKs and platelets can be derived from pluripotent stem cells, including but not limited to, embryonic stem cells (ESCs) (e.g. human embryonic stem cells) and induced pluripotent stem cell (iPSCs) (e.g. human induced pluripotent stem cells). ESCs are pluripotent stem cells derived from the inner cell mass of an early-stage preimplantation embryo called a blastocyst. iPSCs are a type of pluripotent stem cell that can be generated from adult cells by inducing timed expression of particular transcription factors. iPSCs can be expanded and maintained in culture indefinitely and engineered to produce MKs and platelets.
In some embodiments, MKs and platelets can also be derived from hematopoietic stem cells, including but not limited, to CD34 umbilical cord blood stem cells (UCB cells) (e.g. human CD34+ umbilical cord blood stem cells), CD34+ mobilized peripheral blood cells (MPB cells) (e.g. CD34+ human mobilized peripheral blood), or CD34+ bone marrow cells. UCB cells are multipotent stem cells derived from blood that remains in the placenta and the attached umbilical cord after childbirth. MPB cells are multipotent stem cells derived from volunteers whose stem cells are mobilized into the bloodstream by administration of G-CSF or similar agent.
In some embodiments, MKs and platelets can be derived from other stem cell types, including but not limited to mesenchymal stem cells (MSC) (such as, adipose-derived mesenchymal stem cells (AdMSC)) or mesenchymal stem from other sources.
AdMSCs are derived from white adipose tissue, which is derived from the mesoderm during embryonic development and is present in every mammalian species, located throughout the body. Due to their wide availability and ability to differentiate into other tissue types of the mesoderm-including bone, cartilage, muscle, and adipose-ASCs may serve a wide variety of applications.
In the present disclosure, the stem cell cultures can be maintained independently of embryonic fibroblast feeder cells and/or animal serum. In some embodiments, serum-free, feeder-cell free alternatives can be utilized in the instant methods.
The present disclosure provides methods for producing megakaryocytic progenitors (preMKs) and megakaryocytes (MKs) from stem cells.
In some embodiments, the present disclosure provides a method for megakaryocyte production comprising: expanding pluripotent stem cells under low adherent or non-adherent conditions and under agitation wherein expanded pluripotent stem cells form self-aggregating spheroids; differentiating the pluripotent cells in a first culture medium into hemogenic endothelial cells; differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors. The differentiating of the pluripotent cells into hemogenic endothelial cells can be carried out under adherent conditions on a matrix. In some embodiments, the differentiating of the pluripotent cells into hemogenic endothelial cells is carried out under low-adherent or non-adherent conditions to enable the hemogenic endothelial cells to self-aggregate.
In some embodiments, the present disclosure provides a method for megakaryocyte production comprising: differentiating pluripotent cells in a first culture medium into hemogenic endothelial cells; and differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors, wherein at least one of the differentiating the pluripotent cells and the differentiating the hemogenic endothelial cells is carried out on a matrix coated 3-dimensional structure. The 3-dimensional structure can be a microcarrier or a microcarrier.
In some embodiments, the present disclosure provides a method for megakaryocyte production comprising: differentiating pluripotent cells in a first culture medium into hemogenic endothelial cells; and differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors, wherein at least one of the differentiating the pluripotent cells and the differentiating the hemogenic endothelial cells is carried out under low-adherent or non-adherent conditions to enable the cells to self-aggregate.
The present disclosure further provides methods for producing platelets from MKs.
Methods of Production
Stage 0: Expansion of human induced pluripotent stem cells and preparation for differentiation
Matrix-Dependent Expansion Cultures
For matrix-dependent expansion cultures, clinical grade pluripotent stem cells (PSCs) can be expanded as colonies by culturing without feeder cells on a supportive matrix in a pluripotent stem cell culture medium. The supportive matrix can be a 2-dimensional surface or a 3-dimensional structure that enables cell attachment. In some embodiments, the clinical grade human induced pluripotent stem cells can be human induced pluripotent stem cells (iPSCs), but other types of pluripotent stem cells, such as embryonic stem cells, or other stem cells can be used.
In some embodiments, the supportive matrix can be, by way of a non-limiting example, tissue-culture treated plastic, recombinant vitronectin, recombinant laminin, Matrigel, Geltrex, or any combinations of the foregoing. In some embodiments, the pluripotent stem cell culture medium can be, for example, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (Thermofisher), NutriStem medium (Biological Industries), or other medium able to support the maintenance and growth of pluripotent cells known in the art. In some embodiments, the cells can be cultured to reach confluency. In some embodiments, the cells can be cultured to reach from 30% to 90% % confluency. In some embodiments, the cells are cultured to reach up to 60%, up to 65%, up to 70%, up to 75% confluency. For example, the cells are cultured to reach about 70% confluency. Upon reaching a predetermined maximum percent confluency, the cells are harvested. In some embodiments, the cells can be harvested as clumps by dissociation using from 0.1 mM to 5 mM EDTA or similar chelating agent or reagent. For example, the cells can be harvested using about 0.5 mM EDTA. In some embodiments, the cells can be harvested as single cells, such as, for example, by dissociation with proteolytic enzymes, collagenolytic enzymes, or combinations thereof. For example, the cells can be harvested as single cells by dissociation with, for example, recombinant trypsin such as TrypLE™, or Accutase™. For maintenance/expansion of PSCs, the harvested cells can be resuspended in pluripotent stem cell culture medium.
A high-efficiency single cell passaging technique can be used to support scaled expansion of undifferentiated hiPSC cultures. The same methodology is intended for cell banking and scaled hiPSC seed-trains leading to pre-MK manufacturing. The approach provides rapid expansion for overall manufacturing capacity, undifferentiated pluripotent cultures with capacity to produce pre-MK, and uniformity of harvest yields and culture performance in a system compatible with cGMP manufacturing and clinical entry. Briefly, a single cell iPSC suspension is generated using one or more cell-dissociation enzymes (such as, for example, TrypLE (Thermo Fisher), followed by plating at a defined density in a feeder free culture medium (for example, NutriStem hPSC XF (Biological Industries). In some embodiments, the culture medium may be further supplemented, such as, for example, with a ROCK inhibitor and a one or more growth factors. In some embodiments, the cultures are plated at a density between 5×103 cells/cm2 and 5×104 cells/cm2 for 3-day or 4-day culture interval. For example, in some embodiments, cultures are plated at a density of about 5×103, cells/cm2 about 1×104 cells/cm2, about 2×104 cells/cm2, about 3×104 cells/cm2, about 4×104 cells/cm2, or about 5×104 cells/cm2, for a 3-day or 4-day culture interval, or 2×104 cells/cm2 for a 3-day culture interval. In some embodiments, cultures are plated at a density of about 1×104 cells/cm2 for a 4-day culture interval or about 2×104 cells/cm2 for a 3-day culture interval. In some embodiments, cell attachment to untreated surfaces can be mediated by human serum. In some embodiments, 18-22 hours post-plating, cultures can be fed with a feeder-free culture medium, without supplementation. Cultures can be passaged at 3- or 4-day intervals, achieving predictable and consistent harvest yields over multiple passages.
Matrix-Independent 3D Expansion Cultures
The cells can be subjected to continuous motion by slow stirring or gentle shaking in low-adherent or non-adherent conditions in a pluripotent stem cell culture medium. In some embodiments, feeder free, serum free medium can be used. The pluripotent stem cell culture medium can be, for example, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (Thermofisher), NutriStem medium (Biological Industries), or other similar medium able to support the maintenance and growth of pluripotent cells known in the art. In some embodiments, the culture medium can be supplemented with Rock inhibitor (e.g. H1152). In some embodiments, the medium may include an epidermal growth family member, for example, Heregulin-beta-1. In some embodiments, Heregulin-beta-1 medium is used for less than 24 hours (e.g., 18-22 hours). In some embodiments, the PSC spheroids are cultured until reaching an overall cell density of from about 3 to about 10 million cells/ml and/or attain a median spheroid size of about 150 to about 350 μm, for approximately 5-7 days. In some embodiments, the PSC spheroids are cultured until reaching an overall cell density of 5 million cells/ml. In some embodiments, the PSC spheroids are cultured until the cells attain a median spheroid size of about 250 μm. The culturing step may last for 3, 4, 5, 6, 7, or 8 days. When applicable, PSCs can be harvested as single cells by dissociation with proteolytic enzymes, collagenolytic enzymes, or combinations thereof. For example, the cells can be harvested as single cells by dissociation with, but not limited to trypsin, recombinant trypsin such as TrypLE™, Accutase™, or similar reagent known in the art. In some embodiments, the single cells are used to initiate another 3D expansion culture and/or directed differentiation culture.
Preparation for Differentiation
In some embodiments, to prepare for differentiation, PSC aggregates can be generated by partial dissociation of PSC colonies from matrix-dependent 2D cultures, by partial dissociation of PSC spheroids from matrix-independent 3D cultures, or by self-aggregation of single PSCs generated by any method known in the art. In some embodiments, prior to initiation of differentiation, these aggregates can be generated in a pluripotent stem cell culture medium, for example, but not limited to, Essential 8 medium (ThermoFisher), StemFlex medium (Thermofisher), or NutriStem medium (Biological Industries). In some embodiments, the medium may include a ROCK inhibitor, such as, for example, but not limited to, Y27632, H1152, or combination thereof. In some embodiments, the medium may include soluble Laminin, for example, recombinant Laminin 521. In some embodiments, the medium may include an epidermal growth family member, for example, Heregulin-beta-1. In some embodiments, Heregulin-beta-1 medium is used for less than 24 hours (e.g., 18-22 hours). In some embodiments, the cells can be cultured for between 0 and 72 hours at 37° C., 5% CO2, 20% O2 prior to initiation of differentiation.
For matrix-dependent differentiation cultures, the aggregates can be allowed to attach to a surface. In some embodiments, the step of attachment may be allowed to proceed for about 24 hours, although any time between 1 hour and 24 hours or longer may be used. In some embodiments, the surface can be pre-coated with collagen, laminin, or any other extracellular matrix protein. In some embodiments, human collagen IV can be used for coating the surface. In some embodiments, the matrix-coated surface can be 2D (for example, the bottom of a plastic dish or flask). In some embodiments, the matrix-coated surface can be 3D (for example, smooth or textured spherical microcarriers, or macrocarriers, such as, Rauchig rings). The cells on the 3D matrix coated surfaces can then be cultured with or without continuous motion. For example, the cells can be cultured under ultra-low-adherent static conditions, in roller bottles, spinner flasks, stir tank bioreactors, vertical wheel bioreactors, packed bed bioreactors, or fluidized bed systems.
For matrix-independent differentiation cultures, the aggregates can be subjected to continuous motion by slow stirring or gentle shaking in a low-adherent vessel, such as, but not restricted to, plates or flasks on an orbital shaker, spinner flasks, roller bottles, vertical wheel bioreactors, or stir tank bioreactors). The cells can be transitioned into Stage 1 of differentiation after between 0 and 72 hours, for example after about 24 hours.
Stage 1. Generation of Hemogenic Endothelial Cells
In Stage 1, prepared PSC aggregates can be differentiated into hemogenic endothelial cells. Briefly, some or all of the pluripotent stem cell culture medium is removed and replaced with Stage 1 differentiation medium. In some embodiments, the Stage 1 differentiation medium can be an animal-component free medium (ACF) comprising StemSpan™-ACF (STEMCELL Technologies, Cat. No. 09855) as basal medium, supplemented with one or more growth factors, including, for example, bone morphogenic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). In some embodiments, the basal medium is supplemented with between 1 and 200 ng/ml of one or more each of BMP4 (for example, at 50 ng/ml), bFGF (for example, at 50 ng/ml), and VEGF (for example, at 50 ng/ml). In some embodiments, the Stage 1 medium can be further supplemented with a WNT agonist (such as CHIR98014 or CHIR99021), a Laminin (such as recombinant Laminin 521), or any combination thereof. In some embodiments, cells can be incubated for between 2 and 6 days in low oxygen conditions (for example, 37° C., 5% CO2, 5% O2), followed by between 2 and 6 days in normoxia (37° C., 5% CO2, 20% O2). In some embodiments, BMP4 can be added for the first 6-48 hours (e.g., 24 hours) and can be dispensable for the remainder of Stage 1 (
In 2D matrix-dependent cultures, by day 2, the morphology of the colonies changes to scattered elongated cell clusters (
Stage 2. Generation of Detached Megakaryocytic Progenitors (preMKs) from Hemogenic Endothelial Cells
In some embodiments, initiation of megakaryocytic progenitor (Stage 2) differentiation can be performed following between 4 and 8 days of Stage 1. Briefly, some or all of the Stage 1 medium is removed and replaced with a volume of Stage 2 medium, such as, for example, STEMdiffr™ APEL™2 basal medium (STEMCELL Technologies, Cat. No. 05275). Such Stage 2 medium can be supplemented with between 1 and 200 ng/ml of each of one or more of Stem Cell Factor (SCF) (for example, at 25 ng/ml), Thrombopoietin (TPO) (for example, at 25 ng/ml), Fms-related tyrosine kinase 3 ligand (Flt3-L) (for example, at 25 ng/ml), Interleukin-3 (IL-3) (for example, at 10 ng/ml), Interleukin-6 (IL-6) (for example, at 10 ng/ml), and Heparin (for example, at 5 Units/ml). In some embodiments, the Stage 2 medium can be further supplemented with UM171, UM729, SR-1, SU6656, Laminin (such as recombinant Laminin 521), or any combinations thereof.
Cells are then incubated for at least 3 and up to 12 or more days at 37° C., 5% CO2, 20% O2. For example, the cells can be incubated for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 days. In some embodiments, daily partial media exchanges can be performed, with 10-99% of the spent media removed and replaced with equivalent volumes of fresh Stage 2 media. In some embodiments, additional volumes of fresh media can be added with the net effect of increasing the total volume of the culture. In some embodiments, specific media components can be spiked into the culture in lieu of replacement or addition of fresh Stage 1 media.
Within 1-2 days after initiation of Stage 2, small, round, refractile cells appear within the adherent hemogenic endothelial cells and are eventually released into the supernatant (
Stage 3. Generation of mature Megakaryocytes (MK) from Megakaryocytic Progenitors
In some embodiments, differentiation of mature megakaryocytes can be initiated using PSC-derived preMKs, generated as described above. Fresh or thawed megakaryocytic progenitors can be seeded onto a non-adherent surface in Stage 3 medium, comprising, for example, StemSpan™-ACF. Non-adherent surfaces refer to surfaces such that the majority of cells are not intended to stick or cling to such surfaces, but instead remain mostly in suspension. For example, such surface can be made of “ultra-low adherence plastic” or may not be coated with extracellular matrix proteins to prevent or minimize adhesion of cells to the surface. In some embodiments the Stage 3 medium can be supplemented with between 0 and 200 ng/ml each of one or more of TPO (for example, at 25 ng/ml), SCF (for example, at 25 ng/ml), IL-6 (for example, at 10 ng/ml), IL-9 (for example, at 10 ng/ml), Heparin (for example, at 5 units/ml), and Rock inhibitor (e.g., Y27632 at 5 μM). In some embodiments, the Stage 3 medium can also be supplemented with UM171, UM729, SR-1, SU6656, or any combinations thereof.
Cells can then be incubated at between 37° C. and 40° C. (for example, 39° C.), between 5 and 20% CO2 (for example, 7%-10%), and between 5 and 20% O2 for up to 5 days. In some embodiments, partial daily media exchanges are performed, with 10-95% of the spent media removed and replaced with equivalent volumes of fresh Stage 3 media. In some embodiments, the non-adherent surface is an ultra-low-adherent plate or flask. In some embodiments, the non-adherent surface is a gas permeable membrane (such as the G-Rex®). In some embodiments, the non-adherent surface is a cell culture bag or vessel with gentle agitation. In either case, preMKs (either freshly harvested from Stage 2 culture, or thawed from cryopreserved stocks) are suspended in Stage 3 media at a density of 0.1-10 million per ml, and introduced into the vessel. For example, the preMKs can be at density of 1 to 1.5 million per ml, of 1 to 2 million per ml, of 1 to 3 million per ml, of 1 to 4 million per ml, of 2 to 5 million per ml, of 2 to 6 million per ml, of 3 to 7 million per ml, of 3 to 8 million per ml, of 5 to 9 million per ml, or of 8 to 10 million per ml. In some embodiments, the preMKs can be at a density of 0.1 to 1.5 million per ml, 0.2 to 1.5 million per ml, 0.3 to 1.5 million per ml, 0.4 to 1.5 million per ml, 0.5 to 1.5 million per ml, 0.6 to 1.5 million per ml, 0.7 to 1.5 million per ml, 0.8 to 1.5 million per ml, or 0.9 to 1.5 million per ml. The cells are cultured for a total of 1-5 days (for example, 3 days) to enable differentiation into mature MKs. In some embodiments, daily half media exchanges are performed, with 10-95% of the spent media removed and replaced with equivalent volumes of fresh Stage 3 media. At the end of the Stage 3 cultures, the resulting cells are increased in size and ploidy, and exhibit a host of features indicative of mature megakaryocytes (as for example, shown in
In some embodiments, during Stage 3, the megakaryocytic progenitors differentiate into mature MKs within several days. In some embodiments, cells that are initially uniformly small, round, and refractile (
Stage 4. Platelet and Preplatelet Production from Mature Megakaryocytes
After Stage 3, platelets are produced by mature MKs into the culture medium. These platelets can be harvested, quantified, and assessed by flow cytometry, electron microscopy, and fluorescence microscopy, confirming their identity as bona fide platelets (
Platelets and preplatelets are produced from iPSC-derived megakaryocytes, in some iterations, by subjecting the MKs to shear stresses. In some embodiments, this can be achieved by seeding mature megakaryocytes into a millifluidic bioreactor. (
Additional methods for producing platelets are contemplated herein. For example, platelets can be produced using the methods disclosed in U.S. Pat. No. 9,763,984, the contents of which are incorporated herein by reference in their entirety. Briefly, the megakaryocytes of Stage 3 may be contacted by one or more of the following: thrombopoietin or hematopoietic expansion medium that comprises at least one reagent selected from: Stem Cell Factor (SCF), thrombopoietin (TPO), interleukin-11, at least one ROCK inhibitor, and heparin. In some embodiments, culture mediums and growth factors similar to those of Stage 3 can be used. The platelets produced by the instant methods can be loaded with a therapeutic agent or genetically modified to comprise an agent of interest.
3D Systems
Packed Bed Bioreactor
In some embodiments, a 3D scalable packed-bed bioreactor may be used for the production of one or more of preMKs, megakaryocytes, platelets, or megakaryocytes and platelets. In some embodiments, the packed bed bioreactor can be used for the Stage 1 and Stage 2 culture (
At the completion of Stage 1, the media can be switched to allow Stage 2 differentiation and production and release of preMKs. Appropriately sized and shaped carriers such as the lmm Raschig rings can enable sufficient media flow and channel width to enable the released cells to percolate through the packed bed and out of the reactor for collection and cryostorage. In some embodiments, this design can decrease the shear forces experienced by the cells, can allow for efficient media usage due to its perfusion based design, and can enable the continuous collection of preMKs as they are released.
Self Aggregating Spheroids in Stir Tank Bioreactor
In some embodiments, certain process steps may be carried out using a scalable 3D solution, which can involve performing differentiations using self-aggregating spheroids suspended in stirred or shaken vessels. (
Media can then be partially or completely exchanged with Stage 1 differentiation media to promote the differentiation towards hemogenic endothelium, and agitation can be resumed, with incubation in hypoxic conditions (for example, 37° C., 5% CO2, 5% O2). Partial or complete media exchanges can be performed on a regular basis (for example, daily) during which time the spheroids can grow larger and develop characteristic structure and shape. For example, as shown in
To transition to Stage 2, agitation can be paused and the spheroids can be allowed settle to the bottom of the vessel. Media can then be partially or completely exchanged with Stage 2 differentiation media to promote the differentiation and subsequent release of preMK-containing suspension cells. On a regular basis thereafter (for example, daily), suspension cells can be collected and a partial media exchange can be performed. The released Stage 2 cells can be harvested by pausing agitation, allowing the aggregates to settle to the bottom of the vessel, and collecting the medium along with the released suspension cells. Thereafter, a half-media change can be initiated by adding half the original volume of fresh Stage 2 medium on top of the adherent cell layer or 3D aggregates. An aliquot of the collected cells in medium can be removed for viable cell enumeration and biomarker analysis by flow cytometry. The remainder of the cells can then be concentrated by centrifugation, counter-centrifugal elutriation, acoustic separation, or other related technology. Following concentration, the half-media change can be completed by adding back half the original volume of conditioned media to the adherent cell layer or 3D aggregates. In some embodiments, additional volumes of fresh media can be added with the net effect of increasing the total volume of the culture. In some embodiments, specific media components can be spiked into the culture in lieu of replacement or addition of fresh Stage 2 media. The remainder of the supernatant can be discarded and the preMK-containing cell pellet can be stored or transitioned directly into Stage 3. In some embodiments, the preMK-containing cells can be collected over a 2- to 7-day period (e.g. 3 days) and additionally cultured in Stage 2 medium or other medium in a separate vessel. Once the final harvest is complete, the preMK-containing cells can be pooled together and stored or transitioned directly into Stage 3. In some embodiments, the preMKs can cryopreserved for storage. For example, the preMKs can be stored at −180° C. in Cryostor 10 cryopreservation media.
Upon transition to static Stage 3 cultures, preMKs from 3D self-aggregating spheroid cultures can generate similar MK purities as preMKs from 2D culture systems. Furthermore, Stage 3 differentiation cultures generated from 3D self-aggregating spheroid cultures can contain cells that increased dramatically in size and are able to generate proplatelets, consistent with their identity as bona fide megakaryocytes.
Stage 5. Drug Loading in preMK, MK, Preplatelets, and Platelets
In reference to
In some embodiments, a target cell may be loaded with a therapeutic composition by incubating the therapeutic composition with a cellular suspension. The therapeutic, in some embodiments, is actively taken up by the cell (e.g., receptor dependent uptake), while in other embodiments, the therapeutic is passively taken up by the cell (e.g., receptor independent endocytosis, such as by embedding within the open canalicular system of the platelets and/or by passive diffusion.). In some embodiments, the present methods do not require physical or chemical deformation of the cell for efficient uptake of therapeutic composition. Therapeutics taken up by the cells are stored within the cell, for example, in the cell's secretory granules.
In some embodiments, a therapeutic composition is conjugated to the surface of a cell (i.e., megakaryocyte progenitor, mature megakaryocyte, preplatelet, and platelet). Conjugation, in some embodiments, requires functionalization of the surface of the cell using techniques known to those of skill in the art. In some embodiments, the therapeutic composition will comprise a reactive moiety that is able to bind or otherwise interact with the surface of the cell or a functional group on the surface of the cell.
In some embodiments, the iPSCs may be genetically engineered to express a transgene for the production of a therapeutic polynucleotide or polypeptide, followed by directed differentiation to megakaryocytes and platelets from iPSCs. Viral transduction and other methods for transgene delivery to iPSCs can be used to cause stable expression of biologics that can be used as drugs with efficient transcription and translation in pre-MK, MK, and PLT.
Megakaryocytes and Platelets
In some embodiments, the present disclosure provides a megakaryocytic progenitor, a megakaryocyte, preplatelet, proplatelet or a platelet derived in vitro from a PSC cell or cell line. According to aspects of the present disclosure, the megakaryocytic progenitor, a megakaryocyte, preplatelet, proplatelets or a platelet derived from a PSC cell or cell line are produced using the method of U.S. Pat. No. 9,763,984 or the bioreactor as disclosed in International Application No. PCT/US2018/021354, which are incorporated herein by reference in their entireties.
In some embodiments, the present disclosure provides an isolated population of cells comprising the megakaryocyte or megakaryocytic progenitor.
In some embodiments, the present disclosure provides a composition containing a megakaryocyte or megakaryocytic. In some embodiments of the present disclosure, the composition comprising megakaryocyte, megakaryocytic progenitor or products thereof is disclosed.
According to some embodiments of the present disclosure, the megakaryocyte, megakaryocytic progenitor or products thereof are hom*ogenous in shape, size and/or phenotype. It should be appreciated that the megakaryocyte, megakaryocytic progenitor or products thereof of the present disclosure may comprise a variability in biomarker expression, size, ploidy, number and purity that is characteristically different than the variability in corresponding human cells. In some embodiments, such variability can be significantly lower. In some embodiments, the cell populations may be created to have a desired variability, which may be lower or higher than that of the naturally-occurring cells.
In some embodiments, megakaryocytic progenitors (preMKs) are characterized by the expression of the markers CD43 and CD41, and the lack of CD14 (i.e. CD14−, CD41+, CD43+). Additional expression of CD42b may indicate that the megakaryocytic progenitor is in the process of final maturation towards mature megakaryocytes. In certain embodiments, megakaryocytic progenitors generated in differentiation cultures are non-adherent and may float freely in the culture medium.
In some embodiments, the instant megakaryocytes are one or more of CD42a+, CD42b+, CD41+, CD61+, GPVI+, and DNA+. In some embodiments, the instant megakaryocytes are one or more CD42a+, CD42b+, CD41+, CD61+, and DNA+. In some embodiments, the instant megakaryocytes are one or more of CD42b+, CD61+, and DNA+. In some embodiments, the instant megakaryocytes are one or more of CD42a+, CD61+, and DNA+. In some embodiments, the instant megakaryocytes are one or more of CD42a+, CD41+, and DNA+. In some embodiments, the instant megakaryocytes are one or more of CD42b+, CD41+, CD61+, and DNA+. In some embodiments, the instant megakaryocytes are one or more of CD42b+, CD42a+, CD61+, and DNA+. In some embodiments, the instant megakaryocytes are one or more of CD42b+, CD42a+, CD41+, and DNA+. In some embodiments, the megakaryocyte is CD41+CD61+CD42b+GPVI+. In some embodiments, the megakaryocyte is CD41+CD61+CD42a+GPVI+.
In some embodiments, the instant megakaryocyte is CD61+ and DNA+ and has a diameter of about 10-50 μm. In some embodiments, the megakaryocytes produced by the methods described herein have an average size between 10 and 20 μm, between 11 and 19 μm, between 12 and 18 μm, between 13 and 17 μm, between 14 and 16 μm, between 14 and 15 μm. In some embodiments, the megakaryocytes produced by the methods described herein have an average size of 14.5 μm. In some embodiments, the instant megakaryocyte has a diameter of about 10-20 μm. In some embodiments, the instant megakaryocyte has a diameter of about 10-30 μm. In some embodiments, the instant megakaryocyte has a diameter of about 10-40 μm. In some embodiments, the instant megakaryocyte has a diameter of about 10-50 μm. In some embodiments, the instant megakaryocyte has a diameter of about 20-40 μm. In some embodiments, the instant megakaryocyte has a diameter of about 25-40 μm.
In some embodiments, the instant megakaryocytes produced by the methods described herein have a ploidy of 2N-16N. In some embodiments, the instant megakaryocyte has a ploidy of at least 4N, 8N, or 16N. In some embodiments, instant megakaryocytes have ploidy 4N-16N. In some embodiments, the instant megakaryocytes produced by the methods described herein are 16%+/−11.4% of CD61+ cells at 72 hours of Stage 3 culture with higher than 4N DINA.
In some embodiments, at least 50% of the megakaryocyte population produced by the methods described herein is CD61+ and DNA+, and has a ploidy of 2N to 16N. For example, the megakaryocytes (i.e. beta-1-tubulin positive Stage 3 cells) from a representative iPSCs differentiation culture ranged in size from about 9 μm to about 27 μm, with a median of 15 μm (
In some embodiments, the isolated population of cells or the composition contains at least 50% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 55% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 65% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 60% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 70% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 75% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 80% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 85% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 90% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 95% of CD42b+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 98% of CD42b+ CD61+ DNA+ cells.
In some embodiments, the isolated population of cells or the composition contains at least 50% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 55% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 65% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 60% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 70% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 75% of CD42b+ CD41+ CD61+ cells. In some embodiments, the isolated population of cells or the composition contains at least 80% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 85% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 90% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 95% of CD42b+ CD41+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 98% of CD42b+ CD41+ CD61+ DNA+ cells.
In some embodiments, the isolated population of cells or the composition contains at least 50% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 55% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 65% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 60% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 70% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 75% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 80% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 85% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 90% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 95% of CD42b+ CD42a+ CD61+ DNA+ cells. In some embodiments, the isolated population of cells or the composition contains at least 98% of CD42b+ CD41+ CD61+ DNA+ cells.
In some embodiments, the isolated population of cells or the composition contains at least 50% megakaryocytes having ploidy of 4N or greater. In some embodiments, at least 50% megakaryocytes have ploidy 4N-16N. In some embodiments, at least 60% megakaryocytes have ploidy 4N-16N. In some embodiments, at least 70% megakaryocytes have ploidy 4N-16N. In some embodiments, at least 80% megakaryocytes have ploidy 4N-16N. In some embodiments, at least 90% megakaryocytes have ploidy 4N-16N. In some embodiments, the isolated population of cells or composition contains megakaryocytes having a mean ploidy of 4N.
In some embodiments, the isolated population of cells or the composition contains a proplatelet, preplatelet or platelet generated from a megakaryocyte of the present disclosure. In some embodiments, the proplatelet, preplatelet or platelet is a CD42b+ CD61+ DNA− cell. In some embodiments, the megakaryocyte is produced in vitro by differentiation of hiPSC cell or cell line.
In some embodiments, the megakaryocytes produced by the methods described herein comprise one or more of the following: (a) content of MK granules by immunofluorescence microscopy: PF4 and VFW for alpha-granules, LAMP-1 and serotonin for dense-granules; (b) gene expression data: Oct4-, Nanog-, Sox2-, Zfp42-, Zfpml+, Nfe2+, Runxl+, Meisl+, Gatal+; (c) have low/no fibrinogen, serotonin, and LDL, and (d) can uptake fibrinogen, serotonin, and LDL when incubated with plasma.
In some embodiments, the megakaryocytes produced by the methods described herein have a characteristic expression profile of growth factors, cytokines, chemokines, and related factors (
In some embodiments, the instant platelets derived from iPSCs are 2-5 μm in diameter (e.g., 3 μm in diameter) and preplatelets are greater than 5 μm in diameter (
Human iPSC derived platelets described herein can be distinguished from primary, human donor derived platelets with respect to their lack of GPVI expression and greater thrombin generation over a more acute timeframe (
In some embodiments, the instant platelets are one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P− (in resting state). In some embodiments, the instant platelets are of CD61+, DRAQ−, Calcein AM+, and CD62P−. In some embodiments, the instant platelets are one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P+(in activated state). In some embodiments, the instant platelets are CD61+, DRAQ−, Calcein AM+, and CD62P+ In some embodiments, the instant platelets are distinct from donor platelets in that they do not express GPVI on the cell surface, but are still one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P− (in resting state) and/or one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P+(in activated state). In some embodiments, the instant platelets are distinct from donor platelets in that they do not express GPVI on the cell surface, but are still CD61+, DRAQ−, Calcein AM+, CD42a+, CD62P− (in resting state), and/or CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P+(in activated state). In some embodiments, the instant platelets are distinct from donor platelets in that they do not express GPVI on the cell surface, but are still CD61 and CD62P+.
In some embodiments, the instant platelets have a diameter of about 1 μm. In some embodiments, the instant platelets have a diameter of about 2 μm. In some embodiments, the instant platelets have a diameter of about 3 μm. In some embodiments, the instant platelets have a diameter of about 4 μm. In some embodiments, the instant platelets have a diameter of about 5 μm. In some embodiments, instant proplatelets are 5 μm or greater. In some embodiments, the diameter of the instant platelets is between about 1 μm and about 5 μm. In some embodiments, the diameter of the instant platelets is between about 1 μm and about 4 μm. In some embodiments, the diameter of the instant platelets is between about 1 μm and about 3 μm. In some embodiments, the diameter of the platelet is between about 3 μm and about 4 μm. In some embodiments, the diameter of the platelet is between about 3 μm and 5 μm.
In some embodiments, PLTs differentiated from iPSC-derived megakaryocytes are efficient at generating thrombin when stimulated by tissue factor, with reduced lag time and a greater quantity of thrombin than what is observed in platelets from peripheral blood plasma in a thrombin generation assay (
Thrombin generation is quantified by the enzymatic conversion of a thrombin substrate to a fluorogenic molecule that can be detected by standard methods. The graph shows a rapid generation of thrombin as well as a rapid decrease in signal in the hiPSC-derived platelet sample. This effect is in stark contrast to the thrombin generation observed from peripheral blood isolated platelets over the course of 90 minutes
In some culture conditions, the number of PLTs used to generate thrombin can impact the maximum concentration of thrombin produced and the lag time observed before the maximum concentration is achieved. For example, in some embodiments, a culture comprising 3×106 PLT will generate a higher concentration of thrombin in a shorter period of time compared to a culture comprising 0.5×106 PLT. In some embodiments, thrombin generation in PLT is greater than 2 to 3-fold the thrombin generated in donor derived platelets. In some embodiments, a maximum concentration of thrombin generated by PLT is between 250 nM and 850 nM, between 250 nM and 700 nM, between 250 nM and 650 nM, between about 250 nM and 600 nM, between about 250 and 550 nM, between about 250 and 500 nM, or between about 250 nM and 450 nM. In some embodiments, the maximum concentration of thrombin generated by PLT is between about 300 nM and 850 nM, between about 350 and 850 nM, between about 400 nM and 850 nM, between about 450 nM and 850 nM, between about 500 nM and 850 nM, between about 550 nM and 850 nM, between about 600 nM and 850 nM, between about 650 nM and 850 nM, between about 700 nM and 850 nM, or between about 750 nM and 850 nM. Conversely, donor derived cells generate a maximum concentration of thrombin of less than 200 nM for the same cell density. In some embodiments, the composition may include cell densities of between about 0.5×106 and 3.0×106 to generate the desired concentrations of thrombin.
In some embodiments, the instant PLTs exhibits certain distinctive features that are different than those of the naturally occurring or donor platelets. These features may indicate biological properties or compositions unique to an iPSC-derived MK bioreactor produced product compared to that collected from adult circulation. For example, in some embodiments, the distinctive features comprise being of uniform age post-derivation, being of a developmental age more similar to embryonic, fetal, or neonatal platelets, having a specific or altered cell signaling or activation pathway structure, or containing product-derived non-platelet particles with biological function.
In some embodiments, “being of uniform age post-derivation” means that all PLT coming produced were derived at the same time. In some embodiments, producing PLT occurs in a bioreactor; thus, the produced cells “being of uniform age post derivation” refers to cells that were newly made (ie same “age”, day 0). In contrast, the platelets in donor samples are a mix of different ages (day 0 to day 10). Donor platelets are typically cleared at around day 10. Accordingly, in some embodiments, the instant PLT composition where at least 60% of PLTs are of the same age. In some embodiments, the instant PLT composition where at least 70% of PLTs are of the same age. In some embodiments, the instant PLT composition where at least 80% of PLTs are of the same age. In some embodiments, the instant PLT composition where at least 90% of PLTs are of the same age. In some embodiments, the instant PLT composition where substantially all PLTs are of the same age.
In some embodiments, the cells produced according to the methods described herein are of a developmental age more similar to embryonic, fetal, or neonatal platelets than to adult cells. Without being bound by theory, these cells may be of a younger developmental age than an adult population.
In some embodiments, cells produced via the methods described herein have a specific or altered cell signaling or activation pathway structure. For example, in some embodiments, compared to donor derived platelets, the instant PLTs exhibit reduced CD62p production when exposed to TRAP-6. In some embodiments, the instant PLTs exhibit lack of response to TRAP or Thrombin. In some embodiments, the instant PLTs will produce thrombin in TGA without the signaling trigger of Tissue Factor (which the donor platelets require for activation in the assay). In some embodiments, the cell contain product-derived non-platelet particles with biological function are provided, such as microvesicles or microparticles. In some embodiments, the non-platelet particles are microvesicles or microparticles.
In some embodiments, instant platelets contain secretory granules, the open canalicular system, and the dense tubular system (
In some aspects, compositions are provided that comprise platelets that are one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P− (in resting state). In some embodiments, the instant platelets are one or more of CD61+, DRAQ−, Calcein AM+, CD42a+, and CD62P+(in activated state). In some embodiments, the compositions comprise platelets that do not express GPVI.
Microparticles
Another aspect of the present disclosure provides a composition that includes a population of microparticles or microvesicles derived from induced pluripotent stem cell (iPSC)-derived platelets, where the microparticles exhibit increased thrombin generation relative to a population of microparticles derived from donor derived platelets, and where the populations of microparticles are derived from about the same number of iPSC derived platelets and donor derived platelets. In some embodiments, the thrombogenic activity of the microparticles derived from induced pluripotent stem iPSC derived platelets is greater than the thrombogenic activity present in a microparticle derived from a donor derived platelet or megakaryocyte. In some embodiments, the thrombogenic activity present in the microparticle results in a maximum concentration of about 400 nM thrombin. In some embodiments, an average diameter of microparticles derived from a population of iPSC derived platelets is less than 50% the diameter of the microparticles derived from a population of donor derived platelets having about the same number of platelets as the population of iPSC derived platelets. In some embodiments, the megakaryocyte or platelet is genetically modified to comprise a nucleic acid molecule encoding a therapeutic agent.
Microvesicles, or microparticles (used interchangeably herein), are subcellular sized particles consisting of a membrane lipid bilayer and cellular content. Platelet-derived microvesicles may exert both anti-inflammatory or pro-inflammatory functions and have potential as vehicles for drug delivery. In some embodiments, the instant microvesicles are able to produce thrombin (See
In some embodiments, the diameter of the instant microparticles is 0.1 and 4 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 3 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 2.5 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 1.5 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 1.0 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.9 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.8 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.7 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.6 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.5 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.4 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.3 μm. In some embodiments, the diameter of the instant microparticles is 0.1 and 0.2 μm. In some embodiments, the diameter of the instant microparticles is 0.2 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.3 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.4 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.5 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.6 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.7 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.8 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.9 and 1 μm. In some embodiments, the diameter of the instant microparticles is 0.2 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.3 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.4 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.5 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.6 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.7 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.8 and 2 μm. In some embodiments, the diameter of the instant microparticles is 0.9 and 2 μm. In some embodiments, the diameter of the instant microparticles is 1.0 and 2 μm. In some embodiments, the diameter of the instant microparticles is 1.5 and 2 μm. In some embodiments, the diameter of the instant microparticles is 2.0 and 2.5 μm.
In some embodiments, thrombogenic compositions of microparticles are provided such that the composition has a peak size of less than approximately 2 μm. In some embodiments, the thrombogenic microparticles range in size between 40 nm and 100 nm in diameter. In some embodiments, the thrombogenic microparticles form greater than 50% of the composition. In some embodiments, the thrombogenic microparticles form greater than 60%, 70%, 80%, 90% or 100% of the composition.
Megakaryocytic Progenitors, Megakaryocytes, Proplatelet, Preplatelets and Platelets as Drug Delivery Vehicles
Platelets circulate in the bloodstream and touch every organ in the body, providing them with the potential to serve as part of a versatile, customizable, and targetable drug delivery system. Moreover, because of their immunomodulatory and angiogenic functions, platelets are actively recruited by tumors to aid in immune evasion and support their growth and metastasis. According to some aspects of the present disclosure, the ex vivo PSC-derived megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets described herein can be used as vehicles for delivering a therapeutic composition, such as a drug, small molecule, biologic (such as a protein) or a similar therapeutic agent. The benefits of this type of drug delivery include, but are not limited to, the ability to deliver molecules to tissues that are traditionally hard to target due to limitations imposed by permeability or retention of the drug; localization and concentration of the drug to the targeted tissue; a reduced need to treat a patient systemically by hiding the drug in megakaryocyte/preplatelet/platelet secretory granules until selective release at therapeutic target; and to avoid unwanted toxicity or immunogenicity. In some embodiments, this type of drug delivery may also lower the dosage needed to achieve a desired therapeutic outcome, and to decrease systemic toxicity.
The terms “therapeutic composition,” “drug,” “therapeutic,” and “agent,” are used interchangeably and refer to any small molecule chemical compound, antibody, nucleic acid molecule, polypeptide, or any other biologic or fragments thereof. In some embodiments, the therapeutic composition can be an agent that binds a target of interest, an antibody against a target of interest, an agonist or antagonist of a target of interest, a peptidomimetic of a target of interest, a small RNA directed against or a mimic of a target of interest, and the like. In some embodiments, therapeutic composition can modulate the expression and/or activity of target of interest.
In some embodiments, the therapeutic composition is a polypeptide or a small molecule. For example, the polypeptide can be atezolizumab, a fully humanized monoclonal antibody. Additional polypeptides can be ipilimumab, bevacizumab, cetuximab, or trastuzumab. Small molecule examples include, without limitation, aripiprazole, esomeprazole, or rosuvastatin.
In some embodiments, the therapeutic composition comprises an anti-angiogenic agent or chemotherapeutic agent suitable to treat, inhibit, and/or prevent cancer. Examples of anti-angiogenic agents include, without limitation, doxorubicin, a DNA damaging agent. Additional examples include vincristine, irinotecan, and pacl*taxel.
In some embodiments, the therapeutic composition comprises a growth factor, including, but not limited to VWF, keratinocyte growth factor, coagulation factors (e.g. FVII, FVIII, FIX) epidermal growth factor, or hair growth factor. FVIIa is an activated clotting factor which has shown benefit in patients with uncontrollable bleeding. To achieve this effect, FVIIa is be administered systemically at high concentration which has cost implications and has been shown to lead to thrombotic complications in some patients. Megakaryocytic progenitors, megakaryocytes, preplatelets, or platelets generated according to the process of the present disclosure supercharged with FVIIa may markedly improve hemostasis and survival in the acute period following injury. Factor VIII participates in blood coagulation; it is a cofactor for Factor IXa which, in the presence of Ca2+ and phospholipids, forms a complex that converts factor X to the activated factor Xa. In humans, factor VIII is encoded by the F8 gene. Defects in this gene result in hemophilia A, a recessive X-linked coagulation disorder. In response to injury, coagulation factor VIII is activated and separates from von Willebrand factor to become FVIIIa. Factor IX is a serine protease in the coagulation system. Deficiency of this protein causes hemophilia B. Factor IX is produced as a zymogen, an inactive precursor. It is processed to remove the signal peptide, glycosylated and then cleaved by factor XIa (of the contact pathway) or factor VIIa (of the tissue factor pathway) to produce a two-chain form where the chains are linked by a disulfide bridge. When activated into factor IXa, in the presence of Ca2+, membrane phospholipids, and a Factor VIII cofactor, it hydrolyses one arginine-isoleucine bond in factor X to form factor Xa.
In some embodiments, the therapeutic composition is a chemokine or growth factor, such as platelet derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) and bone morphogenetic protein 2, -4 and -6 (BMP-2, -4, -6).
In some embodiments, the therapeutic composition is a protein. In some embodiments, the protein is a cytokine, such as, for example, Interleukin 1-beta, Interleukin 2, or Interleukin 12. In some embodiments, the protein is an antibody protein. In some embodiments, the antibody is Atezolizumab or Ipilimumab.
Proplatelets, preplatelets and platelets store bioactive factors in secretory granules, which they acquire from megakaryocytes. In some embodiments, the proplatelets, preplatelets and platelets of the present disclosure can be modified to store, or otherwise carry, a therapeutic composition.
In some embodiments, platelets can be engineered to express proteins on their surface, or otherwise tagged with proteins on their surface, or engineered to express or cultured to ‘take-up’ various antibodies or molecules into their secretory granules. Such engineered platelets can be used to transport a therapeutic composition and directed to to the desired tissue (or therapeutic target). In some embodiments, genetic engineering can occur at the PSC level or at the megakaryocytic progenitor level, and expression conditionally regulated to become expressed at the megakaryocytic progenitor, megakaryocytes, proplatelets, preplatelets, or platelets.
Some aspects of the present disclosure relate to modified megakaryocytes, proplatelets, preplatelets or platelets expressing desired characteristics for targeted applications. These tools can be leveraged to generate the specialized outcomes that personalized medicine approaches promise, without the drawbacks that have prevented their commercial implementation (high cost, time intensive, and inability to scale products). Rather than generating custom hPSC lines from individual donors, it is preferential to develop a platform that utilizes cGMP-compliant hPSC lines that are optimized for therapeutic product manufacture. Genetic control of the hPSC lines can then be applied to generate designer products for targeted therapeutics and recipients.
In some embodiments, the modified megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets express a protein (including a polypeptide, peptide) of interest. In some embodiments, the modified megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets express high level of a protein (including a polypeptide, or peptide) of interest. In some embodiments, the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets are genetically modified to reduce expression or suppress the expression of a protein (including a polypeptide or peptide) of interest. In some embodiments, the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets are genetically modified to express or overexpress a protein (including a polypeptide or peptide) of interest, to reduce expression or suppress the expression of a protein (including a polypeptide or peptide) of interest or any combinations of the foregoing. For example, in some embodiments, the modified megakaryocytic progenitors, megakaryocytes, proplatelets preplatelets, or platelets express high levels of clotting factors, e.g. Factor VIIa, VIII, IX, or VWF, in their granules, thereby enhancing clot formation at the site of damage without risk of systemic hypercoagulation. In some embodiments, the modified megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets are targeted for trauma, increasing the effectiveness of platelet transfusion during the first “Golden Hour” following severe traumatic injury. Another potential application for engineered instant megakaryocytic progenitors, megakaryocytes, preplatelets, or platelets is in the treatment of fetal and neonatal alloimmune thrombocytopenia (FNAIT). In this condition, fetal platelets expressing a human platelet antigen (HPA) that their mother does not express are targeted by the mother's immune system, leading to fetal thrombocytopenia and serious potential complications (including fetal intracranial hemorrhage). In some embodiments, this condition is treated using the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets that have been engineered with a single base pair change, such that HPA (negative) platelets or microparticles are administered to a HPA positive child after delivery by HPA-negative women, preventing the HPA-associated clearance.
In some embodiments, the modified megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets deliver growth factors. In some embodiments, the modified megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets loaded or expressing growth factors are used for cell culture, tissue regeneration, wound healing, cosmeceuticals, and hemostatic bandages.
In some embodiments, the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets are modified to deliver immune-checkpoint inhibitor drugs such as, but not limited to, anti-PDL1, anti-PD1, anti-VEGF, anti−CD20 and anti-CTLA4, or anti-cancer drugs like anti-CCR4, or anti-PI3K. In some embodiments, the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets are modified to deliver cytokines such as, but not limited to, interleukin 1 beta, Interleukin 2 or Interleukin 12.
Loading of iPSC-Derived Megakaryocytic Progenitors, Megakaryocytes, Proplatelets, Preplatelets or Platelets with Therapeutic Composition
In reference to
In some embodiments, as shown in
In some embodiments, therapeutics compositions, such as those described above, can be loaded into iPSC-derived platelets, megakaryocytes, megakaryocyte progenitors, and preplatelets by passive loading (also known as “sponge loading”). In some embodiments, passive loading is achieved by adding the therapeutic composition to cellular suspensions of MKs, MK progenitors, preplatelets, and/or platelets in aqueous buffer for 1 to 5 hours, for example 2 hours, at room temperature or 37° C. In some embodiments, the cellular suspensions are washed of excess therapeutic by diluting the cellular suspensions 2 to 10-fold, for example 5-fold. The diluted cellular suspension are then centrifuged suspensions, removing the supernatant, and resuspending the therapeutic loaded cellular suspension into fresh media.
Without being bound to the theory, incubation of the instant megakaryocytic progenitors, megakaryocytes, preplatelets, or platelets with the therapeutic composition results in the sequestration of the therapeutic composition into secretory granules (e.g. alpha-granules, dense granules) of the cell. In some embodiments, the instant megakaryocytic progenitors, megakaryocytes, preplatelets, or platelets loaded with the therapeutic composition can be used to locally deliver the therapeutic composition at a target site, such as sites of inflammation, vascular damage, tissue regeneration, lymphoangiogenesis, cancer development, progression and metastasis.
In reference to
Genetic Engineering of iPSC-Derived Megakaryocytic Progenitors, Megakaryocytes, Proplatelets, Preplatelets or Platelets to Express a Therapeutic Composition
In reference to
In some aspects of the present disclosure, the PSC-derived megakaryocytes are engineered to express at least one peptide, polypeptide or proteins of interest. Yet in other aspects of the present disclosure, the PSC are engineered to express at least one peptide, polypeptide or proteins of interest and megakaryocytes expressing the at least one peptide, polypeptide or proteins of interest or preplatelets or platelets comprising the at least one peptide, polypeptide or proteins of interest can be produced using the methods of U.S. Pat. No. 9,763,984 or the bioreactor as disclosed in International application No. PCT/US2018/021354, which are incorporated herein by reference in their entireties.
In some aspects of the present disclosure, the PSC-derived megakaryocytes are engineered to comprise a DNA or RNA of interest. Yet in other aspects of the present disclosure, the PSC are engineered to comprise a DNA or RNA of interest. In some embodiments, the preplatelets or platelets derived from the modified megakaryocyte or megakaryocytic progenitor comprising the DNA or RNA of interest can deliver the DNA or RNA of interest.
Some embodiments relate to compositions or isolated populations comprising engineered PSCs engineered to express the at least one peptide, polypeptide or protein of interest. In some embodiments, the protein is a cytokine, a chemokine or a growth factor. Some embodiments relate to compositions or isolated populations comprising megakaryocytes engineered to express the at least one peptide, polypeptide or proteins of interest. In some embodiments, the protein is a cytokine, a chemokine or a growth factor. Some embodiments, relate to compositions comprising platelets produced ex vivo from megakaryocytes engineered to express the at least one peptide, polypeptide or proteins of interest. In some embodiments, the protein is a cytokine, a chemokine or a growth factor. Preplatelets or platelets produced by modified megakaryocytes expressing at least one peptide, polypeptide or proteins of interest can be used as a delivery vehicle to deliver the at least one peptide, polypeptide or proteins of interest at site of interest. In some embodiments, the protein is a cytokine, a chemokine or a growth factor.
Some embodiments relate to compositions or isolated populations comprising engineered PSCs engineered to comprise a DNA or RNA of interest. Some embodiments relate to compositions or isolated populations comprising megakaryocytes engineered to comprise a DNA or RNA of interest. Some embodiments relate to compositions comprising platelets produced ex vivo from megakaryocytes engineered to comprise a DNA or RNA of interest of interest. Proplatelets, preplatelets or platelets produced by modified megakaryocytes comprising the DNA or RNA of interest can be used as a delivery vehicle to deliver the DNA or RNA of interest at a target site of interest.
It should be appreciated that modified megakaryocytes (or products thereof) expressing at least one peptide, polypeptide or proteins of interest or comprising a DNA or RNA of interest can be used for the treatment of different diseases.
In some aspects of the present disclosure, the DNA or RNA of interest or the RNA or DNA encoding the protein of interest can be any gene that the skilled practitioner desires to have integrated and/or expressed. In some embodiments, at least one peptide, polypeptide or protein of interest can be expressed in the megakaryocytes by delivering one or more nucleic acid molecules (i.e. gene of interest) encoding at least one peptide, polypeptide or protein of interest to the megakaryocyte or a precursor cell such as an iPSC cell. In some embodiments, the one or more nucleic acid molecules encoding at least one peptide, polypeptide or protein of interest may be contained within an expression vector. In some embodiments, the vector comprises one or more synthetic nucleotides (e.g., locked nucleic acids, peptide nucleic acids, etc.) or nucleoside linkages (e.g., phosphorothioate linkages). The vector may be single-stranded, double-stranded, or contain regions of both single-strandedness and double-strandedness. Exemplary vectors include, but are not limited to, plasmids, retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, a herpes simplex virus vectors, poxvirus vectors, and baculovirus vectors. In some embodiments, the nucleic acid molecule encoding the peptide, polypeptide or protein of interest may be expressed using a megakaryocyte-specific promoter. In some embodiments, the vector comprises a nucleic acid sequence that encodes a therapeutic polypeptide or fragment thereof. In some embodiments, the vector comprises a nucleic sequence that encodes an mRNA. In some embodiments, the vector comprises a therapeutic gene nucleic acid sequence including the promoter, or a fragment thereof. The vector comprising the nucleic acid molecule of interest may be delivered to the cell (e.g., iPS cell, megakaryocytic progenitor, or megakaryocyte) via any method known in the art, including but not limited to transduction, transfection, infection, and electroporation.
Targeted Delivery of Therapeutics Encapsulated in Megakaryocytes and Platelets
In some embodiments, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure are loaded with therapeutics that would be shielded from circulation upon transfusion of the cellular drug product in vivo. Platelets naturally home to cancerous lesions, solid tumors, and circulating tumor cells, in some examples, by receptor interactions with exposed collagen and other extracellular matrix components, in others by receptors that are surface exposed upon platelet activation. Platelets are known to aggregate in response to tumor cells (also known as tumor cell-induced platelet aggregation) as a result of these interactions. Platelets are “activated” by these interactions and will secrete the contents of their secretory granules, including small-molecule and biologic therapeutics that have been loaded into these cells by passive drug loading or by genetic modification of iPSCs, megakaryocytic progenitors, megakaryocytes, and any other cell that produces a “designer” platelet. They will also shed their membranes as part of an exocytotic process that produces microvesicles. In some embodiments, platelets with small molecule and biologic drugs covalently conjugated to the plasma membrane will remain stably bound during microvesicle formation and selectively delivered to cancer as a result of tumor cell-induced platelet aggregation. In some embodiments, genetically modified human iPSCs, megakaryocyte progenitors, or megakaryocytes can be engineered to express recombinant biologic drugs that are fused to a membrane anchoring domain from surface receptors, in some examples CD3 and DAF, and deliver them specifically to the cell surface. Recombinant biologic drugs anchored to the plasma membrane can also be delivered to sites of disease pathology by being incorporated into microvesicles as a result of platelet activation. Recombinant biologic drugs anchored to the plasma membrane can also include a protease cleavage site to allow for enzymes, in some examples matrix metalloproteases, that are abundant at sites of disease pathology, including a solid tumor, cancerous lesion, or a site of vascular injury or angiogenesis, to cleave the recombinant biologic drug in a separate example of targeted drug delivery.
The practice of the present disclosure may employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure, and, as such, may be considered in making and practicing the present disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
Methods of Use
As discussed above, in some embodiments, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure can be modified to include a therapeutic composition for targeted delivery of such therapeutic composition. In particular, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure can be loaded (such as by, passive absorption or covalent conjugation) with or genetically engineered to express a therapeutic composition, either on the surface or within their granules.
In some embodiments, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure can be used in combination with other nanoparticle materials for drug delivery. For example, in some embodiments, a membrane of the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure can be used as an outer shell for a drug delivery system that comprises one or more materials compatible with interacting with and transporting therapeutic compositions. For example, an outer shell platelet membrane can include platelet proteins capable of interacting with cancer celis. In some embodiments, such drug delivery vehicles can be prepared by lysing the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure and filling the outer membrane of the lysed cells with a drug delivery system comprising a therapeutic composition.
In some embodiments, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure can be a source of growth factors, such as human growth factors. In some embodiments, such growth factor can be used for cell culture, tissue regeneration, wound healing, bone regeneration, cosmeceuticals, and hemostatic bandages. In some embodiments, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure or their lysate or compositions thereof can be used in cell culture. In some embodiments, the megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets of the present disclosure or their lysate or compositions thereof can be used as a cosmeceutical.
For example, platelets store bioactive factors in secretory granules, which they acquire from megakaryocytes. Contents include various chemokines and growth factors, such as platelet derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-b (TGF-b), insulin-like growth factor-1 (IGF-1), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF-2), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) and bone morphogenetic protein 2, -4 and -6 (BMP-2, -4, -6). Human platelet lysate dramatically increases the expansion of cells ex vivo, improves bone marrow regeneration in vivo, and increases the survival rates of animals in radiation studies. In some embodiments, the present disclosure provides a composition or pharmaceutical composition comprising a lysate of a proplatelet, preplatelet or platelet generated from the instant megakaryocytes, wherein such compositions can include factors such as platelet derived growth factor isoforms PDGF-AA or PDGF-BB, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), hematopoietic growth factors Flt3L, G-CSF, GM-CSF, interleukins (IL-1RA, IL-8, or IL-16), CXC chemokine family members CXCL1 (GRO alpha) or CXCL12 (SDF-1), TNF superfamily members sCD40L or TRAIL, or CC chemokine family members CCL5 (RANTES), CCL11 (Eotaxin-1), CCL21 (6CKine) or CCL24 (Eotaxin-2).
Administration
Aspects of the present disclosure relate to pharmaceutical compositions comprising instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets according to embodiments of the present disclosure. In some embodiments, pharmaceutical composition comprises instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets according to embodiments of the present disclosure with a pharmaceutically acceptable carrier. For example, the carrier can be a diluent, an adjuvant, a preservative, an anti-oxidant, a solubilizer, an emulsifier, a buffer, water, an aqueous solution, oil, an excipient, an auxiliary agent or vehicle or combinations thereof. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington. In some embodiments, the carrier may be suitable for intravenous administration.
Aspects of the present disclosure relate to methods of treating a subject in need thereof, the method comprising administering compositions of the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets to a subject in need thereof.
Administration of suitable dose and dosage regimen of the compositions comprising instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets according to embodiments of the present disclosure to a subject in need thereof may be determined based on the subject's age, sex, weight, general medical condition, and the specific condition for which the composition is being administered.
Megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets according to embodiments of the present disclosure may be administered by any method. In some embodiments, the instant megakaryocytic progenitors, megakaryocytes, proplatelets, preplatelets, or platelets can be administered by direct injection, for example intravenous injection. Pharmaceutical preparations for injection may be prepared and delivered as known in the art.
Pharmaceutical Compositions
The present disclosure features methods for treating or preventing disease or infection in a subject. The present invention also features methods for treating wounds. The methods include administering to a subject in need thereof a therapeutically effective amount of a composition comprising an induced pluripotent stem cell (iPSC)-derived platelet comprising a therapeutic agent. In an embodiment, the composition is used in a pharmaceutical composition.
In some embodiments, the pharmaceutical compositions described herein comprise a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is affected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.
Administration of the pharmaceutical compositions contemplated herein may be carried out using conventional techniques including, but not limited to, infusion, transfusion, or parenterally. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally.
Kits
The disclosure provides kits comprising a megakaryocyte or differentiated cell of the disclosure. In one embodiment, the kit includes a composition comprising an isolated megakaryocyte. In particular embodiments, the disclosure provides kits for differentiating, culturing, and/or isolating a megakaryocyte of the disclosure or precursor thereof. In certain embodiments, the disclosure provides kits for producing platelets.
In some embodiments, the kit comprises a sterile container which contains a cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired, the kit is provided together with instructions for generating the megakaryocyte. The instructions will generally include information about the conditions and factors required differentiating, culturing, and/or isolating megakaryocytes or precursors thereof. In some embodiments, instructions for producing platelets are included. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.
EXAMPLES Example 1: Expansion of Clinical Grade hiPSCs
Prior to differentiation, hiPSC expansion is required to produce the large number of cells for use in appropriately sized master and working cell banks, as well as generate sufficient cell numbers to initiate differentiation at an appropriate scale for clinical production. A clinical grade hiPSC cell line was obtained from the NINDS Human Cell and Data Repository (NHCDR) depository at NINDS (National Institute of Neurological Disorders and Stroke)/NIH(National Institutes of Health). This cell line (NINDS ID: LiPSC-Gr1.1), which was derived from male CD34+ cord blood (Lonza), could be maintained and expanded in 2D cultures using recombinant vitronectin (VTN), plus cGMP compatible reagents such as Essential 8, NutriStem, or StemFlex (
A high-efficiency single cell passaging technique was also developed to support scaled expansion of undifferentiated hiPSC cultures. The same methodology is intended for cell banking and scaled hiPSC seed-trains leading to large scale differentiations for clinical manufacturing. The approach provides rapid expansion for overall manufacturing capacity, undifferentiated pluripotent cultures with capacity to produce pre-MK, and uniformity of harvest yields and culture performance in a system compatible with cGMP manufacturing and clinical entry. In this example, LiPSC-Gr1.1 cultures were dissociated to a single cell suspension using TrypLE (Thermo Fisher), followed by plating at a defined density in NutriStem hPSC XF (Biological Industries) containing 0.5 μM H1152 (Tocris) and 10 ng/mL heregulin β1 (Peprotech). Cultures were plated at a density of 1×104 cells/cm2 for a 4-day culture interval, and 2×104 cells/cm2 for a 3-day culture interval. Cell attachment to untreated TC-flasks was mediated by 0.5% human AB serum (Valley Biomedical). On the following day 18-22 hours post-plating, cultures were fed with NurtriStem hPSC XF without supplementation. Cultures were passaged at 3- or 4-day intervals, achieving predictable and consistent harvest yields over multiple passages (
To enable large-scale expansion, LiPSC-Gr1.1 cells were harvested from 2D cultures as single cells using TrypLE and allowed to self-aggregate in stirred 3D vessels, in this case a 300 ml DasBOX mini bioreactor system. For the first 24 hours, ROCK inhibitor such as Y27632 was added to the cells to promote cell survival during initial aggregation Over 6-7 days in a stir tank, the resulting spheroids increased their diameter from 50 to 250 microns and the overall cell density increased up to 40-fold within that period of time (
Example 2: Directed Differentiation of hiPSCs to preMKs and MKs Using Collagen IV Matrix in 2D Culture Vessels
The LiPSC-Gr1.1 hiPSC line was differentiated into megakaryocytes using the 2D matrix-dependent directed differentiation protocol summarized in
When harvested with 0.5 mM EDTA and plated as small clumps onto 4.2 ug/cm2 human Collagen IV, PSCs exhibit a characteristic set of morphological changes through the course of 6 days of Stage 1 differentiation (
Within 2-3 days after initiation of Stage 2 (i.e. day 6+2 to 6+3), small, round, refractile cells appear within the adherent hemogenic endothelial cells and are eventually released into the supernatant above the adherent hemogenic endothelial monolayer (
When preMKs from these cultures are transferred to Stage 3 conditions, they differentiate into mature MKs within several days. Cells that are initially uniformly small, round, and refractile (
Example 3: Directed Differentiation in Matrix-Independent 3D Cultures
To enable yields required for clinical production of megakaryocytes and platelets, it is crucial to transition the entire differentiation process from small-scale tissue culture plasticware (2D, matrix dependent) to a 3D scalable solution. An example of a scalable 3D solution involves performing differentiations using self-aggregating spheroids suspended in stirred or shaken ultra-low-adherent vessels (
Cultures expanded and harvested with single cell passaging (
Upon transition to static Stage 3 cultures, preMKs from 3D self-aggregating spheroid cultures generated similar MK purities as preMKs from 2D culture systems (
Example 4: Addition of Soluble Laminin 521 During iPSC Aggregation or at Stage 1-2 Transition Improves Stage 2 preMK Yields in Two Different 3D Differentiation Formats
Addition of Laminin 521 during the initial iPSC aggregation step 24 hours prior to initiation of differentiation (day −1) or at the time of transition between Stage 1 and Stage 2 (day 6) resulted in increased preMK yields in two different 3D differentiation formats. 5000 single-cell dissociated iPSCs were seeded per well of a 96-well U-bottom ultra-low adherent plate containing StemFlex and the Rock inhibitor H1152 (control media), with or without soluble recombinant Laminin 521. 24 hours later, the media was replaced with Stage 1 media and media exchanges were performed for 6 days. Media was then exchanged with Stage 2 media, with or without soluble Laminin 521. 24 hours later, daily half media exchanges were performed for up to 6 additional days. Comparing the preMK yields in Stage 2 revealed that the addition of Laminin 521 at Day −1 or Day 6 of the differentiation process increased the preMK yields compared to control cultures without Laminin 521 addition (
Example 5: Adjustment of Order and Timing of Growth Factor Addition During Stage 1 Increases Differentiation Efficiency and Decreases Overall Growth Factor Usage
The initial specification events that occur during Stage 1 of differentiation are complex and require a unique order and timing of cell signaling events. Therefore, adjusting the order and timing of addition of the Stage 1 media factors BMP4, bFGF and VEGFA could improve the efficiency of the differentiation process and reduce growth factor usage compared to the standard complete St1 media conditions, where all three growth factors are included for the entirety of Stage 1. The first experiment (Experiment A) (
Example 6: WNT Modulators can Affect Stage 1 and Stage 2 Differentiation Efficiency
WNT signaling is important during development. The GSK3 kinase inhibitors CHIR98014 and CHIR99021 act as WNT agonists. When the Stage 1 differentiation conditions described herein were augmented with 0.6 μM CHIR98014 or 6 μM CHIR99021 for the first 48 hours of differentiation only, a dramatic increase in Stage 1 differentiation efficiency was observed at day 6, as determined by immunofluorescence staining of CD31 and CD34 (
Example 7: Packed Bed Bioreactor with Laminin 521-Coated Macrocarriers
Here, evidence is provided demonstrating that a Laminin 521-coated PTFE macrocarrier in the shape of a 1 mm Raschig ring can provide support for the differentiation of iPSCs and that this macrocarrier material would be amenable for use in a packed bed bioreactor, as illustrated in the schematic shown in
Example 8: Detailed Characterization of iPSC-Derived Megakaryocytes
Megakaryocytes generated using the methods described herein demonstrate many features associated with functional mature MKs, including when imaged by immunofluorescence microscopy for the MK-specific protein beta-1-tubulin (
When compared to primary megakaryocytes (natural product) derived from bone marrow CD34+, peripheral blood CD34+, or cord-blood CD34+ cells, iPSCS-derived MKs, it was found that iPSC-derived MKs had a similar average size (
The results described herein demonstrate a robust process for generating clinical grade human iPSC-derived megakaryocytes. Human iPSC-derived megakaryocytes can be isolated and concentrated for further characterization or use in downstream applications, such as the generation of human platelets.
Example 9: Detailed Characterization of Human iPSC-Derived Platelets
Platelets are generated from mature MKs derived from human iPSCs using the culture methods and harvesting techniques described in this filing. The platelets can be collected in static culture (
Human iPSC-derived platelets have some features that distinguish them from primary, donor-derived, human platelets. They lack a surface receptor, glycoprotein VI, that is abundantly expressed on human platelets (
Example 10: Human iPSC-Derived Platelets Take Up Recombinant Biologic Drugs by Passive Drug Loading
Platelets produced by the methods described herein have characteristics akin to platelets that are extracted from whole peripheral blood as well as platelets that are differentiated from a human CD34+ mobilized peripheral blood cell source.
The drawing in
In one example, human IgG was loaded into and/or on donor-derived, human washed platelets. Human IgG was conjugated to NHS-ester Cy5.5 fluorophore according to manufacturer instructions and at 8-fold molar excess. Conjugated preparations were passed through a 40 k molecular weight cut-off (mwco) zeba desalting column and quantified by pierce 660 kit. Preps were kept at 4 degrees Celsius until further use. For drug loading experiments, Cy5.5 conjugated human IgG was brought to room temperature and centrifuged at 15,000 rcf for 1 minute to remove aggregates. The antibody was then added to 1×10e7 platelets in 1 mL of reaction volume for 1 hour at 37 degrees Celsius. PGE1 was added at 1 ug/ml final concentration to inhibit platelet activation and the cells were centrifuged at 1250 rcf for 17 minutes with no brake. In platelet preparations that were gated on CD61 expression (
Human donor platelets were able to uptake concentrations up to 200 μg of labeled anti-PD-L1 antibody (atezolizumab) in a subsequent experiment (
Human iPSC-derived platelets, produced using the methods described herein, were loaded with the anti-CTLA4 antibody drug, Ipilimumab, by co-incubation in aqueous buffer. Ipilimumab was loaded at varying concentrations, resulting in flow cytometry histogram plots that reveal a dose-dependent increase in encapsulated dose. Ipilimumab was conjugated to NHS-ester Cy5.5 fluorophore according to manufacturer instructions and at 8-fold molar excess. Conjugated preparations were passed through a 40 k molecular weight cut-off (mwco) zeba desalting column and quantified by pierce 660 kit. Preps were kept at 4 degrees Celsius until further use. For drug loading experiments, Cy5.5 conjugated Ipilimumab was brought to room temperature and centrifuged at 15,000 rcf for 1 minute to remove aggregates. Ipilimumab was added to 1e6 human iPSC-derived platelets in 100 microliter volume. In one example, 1, 10, 30, and 60 μg of Ipilimumab was added per sample, incubated at 37° C. for 1 hour, and washed and centrifuged to remove non-specifically bound drug. A flow cytometry based histogram plot was generated showing a dose-dependent increase in Ipilimumab encapsulation in the human iPSC-derived platelets (
A further example of recombinant protein loading in platelets was performed in CD34+ derived material using Ipilimumab conjugated to the fluorophore Cy5.5 using the methods described herein. CD34+ derived platelets were analyzed for size and granularity (
To determine whether donor-derived human platelets take up atezolizumab (
Example 11: Covalent Conjugation of Recombinant Proteins in Human iPSC-Derived Platelets
There are many strategies to create covalent linkages of recombinant proteins on the cell membrane (
The protocol described in
Example 12: Passive Loading of Recombinant Drug Biologics in Human iPSC-Derived preMK's and MK's
Induced pluripotent stem cells were differentiated into mature megakaryocytes using the methods described herein. Cells were incubated for 30 minutes with Dylight 488-labeled Atezolizumab and fixed with 4% paraformaldehyde and centrifuged onto prepared poly-l-lysine coated glass coverslips. To confirm mature megakaryocytes within the culture, cells were additionally stained for CD61. CD61 was visualized using a preconjugated CD61-APC antibody. Samples were washed and mounted onto glass coverslips with Aqua-poly/mount (Fisher Scientific). Images were captured using a Zeiss Meta 880 confocal scanning microscope and analyzed using Fiji ImageJ software (
Megakaryocyte progenitors, or preMKs, were loaded with an unconjugated version of the anti-CTLA4 antibody Ipilimumab using 1e6 cells in 1 ml and with 100 ug of Ipilimumab. PreMKs were immobilized to poly-l-lysine coated coverslips and stained with fibrinogen (alpha granule stain) and CD61 (surface marker) (
Example 13: Covalent Conjugation of Recombinant Drug Biologics in Human iPSC-Derived preMK's and MK's
The ability to conjugate recombinant drug biologics to the cellular membrane of megakaryocyte progenitors (preMKs) and mature megakaryocytes (MKs) derived from human iPSCs is demonstrated herein. PreMKs were harvested from Stage 2 cultures and immunophenotyped for CD41 and CD43 co-expression (
This data demonstrates covalent conjugation of recombinant protein biologic drugs to megakaryocyte progenitors (preMKs) and mature megakaryocytes (MKs) derived from human induced pluripotent stem cells (iPSCs).
Example 14: Small-Molecule Loading of Human Washed Platelets by Passive Diffusion
To demonstrate small-molecule loading and retention in a platelet product from human iPSCs, human washed platelets were co-incubated with the DNA intercalating chemotherapeutic, Doxorubicin hydrochloride (Sigma # D1515). As an anucleate cell type, platelets do not contain genomic material that would typically sequester this drug within the cell as a result of co-incubation with a platelet preparation and entry into the cell by passive diffusion. 10 μM of Doxorubicin was used with a preparation of 1e7 platelets in 1 ml of buffer and incubated at ambient temperature for 30, 120, 240, and 1440 minutes under constant agitation on an orbital shaker in a dialysis cassette (Thermofisher #88400). Doxorubicin has intrinsic fluorescent properties that can be detected by flow cytometry (Ex 427 nm/Em 585 nm), and it was found that multiple wash steps could be performed on the platelet preparation and the drug cargo would still be retained after loss of non-specifically bound molecules (
Example 15: Generating Platelets from Genetically Modified Premegakaryocytes
Platelets expressing a therapeutic transgene would represent a significant advancement in treating injuries, illness, and disease. To generate platelets that express a transgene, premegakaryocytes were transduced with a lentiviral vector comprising nucleic acid cassette encoding a reporter protein. Specifically, the cassette encoded an EF1alpha promoter and a ZsGreen fluorescent protein. 42 hours post infection with the lentiviral vector, fluorescence was detected in premegakaryocytes transduced but not in the untransduced (mock) controls (
The premegakaryocytes carrying the transgene were cultured according to the methods described herein to produce platelets. Referring to
Example 16: Manufacturing Platelets Using a Bioreactor System
Platelets were produced using a bioreactor and conditions suitable for loading with therapeutic agents or genetic modification. To produce the platelets, megakaryocytes were seeded into a platelet bioreactor (
Megakaryocytes immobilized on the porous membrane extend processes into the second chamber from which the megakaryocytes release platelets (
Example 18: Generating Platelets from Genetically Modified iPSCs
iPSCs, including the iPSCs described herein, were genetically modified using replication incompetent lentivirus (as outlined in
Example 19: Comparison of PLT Surface Expression of Platelet Specific Markers and Atypical Signaling Pathways Compared to Donor Derived Platelets
PLT and donor derived platelets were compared. Flow analysis of cell surface markers indicated a unique surface profile with several characteristic markers of platelets.
Furthermore, analysis of activation PLT product compared with adult donor platelets demonstrated different biological responses to agonists.
Example 20: PLT Composition and Function Deviate from Donor Platelets
Analyses of the size and proportion of different particles within the PLT product using several approaches indicated the presence of a large population of microparticles.
The results reported in Examples 19 and 20 above were obtained using the following materials and methods:
Donor Platelet Preparation
Whole blood was collected and centrifuged at 150×g for 17 minutes with no brake. Approximately 2 mL of Platelet Rich Platelet (PRP) was then removed and placed in a 15-mL conical tube. The PRP was diluted with a 1:2 dilution of Platelet Wash Buffer and 1:10000 dilution of PGE1 (prostaglandin El). PRP was centrifuged at 460×g for 17 minutes with the brake set to 3. The cells were resuspended in 1 mL of platelet additive solution (PAS) and supplemented with a 1:2 dilution of Platelet Wash Buffer/1:10000 dilution of PGE1. The sample was centrifuged at 460×g for 17 minutes with the brake set to 3. Cells were resuspended in PAS and concentration calculated according to readings taken from MACSQuant Analyzer 10 (Miltenyi).
Bioreactor Product Concentration and Media Exchange
Platelets were collected from the bioreactor in media and a 1:10000 dilution of 1 mg/mL PGE1 was added immediately prior to concentration. Platelets centrifuged at 1250×g for 30 minutes with brake set to 3. The pellet was resuspended in 1 mL PAS. Centrifugation was repeated as a wash step, again adding a dilution of 1:10000 1 mg/mL PGE1 immediately prior to the spin. Cells were resuspended and allowed to rest at 37° C. until further processing.
Staining of Platelets (DRAQ5, Calcein-AM, CD61, CD42a, CD42b, CD36, CD62p)
Platelets/PLT were counted using flow cytometry by staining for DRAQ5 nuclear stain (Cell Signaling Technologies #4084L) and CD61 (VioBlue, Miltenyi #130-110-754), a known platelet-specific surface marker. Platelets were designated as any event that was DRAQ5-negative and CD61-positive. Platelets were also stained with additional markers to further assess cell integrity including Calcein-AM (BioLegend #425201), CD42a (PE, Miltenyi #130-100-966), CD42b (PE, Miltenyi #130-100-199), and CD36 (PE, Miltenyi #130-095-472). All samples were acquired on a MACSQuant Analyzer 10 (Miltenyi) and analyzed using FlowJo Software. Gating strategy was based on forward- and side-scatter parameters as determined by appropriate IgG controls.
Activation of Samples by TRAP6 and Thrombin
Platelets were assessed by flow cytometry for their ability to activate in response to two agonists, TRAP6 (Bachem #4031274) and thrombin (Enzyme Research Laboratories, # HT1002a). Platelet samples were incubated with an agonist for 10 minutes at 37° C. Additional platelet samples were incubated under the same conditions without agonist to determine baseline activation. Samples were then stained for DRAQ5 and CD61 as described above. Platelets were also stained for CD62p (PE-Vio770, Miltenyi #130-120-725), a standard marker of activation, for 15 minutes. Activation was quantified by the change in CD62p expression of DRAQ5-negative, CD61-positive events between baseline and agonist induced conditions. All samples were acquired on a MACSQuant Analyzer 10 (Miltenyi) and analyzed using FlowJo Software. Gating strategy was based on forward- and side-scatter parameters as determined by appropriate IgG controls.
Multisizer
Particle size measurements were performed using the Beckman Multisizer 4 according to manufacturing protocols using the 0-12 μm aperture. Prior to measurements, samples were filtered using a 15 μm filter to prevent clogging. Measurements were performed using the isoton II buffer.
Electron Microscopy
Platelets were placed into 2.5% formaldehyde glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4 and pelleted at 1500×g for 15 minutes. Supernatant was removed and replaced with 1 mL 0.1M sodium cacodylate. Samples were stored at 4° C. until further processing by the Harvard Medical School Electron Microscopy Core (Boston, Mass.). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Tecnai G2 Spirit BioTWIN transmission electron microscope at an accelerating voltage of 80 kV. Images were recorded with an AMT 2 k CCD camera and accompanying proprietary in-house software.
Thrombin Generation Assay (TGA)
Platelet/PLT concentration was adjusted to a density that is approximately ten times the density required for the assay. Microparticles were removed from the George King Pooled Normal Plasma (GK-PNP) control sample by spinning at 20,000×g for 30 minutes in a temperature regulated microcentrifuge. Platelet samples were resuspended in the GK-PNP such that the final concentration was 5×107 platelets/mL. The TGA reagents, Reagent C Low and the TGA Substrate were prepared according to the manufacturer's instructions. The plate reader was set up as directed, ensuring a temperature of 37° C. Samples and controls were measured in duplicate. Thrombin generation is quantified by the enzymatic conversion of a thrombin substrate to a fluorogenic molecule that can be detected by standard methods.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. In particular, various variations useful in the methods and compositions of the present disclosure are described in commonly-owned PCT/US2019/012437, filed on Jan. 5, 2019, as well as U.S. Pat. Nos. 9,763,984, 9,795,965; US2017/0183616; US2018/0334652; WO2018165308, all of which are incorporated herein by reference in their entireties.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.