EP4598549A2 - Verfahren und zusammensetzungen zur t-zell-differenzierung - Google Patents

Verfahren und zusammensetzungen zur t-zell-differenzierung

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Publication number
EP4598549A2
EP4598549A2 EP23875402.2A EP23875402A EP4598549A2 EP 4598549 A2 EP4598549 A2 EP 4598549A2 EP 23875402 A EP23875402 A EP 23875402A EP 4598549 A2 EP4598549 A2 EP 4598549A2
Authority
EP
European Patent Office
Prior art keywords
cells
population
cell
differentiation
hemogenic endothelium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23875402.2A
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English (en)
French (fr)
Inventor
George Q. Daley
Rubul MOUT
Ran JING
David Baker
Stephen Blacklow
Emily D. EGAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Washington
Boston Childrens Hospital
Harvard University
Original Assignee
University of Washington
Boston Childrens Hospital
Harvard University
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Publication date
Application filed by University of Washington, Boston Childrens Hospital, Harvard University filed Critical University of Washington
Publication of EP4598549A2 publication Critical patent/EP4598549A2/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/42—Notch; Delta; Jagged; Serrate

Definitions

  • the technology described herein relates to immune cell differentiation methods.
  • T cells are key components of the human adaptive immune system and have great therapeutic potential.
  • current T cell-mediated therapy relies on autologous T cells, which limits its broader application.
  • Human induced pluripotent stem cells (iPSCs) represent an ideal source for scalable manufacture of off-the-shelf products for cell therapy.
  • iPSCs Human induced pluripotent stem cells
  • the generation of mature and functional T cells from iPSCs has proven to be difficult.
  • the differentiation of iPSC routinely requires co-culture with mouse stromal cells, which limits the translational potential of iPSC-derived T cells. As such there is a need for high- yield, clinically applicable T cell differentiation methods.
  • Embodiments of the technology described herein include methods of differentiating T cells using a novel formulation of soluble Notch ligand.
  • the method described herein is a stroma-free T cell differentiation method, i.e., a method that does not comprise co-culturing with stromal cells or any other type of supporting cell, using a soluble Notch ligand.
  • Co-culture with stromal cells such as mouse stromal cells limits the translational potential of iPSC-derived T cells; for example, by complicating manufacturing protocols and adding the risk of transplantation rejection due to the presence of stromal cells.
  • T cells differentiated using stromal cells in the past have exhibited an innate-like phenotype (e.g., as measured by TCRgd expression, which is a marker for gamma delta T cells). It is preferred that T cells exhibit an adaptive phenotype, for example characterized by expression of TCR a and p.
  • One aspect provided herein describes a soluble notch ligand oligomer complex comprising at least two Notch ligand monomers and a scaffold.
  • the Notch ligand monomer is selected from the group consisting of Delta-like- 1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged 1 (JAG1), and Jagged 2 (JAG2).
  • the Notch ligand monomer is a DLL4 ligand.
  • the complex comprises a single type of Notch ligand monomer, e.g., the complex comprises only a plurality of DLL4 monomers.
  • the complex comprises at least two types of Notch ligand monomers e.g., the complex comprises both DLL1 and DLL4 monomers.
  • the scaffold is naturally existing scaffold or a synthetic scaffold.
  • the complex is dimeric, trimeric, tetrameric, pentameric, hexameric, octameric, icosahedral or any other higher oligomeric state. In one embodiment of any aspect described herein, the complex is a 60-mer or a 120-mer. In one embodiment of any aspect described herein, the complex is trimeric.
  • the DLL4 ligand monomer comprises a sequence selected from SEQ ID NO: 6-9.
  • composition comprising the any of the soluble notch ligand oligomer complexes described herein.
  • Another aspect provided herein describes a method of producing a soluble Notch ligand oligomer complex, the method comprising contacting a plurality of Notch ligand monomers with a population of scaffolds for a time sufficient to promote formation of a complex.
  • the plurality of Notch ligand monomers further comprises a GS linker.
  • the plurality of Notch ligand monomers is fused to SpyTag via the GS linker.
  • the population of scaffolds further comprises a GGSGGS linker (SEQ ID NO: 49).
  • the population of scaffolds is fused to SpyCatcher via GGSGGS linker (SEQ ID NO: 49).
  • Another aspect provided herein describes a soluble Notch ligand oligomer complex produced from any of the methods described herein. [0019] Another aspect provided herein describes a composition of the soluble Notch ligand oligomer produced from any of the methods described herein.
  • Another aspect provided herein describes a method of activating Notch signaling in a population of cells, the method comprising contacting a population of cells with any of the soluble Notch ligand oligomer complexes or compositions thereof described herein.
  • the population of cells are not adhered to a substrate.
  • the population of cells are adhered to a substrate.
  • the contacting is for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more.
  • the cell is a mammalian cell. In one embodiment of any aspect described herein, the cell is a non-mammalian cell.
  • Another aspect provided herein describes a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; b) inhibiting a histone methyltransferase in the resultant population of CD34+ hemogenic endothelium; and c) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • Another aspect provided herein describes a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; b) inhibiting an epigenetic regulator in the resultant population of CD34+ hemogenic endothelium; and c) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • Another aspect provided herein describes a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; b) inhibiting G9a and/or GLP in the resultant population of CD34+ hemogenic endothelium; and c) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • Another aspect provided herein describes a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and b) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell-differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not comprise co-culturing with a stromal cell expressing a Notch ligand.
  • differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not comprise co-culturing with OP9-DL1 cells or OP9-DL4 cells.
  • the sufficient time to promote differentiation into a population of CD3+ T cells is at least 4 weeks.
  • the CD3+-T-ce 11 -differentiation media is serum -free.
  • the CD3+-T-ce 11 -differentiation media comprises 15 ng/ml FLT3 and 25 ng/ml IL7.
  • the CD3+-T-ce 11 -differentiation media further comprises 5 ng/mL thrombopoietin (TPO) and/or 30 ng/ml SCF for at least the first 2 weeks of differentiating in the CD3+-T-cell-differentiation media.
  • TPO thrombopoietin
  • CD3+-T-cell-differentiation media comprising TPO promotes differentiation into a population of CD5+ CD7+ ProT cells.
  • the method further comprising differentiating the population of CD4+CD8+ T cells in a single-positive-T-cell-differentiation media for a sufficient time to promote differentiation into a population of CD4+ cells and a population of CD8+ cells.
  • the T cell activator comprises a lOul/ml CD3/CD28 T cell activator.
  • the induced pluripotent stem cells are produced by introducing only reprogramming factors OCT4, SOX2, KLF4 and optionally c-MYC or nanog and LIN28 into mature cells.
  • the sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium is at least 8 days.
  • the method further comprises selecting or isolating the resultant population of CD34+ hemogenic endothelium using expression of surface markers on the population of CD34+ hemogenic endothelium.
  • the population of CD34+ hemogenic endothelium is CD45 negative/low. In one embodiment of any aspect described herein, the population of CD34+ hemogenic endothelium is CD38 negative/low. [0054] In one embodiment of any aspect described herein, the method further comprises the step of genetically modifying the resultant population of CD34+ hemogenic endothelium or the resultant population of CD3+ T cells.
  • the genetic modification is editing an endogenous HLA, removing an endogenous TCR, and/or expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the histone methyltransferase catalyzes the addition of methyl group to the histone 3 lysine residue 9 (H3K9) and/or histone 3 lysine residue 27 (H3K27).
  • the histone methyltransferase H3K9 and/or H3K27 is inhibited by a small molecule inhibitor or a nucleic acid inhibitor.
  • the histone methyltransferase H3K9 and/or H3K27 small molecule inhibitor is a heterorganic compound or an organometallic compound.
  • the histone methyltransferase H3K9 and/or H3K27 small molecule inhibitor is selected from the group consisting of BIX-01294, UNC0638, E72, BRD4770, A-366, chaetocin, UNC0224, UNC0631, UNC0646, EPZ005687, EPZ- 6438 (E7438), 3-deazaneplanocin A (DZNep), Ell, GSK343, GSK126, and UNC1999.
  • the nucleic acid inhibitor is a nucleic acid targeting the expression of histone methyltransferase.
  • the nucleic acid inhibitor is a RNA interference inhibitor or agent.
  • the nucleic acid inhibitor is a EZH1 specific nucleic acid that is selected from the group consisting of an aptamer that binds EZH1, a EZH1 specific RNA interference agent, and a vector encoding a EZH1 specific RNA interference agent, wherein the RNA interference agent comprises one or more of the nucleotide sequences selected from SEQ ID NO: 11-19.
  • the epigenetic regulator is a DNA- methyltransferase (DNMT); a methyl-CpG-binding domain (MBD) protein; a DNA demethylase; a histone methyl transferase (HMT); a methyl-histone binding protein; a histone demethylase; a histone acetyl transferase (HAT); an acetyl -binding protein; or a histone deacetylase (HDAC).
  • DNMT DNA- methyltransferase
  • HMT histone methyl transferase
  • HAT histone acetyl transferase
  • HDAC histone deacetylase
  • the inhibitor of an epigenetic regulator is selected from the group consisting of: UNC0224; MC1568; and CAY10591.
  • the inhibitor of an epigenetic regulator is provided at a concentration of at least 500 nM.
  • the sufficient time to promote differentiation from the population of CD34+ cells into a population of CD5+CD7+ proT cells is about 14 days.
  • the G9a and/or GLP inhibitor is selected from the group consisting of: UNC0224; UNC0638; A366; BRD4770; B 1X01294; UNC0642; UNC063I; UNC0646; UNC032I; E72; BIX-01338; BRD9539; Chaetocin; and DCG066.
  • the G9a and/or GLP inhibitor is UNC0224.
  • the G9a and/or GLP inhibitor is provided at a concentration of 300 nM - 5uM.
  • the sufficient time to promote differentiation from the population of CD34+ cells into a population of CD5+CD7+ proT cells is about 14 days.
  • Another aspect provided herein describes a describing a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and b) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell-differentiation media comprising 15 ng/ml LLT3 and 25 ng/ml IL7 in the presence of a soluble Notch ligand for at least 4 weeks to promote differentiation into a population of CD3+ T cells; wherein the CD3+-T-cell- differentiation media further comprises 5 ng/mL TPO and 30 ng/ml SCL for at least the first two weeks.
  • Another aspect provided herein describes a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and b) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell-differentiation media comprising 15 ng/ml LLT3 and 25 ng/ml IL7 in the presence of a soluble Notch ligand for at least 4 weeks to promote differentiation into a population of CD3+ T cells; wherein the CD3+-T-cell- differentiation media further comprises 5 ng/mL TPO, 30 ng/ml SCL, and a G9a/GLP inhibitor for at least the first two weeks.
  • the population of CD3+ T cells exhibits a gene expression profile that is most similar to alpha beta T cells. In one embodiment of any aspect described herein, the population of CD3+ T cells exhibits a gene expression profile that is at least 10%, 20%, 30%, 40% or more similar to alpha beta T cells.
  • Another aspect provided herein describes an immune cell produced by any of the methods described herein.
  • the immune cell exhibits a Productive Simpson Clonality value of about 0.025.
  • composition comprising any of the immune cell produced by methods described herein or population thereof.
  • Another aspect provided herein describes a method of cellular replacement therapy, the method comprising administering an immune cell produced by any method described herein, or a population thereof, or any composition or pharmaceutical composition described herein to a recipient subject in need thereof.
  • the recipient subject has undergone chemotherapy and/or irradiation.
  • Notch expression in the cell increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or greater as compared a suitable control.
  • Another aspect provided herein describes a method comprising a) inhibiting a histone methyltransferase in a population of CD34 + hemogenic endothelium; and b) differentiating the population of CD34 + hemogenic endothelium in Natural Killer (NK)-ce 11 -differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • NK Natural Killer
  • FIG. 1A Engineering Notch ligand Delta-like ligand 4 (DLL4) protein complex: DLL4 is genetically fused with SpyTag (ST) peptide, while the oligomeric protein complexes are genetically fused to a SpyCatcher (SC) protein. SpyTag and SpyCatcher form covalent conjugation upon mixing, and hence oligomeric DLL4 protein complexes can be generated by simple mixing of purified DLL4-ST with oligomer-SC. The resultant protein complexes are oligomeric DLL4.
  • Fig. 1A Engineering Notch ligand Delta-like ligand 4 (DLL4) protein complex: DLL4 is genetically fused with SpyTag (ST) peptide, while the oligomeric protein complexes are genetically fused to a SpyCatcher (SC) protein. SpyTag and SpyCatcher form covalent conjugation upon mixing, and hence oligomeric DLL4 protein complexes can be generated by simple mixing of purified DLL4-ST with
  • IB Cartoon showing cellcell interaction facilitated by soluble oligomer-DLL4, which brings adjacent cells together and triggers a Notch synapse formation to activate the downstream signaling. This results in the efficient production of T cells from human induced pluripotent stem cells (iPSC). The red arrows show the direction of signaling.
  • Fig. 1C PDB models showing all the oligomeric protein complexes (scaffold) used forthis study. C3: trimeric; C5: pentameric; C6: hexameric; C8: octameric; Icos: icosahedral which could be either 60-mer or 120-mer; DLL4: monovalent.
  • Figure 2 presents a bar graph showing soluble DLL4 ligand-mediated activation of Notch signaling in a reporter cell line (U20S-Notchl-Gal4).
  • Activation of Notch was measured through firefly luciferase activity upon treating the U2OS-Notchl-Gal4 cells with different soluble oligomer DLL4 protein complexes at varied concentrations.
  • the trimeric complex C3-DLL4 activated Notch signaling to a degree comparable to plate-bound DLL4 (2D-DLL4) activation, with an inverse dose-dependence suggesting precise tuning of the signal.
  • Stromal control is the coculture of U2OS-Notchl-Gal4 with U2OS-DLL4 cells.
  • FIG. 3A Schematic showing in vitro T cell differentiation experiment.
  • Cord Blood (CB)-CD34+ HSPCs were differentiated into T cell progenitors (proT) cells and seeded in 96-well plates for suspension culture. The proTs were then treated with different types of soluble DLL4 ligands and induced to differentiate into CD4/CD8 double positive (DP) T cells.
  • Fig. 3B Screening of soluble DLL4 protein complexes indicates that C3-DLL4 activates Notch signaling to efficiently produce DP T cells from proT cells in a concentration-dependent manner. All other constructs failed to produce DP T cells.
  • Fig. 3C Flow cytometry result showing the production of DP T cells using engineered soluble C3- DLL4 ligands, while free DLL4 ligands alone failed to support proT differentiation.
  • Figures 4A-4F show cell-cell interaction, induced by the C3-DLL4 complex, leads to the formation of cellular clusters.
  • Fig. 4A Soluble C3-DLL4 complex at various concentrations was added to K562 cells that express engineered Notch receptor (K562-Nl-Gal4 reporter line) and the cell interaction was monitored. After 24h of incubation, the cellular cluster was observed in a concentration-dependent manner, the highest clustering formed at a C3-DLL4 concentration of 10 nM.
  • Fig. 4B Cells grown without soluble DLL4 complex, or Fig. 4C) grown in 2D-DLL4 coated plates showing no cluster formation.
  • D-F Underlying mechanism of concentration-dependent cluster formation.
  • Fig. 4A Soluble C3-DLL4 complex at various concentrations was added to K562 cells that express engineered Notch receptor (K562-Nl-Gal4 reporter line) and the cell interaction was monitored. After 24h of incubation, the cellular
  • Figure 5A-5C show confocal microscopy investigation reveals Notch synapse formation induced by C3-DDL4 mediated cell-cell interaction.
  • the expression of Notchl in K562- Nl-Gal4 cells was induced by doxycycline (Dox) and subsequently, cells were treated with C3-DLL4 at 10 nM concentration for either Ih or 24h. Cells were then fixed and stained for C3-DLL4 (Anti-His tag) and Notchl-ECD (Anti-Flag tag) and imaged by confocal microscopy.
  • Fig. 5A Dox-treated cells exhibiting Notchl expression (middle panel) but no C3-DLL4.
  • Figure 6A-6F show endogenous Notch activation by C3-DLL4.
  • Figure 6A-6D MDA- MB-231 cells.
  • Figure 6A Upregulation of Notch targeting genes (Hesl, Heyl, Hey2, HeyL, and NRARP) in MDA-MB-231 cells, measured by RT-qPCR.
  • Figure 6B Western blotting shows the time-dependent Notchl intracellular domain (N1ICD) cleavage in MDA-MB-231 cells upon C3- DLL4 mediated activation.
  • Figure 6C Confocal microscopy showing Notch synapse formation in MDA-MB-231 cells mediated by C3-DLL4.
  • Figure 6D-6F SVG-A cells.
  • Notch signaling is indispensable for the development of long-term, definitive Hematopoietic Stem and Progenitor Cells (HSPCs) and T-lymphocytes (T-cells). Soluble Notch ligands
  • haematopoietic stem cells (HSCs) in the bone marrow give rise to multipotent progenitors (MPPs) before differentiating into common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs).
  • CLPs migrate from the bone marrow to the thymus, where thymic epithelial cells that express Delta-like ligand 4 (DLL4) trigger canonical Notch 1 signaling in early thymic progenitors (ETPs). This Notch 1 signal is essential for T cell lineage commitment and is further required during early phases of thymocyte differentiation up to the double -negative 3 (DN3) stage.
  • DLL4 Delta-like ligand 4
  • Notch signaling is also involved in cellular processes throughout adulthood. Signaling via Notch occurs between neighboring cells and both the receptor and its ligands are transmembrane proteins. See, e.g., Schmitt T.M., Zuniga-Pflticker J.C. (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17:749-756; Mohtashami M. (2010) Direct Comparison of Dill - and D114-Mediated Notch Activation Levels Shows Differential Lymphomyeloid Lineage Commitment Outcomes.
  • Notch ligands are single-pass transmembrane proteins with a DSL (Delta, Serrate, LAG- 2)-domain and varying numbers of EGE-like repeats.
  • DSL Delta, Serrate, LAG- 2
  • canonical Notch ligands There are two classes of canonical Notch ligands, the Delta/Delta-like and the Serrate/Jagged class. The later has an additional domain of cysteine rich repeats close to the transmembrane domain.
  • DLL1, also known as Notch Delta ligand, Delta-like 1 is a protein which interacts with a NOTCH2 receptor.
  • DLL1 is a protein that in humans is encoded by the DLL1 gene.
  • DLL1 is a human homolog of the Notch Delta ligand.
  • soluble notch ligand oligomer complexes comprising at least Notch ligand monomers and a scaffold.
  • Notch activation requires mechanical pulling of the DLL4 ligand by the Notch receptors, and therefore for signaling to occur, the DLL4 ligand needs to be tethered either to a cell surface or in a plate-bound format (see, e.g., Siebel, C. & Lendahl, U.
  • a cell for example, a stroma cell
  • the oligomer complexes comprise a Notch ligand selected from the group consisting of Delta-like- 1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged 1
  • DLL1 Delta-like- 1
  • DLL3 Delta-like-3
  • DLL4 Delta-like-4
  • the Notch ligand is DLL4.
  • the nucleic acid sequence of the Notch ligand is DLL4 and comprises SEQ ID NO: 6-9 or a sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 6-9, and that maintains the same functions as SEQ ID NO: 6-9 (e.g., binding and/or activating a Notch receptor).
  • the amino acid sequence of the Notch ligand is DLL4 and comprises SEQ ID NO: 4 or an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 4, and that maintains the same functions as SEQ ID NO: 4 (e.g., binding and/or activating a Notch receptor).
  • SEQ ID NO: 9 delta-like protein 4 precursor [Homo sapiens], NCBI Reference Sequence: NP_061947.1, 685 amino acids MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVC LKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHA PGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRL CKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQG RLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSY TCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSP CF
  • the extracellular domain of human DLL4 comprises SEQ ID NO: 10 or an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 5, and that maintains the same functions as SEQ ID NO: 10 (e.g., binding and/or activating a Notch receptor).
  • SEQ ID NO: 10 or an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 5, and that maintains the same functions as SEQ ID NO: 10 (e.g., binding and/or activating a Notch receptor).
  • the nucleic acid sequence of the Notch ligand is DLL1 and comprises SEQ ID NO: 1-3 or a sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the sequence of SEQ ID NO: 1-3 that maintains the same functions as SEQ ID NO: 1-3 (e.g., binding and/or activating a Notch receptor).
  • the amino acid sequence of the Notch ligand is DLL1 and comprises SEQ ID NO: 4 or an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 4 that maintains the same functions as SEQ ID NO: 4 (e.g., binding and/or activating a Notch receptor).
  • SEQ ID NO: 4 delta-like protein 1 precursor [Homo sapiens], NCBI Reference Sequence: NP_005609.3, 723 aa MGSRCALALAVLSALLCQVWSSGVFELKLQEFVNKKGLLGNRNCCRGGAGPPPCACRTFFR VCLKHYQASVSPEPPCTYGSAVTPVLGVDSFSLPDGGGADSAFSNPIRFPFGFTWPGTFSLIIE ALHTDSPDDLATENPERLISRLATQRHLTVGEEWSQDLHSSGRTDLKYSYRFVCDEHYYGEG CSVFCRPRDDAFGHFTCGERGEKVCNPGWKGPYCTEPICLPGCDEQHGFCDKPGECKCRVG WQGRYCDECIRYPGCLHGTCQQPWQCNCQEGWGGLFCNQDLNYCTHHKPCKNGATCTNT GQGSYTCSCRPGYTGATCELGIDECDPSPCKNGGSCTDLENSYSCTCPPGFYGKICELSAMTC ADGPCFNGGR
  • the Notch ligand is Deltalext-IgG and comprises the extracellular domain of human DLL1, which corresponds to approximately amino acids 1-536, or amino acids 22- 544, or amino acids 22-537 of DLL1 (see, e.g., SEQ ID NO: 4 for full-length sequence of DLL1).
  • the extracellular domain of human DLL1 comprises SEQ ID NO: 5, or an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 5, and that maintains the same functions as SEQ ID NO: 5 (e.g., binding and/or activating a Notch receptor).
  • SEQ ID NO: 5 or an amino acid sequence that is at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 5, and that maintains the same functions as SEQ ID NO: 5 (e.g., binding and/or activating a Notch receptor).
  • a Notch ligand for example by providing a purified recombinant form of a Notch ligand or a Notch receptor-binding fragment, the receptor-binding fragment being sufficient to elicit cell signaling events in vivo upon contact and binding with the extracellular Notch receptors on these cells.
  • the Notch ligand is not attached to a solid substrate, for example using a covalent or non-covalent bond or linkage.
  • the Notch ligand is not attached to a cell culture dish.
  • the oligomeric complex is a homo-oligomeric complex. Said another way, the oligomeric complex comprises a single type of Notch monomer. For example, the oligomeric complex comprises only DLL4 monomers.
  • the oligomeric complex is a hetero-oligomeric complex. Said another way, the oligomeric complex comprises at least two types of Notch monomer. For example, the oligomeric complex comprises both DLL1 and DLL4 monomers.
  • the oligomeric complex exists in a dimeric, trimeric, tetrameric, pentameric, hexameric, octameric, icosahedral or any other higher oligomeric state.
  • the oligomeric complex is a 60-mer or a 120-mer.
  • the oligomeric complex comprises a scaffold that binds the ligands of the complex.
  • a scaffold used herein can be a synthetic scaffold.
  • synthetic scaffolds that can be used include dimer (e.g., de novo dimer C2_HD-1092), trimer (e.g., de novo trimer C3_HD-1069), tetramer (e.g., de novo tetramer C4_nat_HF-7900), and pentamer (e.g., de novo pentamer C5_HF-2101).
  • a scaffold used herein can be a naturally occurring scaffold.
  • naturally occurring scaffolds that can be used include any naturally existing protein: a dimer (e.g., IgG Fc fragment), a trimer (e.g., 2-Dehydro-3-deoxy-phosphogluconate aldolase), and a pentamer (e.g. lumazine synthase).
  • Uigand monomers can be immobilized to a scaffold using methods known in the art.
  • the Notch ligand further comprises a domain to immobilize the Notch ligand to the scaffold.
  • the Notch ligand monomer comprises a first member of an affinity pair
  • the scaffold comprises a second member of an affinity pair.
  • the first and second members of the affinity pair are selected from the group consisting of: IgG and protein A; biotin and avidin; biotin and streptavidin; SpyTag and SpyCatcher; a haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., FLAG and anti -FLAG monoclonal antibody, the sequence of which are known in the art); digoxigenin and anti-digoxigenin; mouse immunoglobulin and goat anti -mouse immunoglobulin; a non-immunological binding pair; a hormone and a hormone -binding protein; thyroxine and cortisolhormone binding protein; a receptor and a receptor agonist; a receptor and a receptor antagonist; acetylcholine receptor and acetylcholine or an analog thereof; lectin and carbohydrate; an enzyme and an enzyme cofactor; an enzyme and an enzyme inhibitor; complementary oligonucleotide pairs capable of forming nucleid
  • binding of the affinity pair is not reversible.
  • binding of the affinity pair is reversible.
  • the Notch ligand monomer is immobilized on the scaffold via a synthetic heterodimer, e.g., LHD101A/B, or a coil-coil heterodimer.
  • the affinity pair used to immobilize the monomer on the scaffold is SpyTag and SpyCatcher.
  • the plurality of Notch ligand monomers further comprises a GS linker.
  • SpyTag is fused to the plurality of Notch ligand monomers via the GS linker.
  • population of scaffolds further comprises a GGSGGS linker (SEQ ID NO: 49).
  • population of scaffolds is fused to SpyCatcher via GGSGGS linker (SEQ ID NO: 49).
  • composition of any oligomeric complex descibred herein.
  • Another aspect provided herein is a method producing a Notch ligand oligomeric complex descibred herein, the method comprising contacting a plurality of Notch ligand monomers with a population of scaffolds for a time sufficient to promote formation of a complex.
  • the time sufficient to promote formation of a complex is at least 1 hour, at least 2 hours; at least 3 hours; at least 4 hours; at least 5 hours; at least 6 hours; at least 7 hours; at least 8 hours; at least 9 hours; at least 10 hours; at least 11 hours; at least 12 hours; at least 13 hours; at least 14 hours; at least 15 hours; at least 16 hours; at least 17 hours; at least 18 hours; at least 19 hours; at least 20 hours; at least 21 hours; at least 22 hours; at least 23 hours; at least 24 hours; or longer.
  • composition of any oligomeric complex produced via methods described herein.
  • Soluble Notch ligand complexes and compositions thereof described herein can be used in place of a bound-Notch ligand (e.g., a ligand bound to a substrate) in all applications in which a bound-Notch ligand is used. Exemplary applications are described herein, e.g., for activating Notch signaling, differentiating T cells, and differentiating natural killer (NK) cells. Further, it is fully contemplated herein that Soluble Notch ligand complexes and compositions thereof described herein can be used in applications not described herein known to utilize a bound-Notch ligand.
  • a bound-Notch ligand e.g., a ligand bound to a substrate
  • a method for activating Notch signaling in cells comprising contacting a cell with a soluble Notch oligomer complex described herein for a time sufficient to induce Notch activation.
  • a skilled person can determine if Notch has been activated using standard methods in the art, e.g., methods described herein in the Example.
  • the time sufficient to activate Notch signaling is at least one hour.
  • the time sufficient to activate Notch signaling is no more than 24 hours.
  • the time sufficient to activate Notch signaling is at least 1 hour, at least
  • the cell is a human cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell can be derived from internal organs, skin, bones, blood, and connective tissue of a human.
  • the cell can be a fibroblast, keratinocyte, a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammary cell, a hepatocyte and a pancreatic islet cell.
  • the cell can be an epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, skin, immune cells (e.g., B lymphocytes (B-cells), T lymphocytes (T-cells), hepatic, splenic, lung, peripheral circulating blood cells, gastrointestinal, renal, bone marrow, or pancreatic cells.
  • B-cells B lymphocytes
  • T-cells T lymphocytes
  • lung peripheral circulating blood cells
  • peripheral circulating blood cells gastrointestinal, renal, bone marrow, or pancreatic cells.
  • the cell is a non-human cell.
  • a non-human cell includes any cell derived from a murine, bovine, simian, porcine, equine, or ovine.
  • the cell being contacting is adhered to a substrate, e.g., a solid substrate or cell.
  • the soluble Notch ligand is provided at a concentration of 1 nM to
  • the soluble Notch ligand is provided at a concentration of at least 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM or greater.
  • the soluble Notch ligand is provided at a concentration of at least 0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or greater.
  • the soluble Notch ligand is provided at a concentration of at least 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM or greater.
  • the soluble Notch ligand is provided at a concentration of 10 nM.
  • the levels of Notch activation are at least 10% higher as compared to an appropriate control.
  • an “appropriate control” is an otherwise identical population that is not contacted with the soluble Notch oligomer complexes, or is contacted for a shorter duration, or is contacted with a decreased amount of Notch oligomer complexes.
  • a skilled can determine the level of Notch activation achieved following contacting using standard methods, e.g., methods descibred herein in the Example.
  • the levels of Notch activation are at least 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
  • a method comprising a) inhibiting a histone methyltransferase in a population of CD34 + hemogenic endothelium; and b) differentiating the population of CD34 + hemogenic endothelium in Natural Killer (NK)-ce 11 -differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • NK Natural Killer
  • Also provided herein is a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; b) inhibiting a histone methyltransferase in the resultant population of CD34 + hemogenic endothelium; and c) differentiating the resultant population of CD34 + hemogenic endothelium in Natural Killer (NK)-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • NK Natural Killer
  • Also provided herein is a method comprising a) inhibiting an epigenetic regulator in a population of CD34 + hemogenic endothelium; and b) differentiating the population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • Also provided herein is a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; b) inhibiting an epigenetic regulator in the resultant population of CD34 + hemogenic endothelium; and c) differentiating the resultant population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • Also provided herein is a method comprising a) inhibiting G9a and/or GLP in a population of CD34 + hemogenic endothelium; and b) differentiating the population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • Also provided herein is a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; b) inhibiting G9a and/or GLP in the resultant population of CD34 + hemogenic endothelium; and c) differentiating the resultant population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • Also provided herein is a method comprising differentiating a population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • Also provided herein is a method comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and b) differentiating the resultant population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • Differentiation Methods comprising a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and b) differentiating the resultant population of CD34 + hemogenic endothelium in NK-cell-differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in an aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; and (b) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • the method further comprises inhibiting a histone methyltransferase in the resultant population of CD34+ hemogenic endothelium.
  • Such an inhibition can increase the efficiency of differentiation into T cells.
  • described herein is a method comprising: (a) differentiating a population of pluripotent stem cells in an aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; (b) inhibiting a histone methyltransferase in the resultant population of CD34+ hemogenic endothelium; and (c) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • the CD34 + hemogenic endothelium population is cultured into a CD3 + -T-cell-differentiation media comprising 100 ng/ml SCF, 100 ng/ml FLT3, and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • a CD3 + -T-cell-differentiation media comprising 100 ng/ml SCF, 100 ng/ml FLT3, and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • the CD34 + hemogenic endothelium population is cultured into a CD3 + -T-cell-differentiation media comprising 100 ng/ml FLT3 and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • a CD3 + -T-cell-differentiation media comprising 100 ng/ml FLT3 and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • the CD34 + hemogenic endothelium population is cultured into a CD3 + -T-cell-differentiation media comprising 30 ng/ml SCF, 15 ng/ml FLT3, and 25 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • a CD3 + -T-cell-differentiation media comprising 30 ng/ml SCF, 15 ng/ml FLT3, and 25 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • the CD34 + hemogenic endothelium population is cultured into a CD3 + -T-cell-differentiation media comprising 15 ng/ml FLT3 and 25 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • a CD3 + -T-cell-differentiation media comprising 15 ng/ml FLT3 and 25 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and (b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-ce 11 -differentiation media comprising, 15 ng/ml FLT3 and 25 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell- differentiation media further comprises 5 ng/mL TPO and 30 ng/ml SCF for at least the first two weeks.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and (b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-cell-differentiation media comprising, 15 ng/ml FLT3 and 25 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises 5 ng/mL TPO, 30 ng/ml SCF, and a G9a inhibitor for at least the first two weeks.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and (b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-cell-differentiation media comprising 100 ng/ml SCF, 100 ng/ml FLT3, and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises TPO (50 ng/mL) for at least the first two weeks.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and (b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-cell-differentiation media comprising 100 ng/ml SCF, 100 ng/ml FLT3, and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises TPO (50 ng/mL) and a G9a/GLP inhibitor for at least the first two weeks.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and (b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-cell-differentiation media comprising, 100 ng/ml FLT3 and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises 50 ng/mL TPO and 100 ng/ml SCF for at least the first two weeks.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and (b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-cell-differentiation media comprising, 100 ng/ml FLT3 and 50 ng/ml IL7 in the presence of soluble Notch ligand, e.g., at a lOnM concentration, for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises 50 ng/mL TPO, 100 ng/ml SCF, and a G9a inhibitor for at least the first two weeks.
  • the stroma-free T cell differentiation method descibred herein comprises differentiating a population of pluripotent stem cells.
  • Pluripotent stem cells have the potential to give rise to all the somatic tissues.
  • the population of pluripotent stem cells is induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESC).
  • iPSCs induced pluripotent stem cells
  • ESC embryonic stem cells
  • IPSC and ESC can be produced by any method known in the art.
  • the population of pluripotent stem cells comprises embryonic stem cells (ESC).
  • Embryonic stem cells (ESCs) are stem cells derived from the undifferentiated inner mass cells of a human embryo.
  • Directed differentiation of PSCs aims to recapitulate embryonic development to generate patient-matched tissues by specifying the three germ layers.
  • a common theme in directed differentiation across all germ layers is the propensity of PSCs to give rise to embryonic- and fetal- like cell types, which poses a problem for integration and function in an adult recipient. This distinction is particularly striking in the hematopoietic system, which emerges in temporally and spatially separated waves at during ontogeny.
  • the earliest “primitive” progenitors emerge in the yolk sac at 8.5 dpc and give rise to a limited repertoire of macrophages, megakaryocytes and nucleated erythrocytes.
  • HSC hematopoietic stem cell
  • AGM aorta-gonad- mesonephros
  • the population of pluripotent stem cells comprises induced pluripotent stem cells (iPS cells).
  • the induced pluripotent stem cells are produced by introducing only reprogramming factors OCT4, S0X2, KLF4 and optionally c-MYC or nanog and LIN28 into mature cells.
  • the induced pluripotent stem cells are produced by introducing the reprogramming factors two or more times into the mature cells.
  • the pluripotent stem cells (PSCs) described herein are induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • An advantage of using iPSCs is that the cells can be derived from the same subject to which the eventual immune cells would be reintroduced. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then transfected and differentiated into a modified immune cell to be administered to the subject (e.g., autologous cells). Since the progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic responses is reduced compared to the use of cells from another subject or group of subjects.
  • the cells for generating iPSCs are derived from non-autologous sources.
  • the use of iPSCs negates the need for cells obtained from an embryonic source.
  • the PSCs used in the disclosed methods are not embryonic stem cells.
  • reprogramming refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). Stated another way, reprogramming refers to a process of driving the differentiation of a cell backwards to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics.
  • differentiated cells does not render these cells non-differentiated cells (e.g., undifferentiated cells) or pluripotent cells.
  • the transition of a differentiated cell to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character in culture.
  • Reprogrammed cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.
  • the cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming.
  • reprogramming encompasses complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state.
  • reprogramming encompasses complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming.
  • reprogramming of a differentiated cell causes the differentiated cell to assume an undifferentiated state (e.g., is an undifferentiated cell).
  • the resulting cells are referred to as “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs or iPS cells).”
  • Reprogramming can involve alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation.
  • Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent or maintaining the existing less than fully differentiated state of a cell that is already a multipotent cell (e.g., a common myeloid stem cell).
  • Reprogramming is also distinct from promoting the self-renewal or proliferation of cells that are already pluripotent or multipotent, although the compositions and methods described herein can also be of use for such purposes, in some embodiments.
  • reprogramming The specific approach or method used to generate pluripotent stem cells from somatic cells (broadly referred to as “reprogramming”) is not necessarily critical to the methods described. Thus, any method that re-programs a somatic cell to the pluripotent phenotype would be appropriate for use in the methods described herein.
  • mouse iPSCs satisfy all the standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, contribution to chimeras, germline transmission, and tetrapioid complementation.
  • iPS cells can be obtained using similar transduction methods, and the transcription factor trio, OCT4, SOX2, and NANOG, has been established as the core set of transcription factors that govern pluripotency.
  • the production of iPS cells can be achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell, using viral vectors.
  • OCT4, SOX2, KLF4 and c-MYC are the original four transcription factors identified to reprogram mouse fibroblasts into iPSCs. These same four factors were also sufficient to generate human iPSCs.
  • OCT3/4 and SOX2 function as core transcription factors of the pluripotency network by regulating the expression of pluripotency-associated genes.
  • Kriippel-like factor 4 (KLF4) is a downstream target of LIF-STAT3 signaling in mouse ES cells and regulates self-renewal.
  • Human iPSCs can also be generated using four alternative factors; OCT4 and SOX2 are required but KLF4 and c-MYC could be replaced with NANOG, a homeobox protein important for the maintenance of pluripotency in both ES cells and early embryos, and LIN28, an RNA binding protein.
  • OCT4, SOX2, NANOG and LIN28 reprogramming factors have been reported to be also sufficient to generate human iPSCs.
  • the iPSCs are produced, for example, by introducing exogenous copies of only three reprogramming factors OCT4, SOX2, and KLF4 into mature or somatic cells.
  • cells, or composition described herein c-MYC, or nanog and/or LIN28 are further introduced to iPSCs having exogenous gene coding copies of OCT4, SOX2, and KLF4 to differentiate into mature or somatic cells.
  • the iPSCs are produced by introducing exogenous copies of reprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYC or nanog and/or LIN28 to differentiate into mature or somatic cells.
  • the iPSCs are produced by contacting mature cells with at least one vector, wherein the at least one vector carries an exogenous gene coding copy of reprogramming factors OCT4, SOX2, and KLF4, and optionally with c-MYC, or nanog and/or LIN28 to differentiate into mature or somatic cells, and wherein the reprogramming factors are expressed in vivo in the contacted mature or somatic cells.
  • the contacting is in vitro or ex vivo.
  • the reprogramming factors needed for differentiation can all be expressed by one vector (e.g., a vector that carries an exogenous gene coding copy of OCT4, SOX2, KLF4, and c- MYC).
  • the reprogramming factors can be expressed in more than one vector that is each used to contact the iPSCs.
  • an iPSCs can be contacted by a first vector that carries an exogenous gene coding copy of OCT4, SOX2, and a second vector that carries an exogenous gene coding copy KLF4 and c-MYC.
  • the iPS cell comprises at least an exogenous copy of a nucleic acid sequence encoding a reprogramming factor selected from the group consisting of genes Oct4 (Pou5fl), Sox2, cMyc, Klf4, Nanog, Lin 28 and Glisl.
  • a reprogramming factor selected from the group consisting of genes Oct4 (Pou5fl), Sox2, cMyc, Klf4, Nanog, Lin 28 and Glisl.
  • combinations of reprogramming factors are used. For example, a combination of four reprogramming factors consisting of Oct4, Sox2, cMyc, and Klf4, or a combination of four reprogramming factors consisting of Oct4, Sox2, Nanog, and Lin 28.
  • the iPSCs are produced by introducing the disclosed reprogramming factors, or any combination of the reprograming factors two or more times into the mature or somatic cells.
  • the combination of reprograming factors is different when a combination is introduced to the iPSC more than once, for example, the combination of Oct4 (Pou5fl), Sox2, cMyc, Klf4, Nanog is first introduced to the iPSCs, and the combination of Oct4 (Pou5fl), Sox2, cMyc is subsequently introduced to the iPSCs.
  • the iPSCs are produced by contacting mature cells with the disclosed vector(s) factors two or more times into the mature/somatic cells.
  • the population of pluripotent stem cells e.g., iPSCs
  • the population of pluripotent stem cells are not differentiated in the presence of a Notch ligand, e.g., a soluble Notch ligand.
  • the aggregation media used to promote the differentiation of the population of pluripotent stem cells (e.g., iPSCs) into a population of CD34+ hemogenic endothelium does not comprise a Notch ligand, e.g., a soluble Notch ligand.
  • the cell culture vessel used during the differentiation of the population of pluripotent stem cells (e.g., iPSCs) into the population of CD34+ hemogenic endothelium does not comprise a Notch ligand, e.g., a soluble Notch ligand.
  • iPS cells can be generated or derived from terminally differentiated somatic cells, as well as from adult stem cells, or somatic stem cells. That is, a non-pluripotent progenitor cell can be rendered pluripotent or multipotent by reprogramming. In such instances, it may not be necessary to include as many reprogramming factors as required to reprogram a terminally differentiated cell.
  • reprogramming can be induced by the non- viral introduction of reprogramming factors, e.g., by introducing the proteins themselves, or by introducing nucleic acids that encode the reprogramming factors, or by introducing messenger RNAs that upon translation produce the reprogramming factors (see e.g., Warren et al., Cell Stem Cell, 2010 Nov 5;7(5):618-30, this reference is incorporated herein by reference in its entirety).
  • the methods and compositions described herein further comprise introducing one or more of each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming.
  • the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein.
  • the reprogramming is not effected by a method that alters the genome.
  • reprogramming is achieved, e.g., without the use of viral or plasmid vectors.
  • the efficiency of reprogramming i.e., the number of reprogrammed cells derived from a population of starting cells can be enhanced by the addition of various small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3: 132-135, the contents of each of which are incorporated herein by reference in its entirety.
  • an agent or combination of agents that enhance the efficiency or rate of induced pluripotent stem cell production can be used in the production of patient-specific or disease-specific iPSCs.
  • agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5 '-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.
  • reprogramming enhancing agents include: Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC Toxin, Nullscript (4-(l,3-Dioxo-lH,3H- benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pivaloyloxymethyl butyrate (Pivanex, AN- 9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228), SAA, Trichostatin A
  • reprogramming enhancing agents include, for example, dominant negative forms of the HDACs (e.g., catalytically inactive forms), siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs.
  • HDACs e.g., catalytically inactive forms
  • siRNA inhibitors of the HDACs e.g., anti-viral agents
  • antibodies that specifically bind to the HDACs.
  • Such inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.
  • isolated clones can be tested for the expression of a stem cell marker.
  • a stem cell marker can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl.
  • a cell that expresses Oct4 or Nanog is identified as pluripotent.
  • Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, detection does not involve only RT-PCR, but also includes detection of protein markers. Intracellular markers may be best identified via RT-PCR, while cell surface markers are readily identified, e.g., by immunocytochemistry.
  • the pluripotent stem cell character of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate to cells of each of the three germ layers.
  • teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones.
  • the cells are introduced to nude mice and histology and/or immunohistochemistry is performed on a tumor arising from the cells.
  • the growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.
  • Many US Patents and Patent Application Publications teach and describe methods of generating iPSCs and related subject matter.
  • the iPSCs can be derived from somatic cells.
  • Somatic cells as that term is used herein, refer to any cells forming the body of an organism, excluding germline cells.
  • the cells from which they are made is a differentiated somatic cell.
  • the mature cells from which iPS cells are made include any somatic cells such as B lymphocytes (B-cells), T lymphocytes, (T-cells), and fibroblasts and keratinocytes.
  • a fibroblast e.g., a primary fibroblast
  • a muscle cell e.g., a myocyte
  • a cumulus cell a neural cell, a mammary cell, a hepatocyte and a pancreatic islet cell.
  • the somatic cell is a primary cell line or is the progeny of a primary or secondary cell line.
  • the somatic cell is obtained from a human sample, e.g., a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g., an oral swab sample), and is thus a human somatic cell.
  • a human sample e.g., a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g., an oral swab sample), and is thus a human somatic cell.
  • differentiated somatic cells include, but are not limited to, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, skin, immune cells, hepatic, splenic, lung, peripheral circulating blood cells, gastrointestinal, renal, bone marrow, and pancreatic cells.
  • a somatic cell can be a primary cell isolated from any somatic tissue including, but not limited to brain, liver, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc.
  • the somatic cell can be from any mammalian species, with nonlimiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. In some embodiments, the somatic cell is a human somatic cell.
  • somatic cells isolated from the patient being treated.
  • somatic cells involved in diseases, and somatic cells participating in therapeutic treatment of diseases and the like can be used.
  • a method for selecting the reprogrammed cells from a heterogeneous population comprising reprogrammed cells and somatic cells they were derived or generated from can be performed by any known means.
  • a drug resistance gene or the like, such as a selectable marker gene can be used to isolate the reprogrammed cells using the selectable marker as an index.
  • markers can include Dnmt3L; Soxl5; Stat3; Grb2; p-catenin, and Bmil.
  • Such cells can also be characterized by the down-regulation of markers characteristic of the somatic cell from which the induced pluripotent stem cell is derived.
  • the iPSCs are derived from mature, differentiated, somatic cells.
  • the population of pluripotent stem cells used in the differentiation methods described herein does not comprise CD34+ HSPCs or multipotent lymphoid progenitors (MLPs) purified from a patient sample.
  • the population of pluripotent stem cells does not comprise stem cells purified or isolated from cord blood or bone marrow samples.
  • the population of pluripotent stem cells is not derived from stem cells isolated from a patient sample (e.g., cord blood or bone marrow).
  • the population of pluripotent stem cells comprise iPSCs, such as those derived from a somatic cell sample from a patient. See e.g., Tabatabaei-Zavareh et al., J Immunol May 1, 2017, 198 (1 Supplement) 202.9.
  • the methods described herein comprise differentiating a population of pluripotent stem cells (e.g., iPSCs) into a population of cells with hematopoietic potential.
  • the population of cells with hematopoietic potential comprises hemogenic endothelium and/or hematopoietic stem cells (HSCs).
  • the cells with hematopoietic potential e.g., hemogenic endothelium, HSCs
  • One exemplary approach to generate HSCs from hPSCs is to specify HSCs from its ontogenetic precursors.
  • HSCs originate from hemogenic endothelium (HE) in the aorta-gonad-mesonephros (AGM) and arterial endothelium in other anatomical sites.
  • HE hemogenic endothelium
  • AGM aorta-gonad-mesonephros
  • HE hemogenic endothelium
  • AGM aorta-gonad-mesonephros
  • hemogenic endothelium refers to a unique subset of endothelial cells scattered within blood vessels that can differentiate into haematopoietic cells.
  • HSCs arise beginning embryonic day 10.5 from a small population of endothelial cells with hemogenic potential (hemogenic endothelium) located within the aorta-gonad-mesonephros region.
  • EHT endothelial to hematopoietic transition
  • a population of cells comprising the properties of hemogenic endothelium is differentiated in vitro from a population of pluripotent stem cells (e.g., iPSCs).
  • iPSCs pluripotent stem cells
  • Said “cells comprising the properties of hemogenic endothelium” can also be referred to herein as hemogenic endothelium.
  • hematopoiesis consists of two programs, primitive and definitive, but only definitive hematopoiesis generates HSCs and thus the lymphoid lineage.
  • Definitive hematopoiesis as measured by T-lymphoid potential, emerges after the establishment of the primitive hematopoietic program and develops from a progenitor population that displays characteristics of hemogenic endothelium.
  • the T cell differentiation methods described herein comprise differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium.
  • the resultant CD34+ hemogenic endothelium can undergo definitive hematopoiesis and/or exhibits lymphoid potential.
  • the hemogenic endothelium differentiates or is differentiated into hematopoietic stem cells (HSCs).
  • the population of pluripotent stem cells is differentiated into a population of CD34+ hemogenic endothelium using embryoid bodies (EBs) or 2D adherent cultures; see e.g., Pineda et al., Differentiation patterns of embryonic stem cells in two versus three dimensional culture, Cells Tissues Organs. 2013; 197(5): 399-410, which is incorporated herein by reference.
  • EBs are three-dimensional aggregates of pluripotent stem cells produced and cultured in vitro in the presence of serum. The EBs can generate a mixture of primitive and definitive hematopoietic progenitor cell types.
  • the sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium is at least 8 days (e.g., at least 7, at least 8, at least 9, at least 10 days, or more). In some embodiments, the sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium is at most 8 days, at most 9 days, at most 10 days or more.
  • the aggregation media comprises BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO or any combination of the same.
  • the aggregation media comprises 10 ng/ml BMP4, 6 mM SB-431542, 3 mM CHIR99021, 5 ng/ml bFGF, 15 ng/ml VEGF, 10 ng/ml IL-6, 5 ng/ml IL-11, 25 ng/ml IGF-1, 50 ng/ml SCF, and 2 U/ml EPO (see, e.g., Example 2 and Table 1 presented in International Patent No. W02021/150919A1)
  • the components of the aggregation media are varied during the differentiation of pluripotent stem cells into hemogenic endothelium.
  • embryoid bodies are differentiated in the presence of BMP4, followed by stage-specific addition of bFGF, VEGF, and hematopoietic cytokines (e.g., IL-6, IL-11, IGF-1, SCF, and EPO).
  • Activin-nodal signaling can be manipulated (e.g., using SB-431542 and CHIR99021) between days 2 and 3.
  • the aggregation media comprises BMP (e.g., 10 ug/mL BMP) during days 0, 1, and/or 2 of differentiation. In some embodiments, the aggregation media does not comprise BMP during days 3, 4, 5, 6, 7, or 8 of differentiation.
  • BMP e.g. 10 ug/mL BMP
  • the aggregation media comprises SB-431542 (e.g., 6 mM SB- 431542) and/or CHIR99021 (e.g., 3 mM CHIR99021) during day 2 of differentiation.
  • SB-431542 is a small-molecule antagonist of activin-nodal signaling.
  • CHIR99021 is a GSK-3 inhibitor and a Wnt agonist. Inhibition of activin-nodal signaling and activation of Wnt signaling has been shown to drive PSC differentiation into definitive progenitors (KDR + CD235a“) with lymphoid potential (see e.g., Sturgeon 2014, supra, which is incorporated herein by reference).
  • the aggregation media comprises does not SB-431542 and/or CHIR99021 during days 0, 1, 3, 4, 5, 6, 7, and/or 8 of differentiation.
  • the aggregation media comprises bFGF (e.g., 5 ng/ml bFGF) during days 1, 2, 3, 4, 5, 6, 7, and/or 8 of differentiation. In some embodiments, the aggregation media does not comprise bFGF during day 0 of differentiation. [00224] In some embodiments, the aggregation media comprises VEGF (e.g., 15 ng/ml VEGF) during days 3, 4, 5, 6, 7, and/or 8 of differentiation. In some embodiments, the aggregation media does not comprise VEGF during days 0, 1, or 2 of differentiation.
  • bFGF e.g., 5 ng/ml bFGF
  • VEGF e.g., 15 ng/ml VEGF
  • the aggregation media does not comprise VEGF during days 0, 1, or 2 of differentiation.
  • the aggregation media comprises hematopoietic cytokine(s) during days 6, 7, and/or 8 of differentiation. In some embodiments, the aggregation media does not comprise hematopoietic cytokine(s) during days 0, 1, 2, 3, 4, or 5 of differentiation.
  • the hematopoietic cytokines are selected from the group consisting of: IL-6 (e.g., 10 ng/ml IL-6), IL-11 (e.g., 5 ng/ml IL-11), IGF-1 (e.g., 25 ng/ml IGF-1), SCF (e.g., 50 ng/ml SCF), and EPO (e.g., 2 U/ml EPO).
  • IL-6 e.g., 10 ng/ml IL-6
  • IL-11 e.g., 5 ng/ml IL-11
  • IGF-1 e.g., 25 ng/ml IGF-1
  • SCF e.g., 50 ng/ml SCF
  • EPO e.g., 2 U/ml EPO
  • the differentiation method further comprises selecting or isolating the resultant population of CD34+ hemogenic endothelium using expression of surface markers on the population of CD34+ hemogenic endothelium.
  • Non-limiting examples of methods for selecting or isolating hemogenic endothelium include magnetic -activated cell sorting (MACS) and fluorescence- activated cell sorting (FACS).
  • the surface marker for hemogenic endothelium is CD34 (e.g., high CD34 surface expression).
  • additional positive or negative markers for hemogenic endothelium can include, but are not limited to, CD45, CD38, KDR, CD235, and CD43.
  • the population of CD34+ hemogenic endothelium is CD45 negative/low.
  • the population of CD34+ hemogenic endothelium is CD38 negative/low.
  • the population of CD34+ hemogenic endothelium is KDR+.
  • the population of CD34+ hemogenic endothelium is CD235 negative/low.
  • the population of CD34+ hemogenic endothelium is CD43 negative/low.
  • the hemogenic endothelium and/or HSCs are produced using any method known in the art.
  • the method of differentiating PSCs into hemogenic endothelium can comprise the introduction of transcription factors such as ERG, H0XA5, H0XA9, HOXA10, LCOR, RUNX1, and/or SPI1; see e.g., International Application No. WO 2018/048828, US Patent Application No. 2019/0225940, Doulatov et al., Cell Stem Cell. 2013 October 3, 13(4); Vo et al., Nature 2018, 553(7689): 506-510; the contents of each of which are incorporated herein by reference in their entireties.
  • the hemogenic endothelium is not derived from PSCs but is rather derived directly from endothelial cells.
  • endothelial cells e.g., from lung, brain, and other tissues
  • transcription factors e.g., Fosb, Gfil, Runxl, and Spil
  • cell-extrinsic factors e.g., serum, SB-431542, and/or endothelial mitogen.
  • a T-cell differentiation method comprising a step of inhibiting at least one epigenetic regulator.
  • epigenetic regulator refers to a factor, e.g., a polypeptide, e.g., an enzyme, that influences DNA methylation and/or histone modifications (e.g., histone acetylation, histone methylation), and as such affect the transcription levels of genes without an alteration (e.g., substitution or deletion) to the nucleotide sequence of the genome.
  • Non-limiting examples of epigenetic regulators include: DNA-methyltransferase (DNMT; e.g., DNMT1; DNMT3a; DNMT3b); methyl-CpG-binding domain (MBD) protein (e.g., MeCP2;
  • DNMT DNA-methyltransferase
  • MBD methyl-CpG-binding domain
  • HMT histone methyl transferase
  • methyl-histone binding protein e.g., HP1; Chdl; BPTF; L3MBTL1; ING2; BHC80; JMJD2A
  • histone demethylase e.g., KDMs; e.g., LSDs; JHDMs; JMJDs; JARID; Uts; PHFs
  • HAT histone acetyl transferase
  • HDAC11 HDAC11; Sirtl; Sirt2; Sirt3; Sirt4; Sirt5; Sirt6; Sirt7. See e.g., Cheng et al., Signal Transduction and Targeted Therapy volume 4, Article number: 62 (2019); the content of which is incorporated herein by reference in its entirety.
  • the method comprises the step of, after the step of differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium, inhibiting an epigenetic regulator in the resultant population of CD34+ hemogenic endothelium.
  • the method comprises the step of, prior to the step of differentiating a population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells, inhibiting an epigenetic regulator in the population of CD34+ hemogenic endothelium.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium; (b) inhibiting an epigenetic regulator in the resultant population of CD34+ hemogenic endothelium; and (c) differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • CD34+ hemogenic endothelium is treated with an inhibitor of an epigenetic regulator.
  • exemplary inhibitors of an epigenetic regulator include an inhibitor of at least one of the following: DNMT; MBD; DNA demethylase; HMT; methyl-histone binding protein; histone demethylase; HAT; acetyl-binding protein; or HDAC.
  • the epigenetic regulator is an H3K9 methyltransferase.
  • Methylation of H3K9 in humans relies mostly on members of the Suv39 family, namely EHMT1/GLP, EHMT2/G9a, SUV39H1, SUV39H2, SETDB1 and SETDB2, as well as then non-Suv39 enzymes PRDM2 and ASH1L.
  • Non-limiting examples of DNMT inhibitors include azacitidine; decitabine; guadecitabine; hydralazine.
  • Non-limiting examples of HMT inhibitors include pinometostat; tazemetostat; GSK2816126; CPI-1205; TCP; ORY-2001; GSK2879552; 4SC-202.
  • HDAC inhibitors include valproic acid, phenylbutyrate; vorinostat; trichostatin A; belinostat; entinostat; panobinostat; mocetinostat; CI-994; romidepsin; nicotinamide; suramin; PRI-724; GSK525762; CPI-0610; R06870810; MK-8628.
  • the inhibitor of an epigenetic regulator is selected from Table 1.
  • the inhibitor of an epigenetic regulator is selected from the group consisting of: SB939 (Pracinostat); 4-iodo-SAHA; Scriptaid; Oxaflatin (i.e., Oxamflatin); s-HDAC-42; UNC0224; Pyroxamide; MC1568; CAY10398; CAY10591; SAHA (Vorinostat) (SIH-359); SGI-1027; and Rucaparib (RubracaTM).
  • the inhibitor of an epigenetic regulator is selected from the group consisting of: SB939 (Pracinostat); 4-iodo-SAHA; Scriptaid; Oxaflatin (i.e., Oxamflatin); s-HDAC-42; UNC0224; Pyroxamide; MC1568; CAY10398; CAY10591; and SAHA (Vorinostat) (SIH-359); see e.g., (see, e.g., Fig. 7 and Table 2 presented in International Patent No. W02021/150919A1).
  • Table 1 Small molecule inhibitors that can promote T cell differentiation
  • the inhibitor of an epigenetic regulator is selected from the group consisting of: UNC0224; MC1568; and CAY10591. In some embodiments, the inhibitor of an epigenetic regulator is UNC0224. In some embodiments, the inhibitor of an epigenetic regulator is MC1568. In some embodiments, the inhibitor of an epigenetic regulator is CAY10591.
  • the inhibitor of an epigenetic regulator is UNC0224 or 5-Methyl-2'- deoxycytidine (see e.g., structure in Formula I below).
  • the inhibitor of an epigenetic regulator is 5 -Methyl -2'-deoxy cytidine.
  • 5-Methyl-2'-deoxycytidine is a pyrimidine nucleoside that when incorporated into single -stranded DNA can act in cis to signal de novo DNA methylation; see e.g., Christman et al. Proceedings of the National Academy of Sciences of the United States of America 92(16), 7347-7351 (1995).
  • the inhibitor of an epigenetic regulator is provided at a concentration of at least 500 nM. In some embodiments, the inhibitor of an epigenetic regulator is provided at a concentration of at least 1 nM, at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 150 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1.0 u
  • the inhibitor of an epigenetic regulator is provided at a concentration of InM-lOnM, 10nM-50nM, 50nM-100nM, 100nM-500nM, 500nM-luM, 1UM-5UM, or 5uM-10uM.
  • the cells are cultured exposed to an inhibitor of an epigenetic regulator until the development of CD5+CD7+ proT cells.
  • the cells e.g., CD34+ hemogenic endothelium
  • the cells are cultured exposed to an inhibitor of an epigenetic regulator for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at least
  • G9a can also be referred to interchangeably as Euchromatic Histone Lysine Methyltransferase 2 (EHMT2); Histone H3-K9 Methyltransferase 3; KMT1C; Lysine N-Methyltransferase 1C; BAT8; or NG36.
  • EHMT2 Euchromatic Histone Lysine Methyltransferase 2
  • Histone H3-K9 Methyltransferase 3 KMT1C
  • Lysine N-Methyltransferase 1C BAT8; or NG36.
  • G9a is a methyltransferase that methylates lysine residues of histone H3 (see e.g., NCBI Gene ID: 10919; SEQ ID NOs: 45-46 or a sequence that is at least 95% identical and maintains the same function, or a functional fragment thereof).
  • a T-cell differentiation method comprising a step of inhibiting G9a-like protein (GLP).
  • GLP is also referred to interchangeably as Euchromatic Histone Lysine Methyltransferase 1 (EHMT1); KMT1D; Eu-HMTasel; or Histone-Lysine N-Methyltransferase, H3 Lysine-9 Specific 5 (see e.g., NCBI Gene ID: 79813; SEQ ID NOs: 47-48 or a sequence that is at least 95% identical and maintains the same function, or a functional fragment thereof).
  • EHMT1 Euchromatic Histone Lysine Methyltransferase 1
  • KMT1D Eu-HMTasel
  • Histone-Lysine N-Methyltransferase H3 Lysine-9 Specific 5
  • G9a and GLP exist predominantly as a G9a-GLP heteromeric complex.
  • G9a and GLP are the primary enzymes for mono- and dimethylation at Lys 9 of histone H3 (H3K9mel and H3K9me2) in euchromatin.
  • H3K9me represents a specific tag for epigenetic transcriptional repression by recruiting HP1 proteins to methylated histones.
  • G9a/GLP also weakly methylates 'Lys-27' of histone H3 (H3K27me).
  • G9a/GLP is also required for DNA methylation; the histone methyltransferase activity of G9a/GLP is not required for DNA methylation, suggesting that these two activities function independently.
  • G9a/GLP is probably targeted to histone H3 by different DNA-binding proteins, e.g., E2F6, MGA, MAX and/or DPI.
  • E2F6, MGA, MAX and/or DPI DNA-binding proteins
  • G9a/GLP also methylates non-histone proteins, e.g., dimethylation of 'Lys-373' of p53/TP53.
  • G9a also mediates monomethylation of 'Lys-56' of histone H3 (H3K56mel) in G1 phase, leading to promote interaction between histone H3 and PCNA and regulating DNA replication. G9a is also though to methylate histone Hl. G9a also methylates CDYL, WIZ, ACINI, DNMT1, HDAC1, ERCC6, KLF12, and itself. During GO phase, GLP may contribute to silencing of MYC- and E2F- responsive genes, suggesting a role in G0/G1 transition in cell cycle. In addition to the histone methyltransferase activity, GLP also methylates non-histone proteins: mediates dimethylation of 'Lys- 373' of p53/TP53.
  • SEQ ID NO: 45 Homo sapiens Vietnamese histone lysine methyltransferase 2 (EHMT2), transcript variant 1, mRNA, NCBI Reference Sequence: NM_001289413.1 (region 5-3706), 3702 bp ATGCGGGGTCTACCGAGAGGGAGGGGGTTGATGCGGGCCCGGGGGAGGGGTCGTGCGG CCCCTCCGGGCAGCCGAGGCCGCGGAAGGGGGGGGCCCCACAGAGGAAGAGGTAGGCC CCGGAGCCTACTCTCTCTCTTCCCAGGGCCCAGGCATCCTGGACCCCCCAACTCTCTCTACTGG GCTGACCAGCCCTCCTGTCCCTTGTCTCCCCTCCCAGGGGGAGGCCCCCGCTGAGATGGG GGCGCTGCTGCTGGAGAAGGAAACCAGAGGAGCCACCGAGAGAGTTCATGGCTCTTTGG GGGACACCCCTCGTAGTGAAGAAACCCTGCCCAAGGCCACCCCCGACTCCCTGGAGCCT GCTGGCCCCT GCTGGCCT
  • SEQ ID NO: 46 histone-lysine N-methyltransferase EHMT2 isoform c (Homo sapiens), NCBI Reference Sequence: NP_001276342.1, 1233 aa
  • SEQ ID NO: 47 Homo sapiens Vietnamese histone lysine methyltransferase 1 (EHMT1), transcript variant 2, mRNA, NCBI Reference Sequence: NM_001145527.2 (region 25-2451), 2427 bp ATGGCCGCCGCCGATGCCGAGGCAGTTCCGGCGAGGGGGGAGCCTCAGCAGGATTGCTG
  • SEQ ID NO: 48 histone-lysine N-methyltransferase EHMT1 isoform 2 (Homo sapiens), NCBI Reference Sequence: NP_001138999.1, 808 aa MAAADAEAVPARGEPQQDCCVKTELLGEETPMAADEGSAEKQAGEAHMAADGETNGSCE NSDASSHANAAKHTQDSARVNPQDGTNTLTRIAENGVSERDSEAAKQNHVTADDFVQTSVI GSNGYILNKPALQAQPLRTTSTLASSLPGHAAKTLPGGAGKGRTPSAFPQTPAAPPATLGEGS ADTEDRKLPAPGADVKVHRARKTMPKSVVGLHAASKDPREVREARDHKEPKEEINKNISDF GRQQLLPPFPSLHQSLPQNQCYMATTKSQTACLPFVLAAAVSRKKKRRMGTYSLVPKKKTK VLKQRTVIEMFKSITHSTVGSKGEKDLGASSLHVNGESLEMDSDED
  • the method comprises the step of, after the step of differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34+ hemogenic endothelium, inhibiting G9a and/or GLP in the resultant population of CD34+ hemogenic endothelium.
  • the method comprises the step of, before the step of differentiating a population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells, inhibiting G9a and/or GLP in the population of CD34+ hemogenic endothelium.
  • a method comprising: (a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; (b) inhibiting G9a and/or GLP in the resultant population of CD34 + hemogenic endothelium; and (c) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + -T-cell differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3 + T cells.
  • the inhibitor is a G9a/GLP inhibitor.
  • the G9a/GLP inhibitor is selected from a compound listed in Table 3, or a derivative or analog thereof.
  • the G9a/GLP inhibitor is selected from the group consisting of: UNC0224; UNC0638; A366; BRD4770; BIX01294; UNC0642; UNC0631; UNC0646; UNC0321; E72; BIX-01338; BRD9539; Chaetocin; and DCG066.
  • the G9a/GLP inhibitor is selected from the group consisting of: UNC0224; UNC0638; A366; BRD4770; BIX01294; and UNC0642 (see, e.g., Fig. 8, 10B, 12B, 13B, 13D-13F presented in International Patent No. W02021/150919A1).
  • the G9a/GLP inhibitor is selected from the group consisting of: UNC0224; UNC0638; BRD4770; BIX01294; and UNC0642 (see, e.g., Fig. 8, 10B, 12B, 13B, 13D-13F presented in International Patent No. W02021/150919A1).
  • the G9a/GLP inhibitor is a Type I G9a/GLP inhibitor (e.g., a BIX- 01294 derivative) selected from the group consisting of: UNC0224; UNC0638; A366; B 1X01294;
  • the G9a/GLP inhibitor is a Type II G9a/GLP inhibitor (e.g., a BIX-01338 derivative) selected from the group consisting of: BRD4770; BIX-01338; and BRD9539.
  • the G9a/GLP inhibitor is a Type III G9a/GLP inhibitor such as Chaetocin.
  • the G9a/GLP inhibitor is a Type IV G9a/GLP inhibitor selected from the group consisting of: DCG066.
  • Table 2 G9a/GLP inhibitors that can promote T cell differentiation, (see, e.g., Fig.
  • the G9a/GLP inhibitor is provided at a concentration of at least 500 nM. In some embodiments, the G9a/GLP inhibitor is provided at a concentration of at least 1 nM, at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 150 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1.0 u
  • the G9a/GLP inhibitor is provided at a concentration of InM-lOnM, 10nM-50nM, 50nM-100nM, 100nM-500nM, 500nM-luM, 1UM-5UM, or 5uM-10uM.
  • the G9a/GLP inhibitor (e.g., UNC0224) is provided at a concentration of at least 312 nM, at least 625 nM, at least 1.25 uM, at least 2.5 uM, or at least 5 uM. In some embodiments, the G9a/GLP inhibitor (e.g., UNC0638) is provided at a concentration of at least 8 nM. In some embodiments, the G9a/GLP inhibitor (e.g., BRD4770) is provided at a concentration of at least 200 nM. In some embodiments, the G9a/GLP inhibitor (e.g., BIX01294) is provided at a concentration of at least 200 nM. In some embodiments, the G9a/GLP inhibitor (e.g., UNC0642) is provided at a concentration of at least 40 nM.
  • the cells are cultured exposed to a G9a/GLP inhibitor until the development of CD5+CD7+ proT cells.
  • the cells are cultured exposed to a G9a/GLP inhibitor for about 14 days.
  • the cells are cultured exposed to a G9a/GLP inhibitor for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at
  • culturing cells increases the number of resultant cells (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells) by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at
  • culturing cells decreases the number of erythroid or myeloid lineage cells (e.g., erythroid cell; macrophage; granulocyte; megakaryocyte) by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500
  • erythroid or myeloid lineage cells e.g., erythroid cell; macrophage; granulocyte; megakaryocyte
  • culturing cells decreases the total number of differentiated cells by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or more, or at least lOx,
  • culturing cells e.g., CD34+ hemogenic endothelium
  • a G9a/GLP inhibitor increases the percentage of resultant cells of interest (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells) amongst the total number of differentiated cells by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 20
  • a method for differentiating T cells as described herein produces a population that comprises at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells of interest (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells).
  • a method for differentiating T cells as described herein e.g., G9a/GLP inhibition and stromal-free T cell differentiation
  • the differentiation method can comprise inhibiting a histone methyltransferase.
  • the step of inhibiting a histone methyltransferase e.g., EZH1 knockdown
  • the differentiation method comprises inhibiting a histone methyltransferase, e.g., in the resultant population of CD34+ hemogenic endothelium.
  • Methods of inhibiting a histone methyltransferase are known in the art; see e.g., International Application No. WO 2018/048828, US Application No. 2019/0225940, Doulatov et al., Cell Stem Cell.
  • the differentiation method does not comprise inhibiting a histone methyltransferase, e.g., in the resultant population of CD34+ hemogenic endothelium.
  • histone modifying enzymes targeting H3K9 and H3K27 promotes lymphoid potential of hematopoietic progenitors derived from pluripotent stem cells.
  • the histone modifying enzymes are histone lysine methyltransferases. Post-translational modifications of histone proteins regulate chromatin compaction, mediate epigenetic regulation of transcription, and control cellular differentiation in health and disease. Methylation of histone tails is one of the fundamental events of epigenetic signaling. Tri-methylation of lysine 9 of histone H3 (H3K9) mediates chromatin recruitment of HP1, heterochromatin condensation and gene silencing.
  • H3K27 and H4K20 are associated with a repressed state of chromatin, whereas expressed genes are methylated at H3K4, H3K36 and H3K79.
  • Methylation of H3K9 in humans relies mostly on members of the Suv39 family, namely EHMT1/GLP, EHMT2/G9a, SUV39H1, SUV39H2, SETDB1 and SETDB2, as well as then non-Suv39 enzymes PRDM2 and ASH1L (see e.g., Hong Wu et al., Structural Biology of Human H3K9 Methyltransferases, 2010, PLoS ONE, 5(2): e8570, which is incorporated herein by reference).
  • the methylation of H3K27 is carry out by the polycomb repressive complex 2 (PRC2).
  • H3K9 Di/trimethylation of H3K9 is mainly catalyzed by the conserved SUV39H1/2 histone methyltransferases, while the polycomb repressive complex 2 (PRC2) ensures di/trimethylation of H3K27 (see e.g., Rea S, 2000. Nature 406:593-599; Margueron R, and Reinberg D. 2011. Nature 469:343-349).
  • PRC2 comprises the EZH1/2 catalytic subunit, SUZ12, EED, and RBBP7/4 (see e.g., Margueron R, and Reinberg D, 2011).
  • the histone methyltransferase catalyzes the addition of methyl group to the histone H3 lysine residue 9 (H3K9) and/or histone H3 lysine residue 27 (H3K27).
  • the histone methyltransferase inhibitor inhibits the G9a/GLP heteromeric complex.
  • G9a (EC 2. 1. 1.43) (UniProtKB: Q96KQ7) is also known as EHMT2, (Euchromatic Histone-Lysine N-Methyltransferase 2), G9A Histone Methyltransferase and protein G9a.
  • GLP (EC 2.1.1.43) (UniProtKB: Q9H9B 1) is also known as EHMT1 (Euchromatic Histone-Lysine N-Methyltransferase 1), G9a-Like Protein 1 and GLP1.
  • EHMT1 Euchromatic Histone-Lysine N-Methyltransferase 1
  • G9a-Like Protein 1 GLP1.
  • the histone methyltransferase inhibitor inhibits EZH1 (Enhancer of Zeste 1 Polycomb Repressive Complex 2 Subunit).
  • the H3K27 histone methyltransferase is EZH1 (EC:2. 1.1.43) (UniproKB Q92800-1).
  • the H3K27 histone methyltransferase is not EZH2 (EC:2. 1.1.43) (Unipro QI59I0-I).
  • the inhibitor of histone methyltransferase inhibits the gene expression or protein catalytic activity of the histone methyltransferase.
  • the histone methyltransferase H3K9 and/or H3K27 is inhibited by a small molecule or a nucleic acid or a CRISPR-mediated target genetic interference.
  • the histone methyltransferase H3K9 and/or H3K27 is inhibited by a small molecule inhibitor or a nucleic acid inhibitor.
  • the histone methyltransferase small molecule inhibitor is a chemical agent including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salt
  • the histone methyltransferase small molecule inhibitor include but are not limited to AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, E72, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438 (E7438), 3- deazaneplanocin A (DZNeP) HC1, UNCI 999, MM- 102, SGC 0946, Entacapone, EPZ015666, UNC0379, Ell, MI-2 (Menin-MLL Inhibitor), MI-3 (Menin-MLL Inhibitor), PFI-2, GSK126, or EPZ004777.
  • the histone methyltransferase small molecule inhibitor is selected from the group consisting of UNC0631, BRD4770, UNC1999, CPI-360, and BIX 01294.
  • the nucleic acid inhibitor is a nucleic acid targeting the expression of histone methyltransferase.
  • histone methyltransferase targeting the mRNA or primary transcript of the histone methyltransferase, EZH1, thereby inhibiting protein expression of the enzyme.
  • Histone-lysine N- methyltransferase aka Enhancer of Zeste 1 Polycomb Repressive Complex 2 Subunit (EZH1) or EC 2.1. 1.43, is a component of a noncanonical Polycomb repressive complex-2 (PRC2) that mediates methylation of histone H3 (see MIM 602812) lys27 (H3K27) and functions in the maintenance of embryonic stem cell pluripotency and plasticity.
  • PRC2 noncanonical Polycomb repressive complex-2
  • the external identification for the human EZH1 gene are as follows: HGNC: 3526; Entrez Gene: 2145; Ensembl: ENSG00000108799; OMIM: 601674; UniProtKB: Q92800; EMBL: AB002386 mRNA and the corresponding mRNA translation: BAA20842.2; GENBANK: BT009782 mRNA and the corresponding mRNA translation: AAP88784.1.
  • the nucleic acid inhibitor targets the human EZH1 mRNA.
  • the nucleic acid inhibitor is a RNA interference inhibitor or CRISPR- mediated genetic interference inhibitor.
  • the RNA interference inhibitor can be designed using the predictor RNAi softwares found at the Whitehead Institute, MIT, siRNA website, BLOCK-iTTM RNAi Designer at Invitrogen / ThermoFisher, and other online siRNA design tools at The RNAi Web using the mRNA of EZH1 as the target.
  • Crisper guide RNA can be designed using the Broad Institute (MIT) CRISPR software (available on the world-wide web at, for example, portals.broadinstitute.org/gpp/public/analysis-tools/sgma-design), dna20, Clontech, AddGene, e-crisp, and innovative Genomic using the mRNA or genomic gene of EZH1 as the target.
  • MIT Broad Institute
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas9-mediated gene disruption has been widely used in generating loss-of-function mutations in diverse organisms including mammals (Cong et al., 2013, Science, 339(6121): 819-23; reviewed in Hsu et al., 2014, Cell, 157(6): 1262-78)).
  • Cas9-based knockout screens have been applied in identifying essential genes and genes involved in drug resistance in various cell lines.
  • the CRISPR/Cas system envisaged for use in the context of the invention can make use of any suitable CRISPR enzyme.
  • the CRISPR enzyme is a type II CRISPR system enzyme.
  • the CRISPR enzyme is a Cas9 enzyme.
  • the Cas9 enzyme is .S', pneumoniae, S. pyogenes, or .S', thermophilus Cas9, and may include mutated Cas9 derived from these organisms.
  • the enzyme may be a Cas9 homolog or ortholog.
  • the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.
  • the CRISPR/Cas system is used to specifically target a multitude of sequences within the continuous genomic region of interest.
  • the targeting typically comprises introducing into each cell of a population of cells a vector system of one or more vectors comprising an engineered, non-naturally occurring CRISPR-Cas system comprising: at least one Cas protein, and one or more guide RNAs of the guide RNA library described herein.
  • the Cas protein and the one or more guide RNAs may be on the same or on different vectors of the system and are integrated into each cell, whereby each guide sequence targets a sequence within the continuous genomic region in each cell in the population of cells.
  • the Cas protein is operably linked to a regulatory element to ensure expression in said cell, more particularly a promoter suitable for expression in the cell of the cell population.
  • the promoter is an inducible promoter, such as a doxycycline inducible promoter.
  • the guide RNA comprising the guide sequence directs sequence -specific binding of a CRISPR-Cas system to a target sequence in the continuous genomic region. Typically binding of the CRISPR-Cas system induces cleavage of the continuous genomic region by the Cas protein.
  • RNA interference mediated by short interfering RNAs (siRNA) or microRNAs (miRNA) is a powerful method for post-transcriptional regulation of gene expression.
  • siRNA short interfering RNAs
  • miRNA microRNAs
  • RNAi has been extensively used for the study of biological processes in mammalian cells and could constitute a therapeutic approach to human diseases in which selective modulation of gene expression would be desirable.
  • loss of gene expression occurs by inducing degradation of the cognate mRNA or by translational attenuation.
  • Endogenous miRNAs are transcribed as primary transcripts and subsequently processed by the RNAse III enzyme Droshato create a stem loop structure.
  • RNA induced silencing complex RISC
  • the loading of guide versus passenger strands into RISC largely depends on the 5’ end stability of the siRNA, with the less stable strand preferentially incorporated into RISC, although the exact regulation in mammalian cells is incompletely understood.
  • the 5’ end of the guide strand contains the “seed region,” which is critical for target identification.
  • Precise cleavage by Drosha and Dicer is critical for the generation of guide RNAs with defined seed regions that mediate efficient binding to the appropriate target mRNAs. Inaccurate processing results in binding to off-target molecules but a shift in cleavage sites also alters the nucleotide composition of duplex ends, which may have a profound effect on strand loading into RISC.
  • RNA interference uses small interfering RNA (siRNA) duplexes that target the messenger RNA encoding the target polypeptide for selective degradation.
  • siRNAdependent post-transcriptional silencing of gene expression involves cleaving the target messenger RNA molecule at a site guided by the siRNA.
  • RNAi is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see e.g., Cobum, G.
  • PTGS sequence specific degradation or specific post-transcriptional gene silencing
  • RNA double stranded RNA
  • dsRNA double stranded RNA
  • RISC RNA induced silencing complex
  • RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes.
  • “inhibition of target gene expression” includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to a situation wherein no RNA interference has been induced. The decrease will be of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an RNA interfering agent.
  • RNA interference agent and “RNA interference” as they are used herein are intended to encompass those forms of gene silencing mediated by double-stranded RNA, regardless of whether the RNA interfering agent comprises an siRNA, miRNA, shRNA or other double -stranded RNA molecule.
  • siRNA is defined as an RNA agent which functions to inhibit expression of a target gene, e.g., by RNAi.
  • An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell.
  • siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides.
  • the length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
  • the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
  • PTGS post-transcriptional gene silencing
  • siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).
  • shRNAs small hairpin (also called stem loop) RNAs
  • these shRNAs are composed of a short (e.g., about 19 to about 25 nucleotide) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand.
  • the sense strand may precede the nucleotide loop structure and the antisense strand may follow.
  • shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al.
  • the target gene or sequence of the RNA interfering agent may be a cellular gene or genomic sequence, e.g., the G9a/GLP or EZH1 sequence.
  • An siRNA may be substantially homologous to the target gene or genomic sequence, or a fragment thereof.
  • the term “homologous” is defined as being substantially identical, sufficiently complementary, or similar to the target mRNA, or a fragment thereof, to effect RNA interference of the target.
  • RNA suitable for inhibiting or interfering with the expression of a target sequence include RNA derivatives and analogs.
  • the siRNA is identical to its target.
  • the siRNA preferably targets only one sequence.
  • Each of the RNA interfering agents, such as siRNAs can be screened for potential off-target effects by, for example, expression profiling. Such methods are known to one skilled in the art and are described, for example, in Jackson et al. Nature Biotechnology 6:635-637, 2003.
  • expression profiling one may also screen the potential target sequences for similar sequences in the sequence databases to identify potential sequences which may have off-target effects. For example, 15, or perhaps as few as 11 contiguous nucleotides, of sequence identity are sufficient to direct silencing of non-targeted transcripts.
  • siRNA sequences are chosen to maximize the uptake of the antisense (guide) strand of the siRNA into RISC and thereby maximize the ability of RISC to target G9a/GLP or EZH1 mRNA for degradation. This can be accomplished by scanning for sequences that have the lowest free energy of binding at the 5'-terminus of the antisense strand.
  • siRNA molecules need not be limited to those molecules containing only RNA, but, for example, further encompasses chemically modified nucleotides and non-nucleotides, and also include molecules wherein a ribose sugar molecule is substituted for another sugar molecule or a molecule which performs a similar function.
  • RNA strand can be derivatized with a reactive functional group of a reporter group, such as a fluorophore.
  • a reporter group such as a fluorophore.
  • Particularly useful derivatives are modified at a terminus or termini of an RNA strand, typically the 3' terminus of the sense strand.
  • the 2'-hydroxyl at the 3' terminus can be readily and selectively derivatizes with a variety of groups.
  • RNA derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2'0-alkylated residues or 2'- O-methyl ribosyl derivatives and 2'-O-fluoro ribosyl derivatives.
  • the RNA bases may also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence may be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated.
  • the bases may also be alkylated, for example, 7-methylguanosine can be incorporated in place of a guanosine residue. Non-natural bases that yield successful inhibition can also be incorporated.
  • Preferred siRNA modifications include 2'-deoxy-2'-fluorouridine or locked nucleic acid (LAN) nucleotides and RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages. Such modifications are known to one skilled in the art and are described, for example, in Braasch et al., Biochemistry, 42: 7967-7975, 2003. Most of the useful modifications to the siRNA molecules can be introduced using chemistries established for antisense oligonucleotide technology. Preferably, the modifications involve minimal 2'-O-methyl modification, preferably excluding such modification. Modifications also preferably exclude modifications of the free 5'-hydroxyl groups of the siRNA.
  • the Examples herein provide specific examples of RNA interfering agents, such as shRNA molecules that effectively target mRNA.
  • the nucleic acid is a G9a/GLP or EZH1 specific RNA interference agent or a vector encoding the RNA interference agent.
  • the RNA interference agent comprises one or more of the nucleotide sequences selected from the group consisting of CTATCTGGCAGTGCGAGAATG (SEQ ID NO: 11), AGACGTGCAAGCAGGTCTTTC (SEQ ID NO: 12), TGGATGACTTATGCGTGATTT (SEQ ID NO: 13), CAACAGAACTTTATGGTAGAA (SEQ ID NO: 14), CCGCCGTGGTTTGTATTCATT (SEQ ID NO: 15), GCTTCCTCTTCAACCTCAATA (SEQ ID NO: 16), CCGCCGTGGTTTGTATTCATT (SEQ ID NO: 17), GCTCTTCTTTGATTACAGGTA (SEQ ID NO: 18), and GCTACTCGGAAAGGAAACAAA (SEQ ID NO: 19).
  • the nucleic acid inhibitor is a EZH1 specific nucleic acid that is selected from the group consisting of an aptamer that binds EZH1, a EZH1 specific RNA interference agent, and a vector encoding a EZH1 specific RNA interference agent, wherein the RNA interference agent comprises one or more of the nucleotide sequences selected from SEQ ID NO: 11-19.
  • the multilineage hematopoietic progenitor cells are contacted with the viral vector or vector carrying a nucleic acid molecule comprising a nucleic acid sequence selected from a group consisting of SEQ ID NO: 11-19.
  • the contacting with the histone methyltransferase inhibitor occurs more than once.
  • the contacted cell is washed to remove that virus or vector, and the washed cell is then contacted for a second time with the same virus or vector used in the first contact.
  • Cas9/CRISPR system of genome editing be employed with the methods, cells and compositions described herein.
  • Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems is useful for RNA-programmable genome editing (see e.g., Jinek, M. et al. Science (2012) 337(6096):816-821).
  • Trans-activating crRNA is a small trans-encoded RNA. It was first discovered in the human pathogen Streptococcus pyogenes. (See Deltcheva E, et al. (2011). Nature 471 (7340): 602-7). In bacteria and archaea, CRISPR/Cas (clustered, regularly interspaced short palindromic repeats/CRISPR-associated proteins) constitute an RNA-mediated defense system which protects against viruses and plasmids. This defensive pathway has three steps. First a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus.
  • crRNAs CRISPR RNAs
  • the crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid.
  • the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid.
  • CRISPR activation There are several pathways of CRISPR activation, one of which requires a tracrRNA which plays a role in the maturation of crRNA.
  • TracrRNA is complementary to and base pairs with a pre-crRNA forming an RNA duplex. This is cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid.
  • This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid, (see e.g., Deltcheva E, et al. supra; Jinek M, et al. (2012), Science 337 (6096): 816-21; and Brouns SJ (2012), Science 337 (6096): 808-9).
  • Cas9/CRISPR system guide RNAs are designed to target the exon 3 of EZH1 gene, which is present in all transcripts of EZH1 known.
  • Exon 3 sequence is ATTACAGCAAGATGGAAATACCAAATCCCCCTACCTCCAAATGTATCACTTACTGGAAAA GAAAAGTGAAATCTGAATACATGCGACTTCGACAACTTAAACGGCTTCAGGCAAATATG GGTGCAAAG (SEQ ID NO: 20).
  • Non-limiting exemplary gRNAs that target exon 3 are TCGACAACTTAAACGGCTTC (SEQ ID NO: 21), TGCGACTTCGACAACTTAAA (SEQ ID NO: 22), CCTCCAAATGTATCACTTAC (SEQ ID NO: 23), TAAACGGCTTCAGGCAAATA (SEQ ID NO: 24) AAACGGCTTCAGGCAAATAT (SEQ ID NO: 25), CATTTGGAGGTAGGGGGATT (SEQ ID NO: 26), CCAGTAAGTGATACATTTGG (SEQ ID NO: 27), GTGATACATTTGGAGGTAGG (SEQ ID NO: 28), AAGTGATACATTTGGAGGTA (SEQ ID NO: 29), AGTGATACATTTGGAGGTAG (SEQ ID NO: 30), TTTCCAGTAAGTGATACATT (SEQ ID NO: 31), and TAAGTGATACATTTGGAGGT (SEQ ID NO: 32) [00299] In other embodiments, Cas9/CRISPR system
  • Exon 4 sequence is GCTTTGTATGTGGCAAATTTTGCAAAGGTTCAAGAAAAAACCCAGATCCTCAATGAAGA ATGGAAGAAGCTTCGTGTCCAACCTGTTCAGTCAATGAAGCCTGTGAGTGGACACCCTTT TCTCAAAAAG (SEQ ID NO: 33).
  • Non-limiting exemplary gRNAs that target exon 4 are GCTTCATTGACTGAACAGGT (SEQ ID NO: 34), ACAGGCTTCATTGACTGAAC (SEQ ID NO: 35), AGAAAAGGGTGTCCACTCAC (SEQ ID NO: 36), TCCATTCTTCATTGAGGATC (SEQ ID NO: 37), CCATTCTTCATTGAGGATCT (SEQ ID NO: 38), CCCAGATCCTCAATGAAGAA (SEQ ID NO: 39), GTATGTGGCAAATTTTGCAA (SEQ ID NO: 40), and CAGTCAATGAAGCCTGTGAG (SEQ ID NO: 41).
  • a vector is used as a transport vehicle to introduce any of the herein described nucleic acid inhibitors of a histone methyltransferase into the target cells selected from the cell populations as described herein (e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs).
  • a vector is used as a transport vehicle to introduce any of the herein described nucleic acid comprising the described nucleic acid inhibitors of a histone methyltransferase into the target cells selected from the cell populations as described herein (e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs).
  • the in vivo expression of the nucleic acid inhibitor is for degrading the mRNA of the targeted histone methyltransferase such as G9a/GLP or EZH1 so as to reduce and inhibit the expression of the respective histone methyltransferase, with the goal being to reduce methylation of the histone H3 in the transfected cells and relief repression of gene expression therein.
  • the targeted histone methyltransferase such as G9a/GLP or EZH1
  • the vector further comprises a spleen focus-forming virus promoter, a tetracycline-inducible promoter, a Doxycycline (Dox)-inducible, or a P-globin locus control region and a P-globin promoter.
  • the promoter provides for targeted expression of the nucleic acid molecule therein.
  • Other examples of promoters include but are not limited to the CMV promoter and EFl -alpha promoters for the various transgenes, and U6 promoter for shRNAs targeting EZH1.
  • the vector is a virus or a non-viral vector.
  • viral vectors for gene delivery and expressions in cells are retrovirus, adenovirus (types 2 and 5), adeno-associated virus (AAV), Helper-dependent adenoviral vector (HdAd), hybrid adenoviral vectors, herpes virus, pox virus, human foamy virus (HFV), and lentivirus.
  • Exemplary vectors useful in the invention described herein include episomal vectors, integrating vectors, non-integrating vectors, and excisable vectors.
  • the differentiation method comprises differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • the method described herein is a stroma-free T cell differentiation method. Compared to differentiation with stromal cells expressing a Notch ligand, stroma-free differentiation unexpectedly results in an increased number of differentiated T cells, with a smaller portion of these T cells being innate-like cells.
  • the soluble Notch ligand is provided at a concentration of 1 nM to 100 nM or a concentration of 5 nM to 15 nM.
  • the soluble Notch ligand is provided at a concentration of at least 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM or greater.
  • the soluble Notch ligand is provided at a concentration of at least 0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or greater.
  • the soluble Notch ligand is provided at a concentration of at least 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM or greater.
  • the cells are cultured exposed to soluble Notch ligand for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 31 days, at least 32 days, at least 33 days, at least 34 days, at least 35 days, at least 36 days, at least 37 days, at least 38 days, at least 39 days, at least 40 days, at least 41 days, at least 42 days, at least 43 days, at least 44 days, at least 45 days, at least 40 days, at least 41 days
  • the method described herein is a stroma-free T cell differentiation method, i.e., a method that does not comprise co-culturing with stromal cells or any other type of supporting cell.
  • Co-culture with stromal cells such as mouse stromal cells limits the translational potential of iPSC-derived T cells; for example, there can be fears of transplantation rejection due to the presence of stromal cells.
  • T cells differentiated using stromal cells exhibit an innate-like phenotype (e.g., as measured by TCRgd expression, which is a marker for gamma delta T cells). It is preferred that T cells exhibit an adaptive phenotype, for example characterized by expression of TCR a and p.
  • stroma-free T cell differentiation methods result in increased numbers of CD3+ T cells (e.g., CD4+CD8+ cells) compared to differentiation methods comprising stromal co-culture.
  • T cells differentiated using stromal-free methods exhibit at least the following unexpected benefits compared to stromal co-culture methods: (1) increased potential for transplantation in humans; (2) decreased number of innate-like T cells; (3) increased number and/or percentage of resultant T cells (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells); (4) gene expression profiles most similar to alpha beta T cells; (5) a more diverse TCR repertoire; and/or (6) increased TCR CDR length.
  • an epigenetic regulator e.g., an HMT; e.g., EZH1, G9a/GLP
  • supporting cell or stromal cell when used in the context of cell differentiation refers to any cells that are capable of creating, promoting, or supporting a microenvironment for the growth, proliferation, differentiation, or expansion of multipotent hematopoietic progenitor cells or T cells or B cells.
  • supporting cells that are not comprised by the differentiation methods described herein include, but are not limited to, stromal cells and fibroblast cells.
  • Supporting cells used previously in co-cultures for cell differentiation purposes are typically stromal cells. However, the methods described herein do not comprise co-cultures comprising stromal cells.
  • stromal cell lines that are not comprised by the differentiation methods described herein include, but are not limited to, murine MS5 stromal cell line; murine bone marrow-derived stromal cell lines, such as S10, S17, OP9 (e.g., OP9-DL1 cells or OP9-DL4 cells) and BMS2 cell lines; human marrow stromal cell lines such as those described in U.S. Patent No.
  • OP9-DL1 cells are a bone-marrow- derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (DLL1).
  • DLL1 Delta-like 1
  • the soluble Notch ligand used herein is not derived from a stromal cell.
  • differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not comprise co-culturing with a stromal cell expressing a Notch ligand.
  • differentiating the hemogenic endothelium in the presence of a soluble Notch ligand does not comprise co-culturing with OP9-DU1 cells or OP9-DU4 cells.
  • the differentiation method comprises differentiating the resultant population of CD34+ hemogenic endothelium in a CD3+-T-cell differentiation media for a sufficient time to promote differentiation into a population of CD3+ T cells.
  • the sufficient time to promote differentiation into a population of CD3+ T cells is at least 3 weeks, at least 3.5 weeks, at least 4 weeks, at least 4.5 weeks, at least 5 weeks, at least 5.5 weeks, at least 6 weeks, or more.
  • the sufficient time to promote differentiation into a population of CD3+ T cells is at most 6 weeks.
  • a polypeptide that can be expressed by the supporting cell or stromal cell can be provided in the cell culture medium.
  • polypeptides that support the differentiation of T cells include IU-7, SCF, Flt3, and TPO.
  • Interleukin-7 IU-7 is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus, and it is involved in B and T cell development.
  • Stem cell factor also known as SCF, KIT-ligand, KU, or steel factor
  • SCF c-KIT receptor
  • FUT3 also referred to as Flit3 or Fms-Uike Tyrosine Kinase 3
  • TPO Thrombopoietin
  • HSCs hematopoietic stem cells
  • the CD3+-T-cell-differentiation media is serum-free.
  • the CD3+-T-cell-differentiation media comprises at least one of SCF, FLT3, and/or IL7.
  • the CD3+-T-cell-differentiation media comprises SCF, FLT3, and IL7.
  • the CD3+-T-cell-differentiation media comprises 30 ng/ml SCF, 15 ng/ml FLT3, and 25 ng/ml IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 100 ng/ml SCF, 100 ng/ml FLT3, and 50 ng/ml IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises FLT3 and IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 15 ng/ml FLT3 and 25 ng/ml IL7. In some embodiments, the CD3+-T-cell-differentiation media comprises 100 ng/ml FLT3 and 50 ng/ml IL7.
  • the concentrations of SCF, FLT3, and/or IL7 should be used such that they promote the differentiation of hemogenic endothelium into a population of CD3+ T cells.
  • the concentration of SCF can range from 1 ng/mL to 200 ng/mL.
  • the concertation of SCF e.g., in the CD3+-T-cell-differentiation media
  • the concertation of SCF is 100 ng/ml.
  • the concentration of FLT3 can range from 1 ng/mL to 200 ng/mL.
  • the concertation of FLT3 (e.g., in the CD3+-T- cell-differentiation media) is 15 ng/ml. In some embodiments, the concertation of FLT3 (e.g., in the CD3+-T-cell-differentiation media) is 100 ng/ml.
  • the concentration of IL7 can range from 1 ng/mL to 200 ng/mL. In some embodiments, the concertation of IL7 (e.g., in the CD3+-T-cell-differentiation media) is 25 ng/ml. In some embodiments, the concertation of IL7 (e.g., in the CD3+-T-cell- differentiation media) is 50 ng/ml.
  • the CD3+-T-cell-differentiation media further comprises thrombopoietin (TPO) for at least the first 2 weeks of differentiating in the CD3+-T-cell- differentiation media.
  • the CD3+-T-cell-differentiation media further comprises thrombopoietin (TPO) for at least the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days of differentiating in the CD3+-T-cell-differentiation media.
  • CD3+-T-cell-differentiation media comprising TPO promotes differentiation into a population of CD5+ CD7+ ProT cells.
  • Such CD5+ CD7+ ProT cells can be detected after at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days of differentiating in the CD3+-T-cell-differentiation media.
  • CD5+ CD7+ ProT cells can be detected after at least 2 weeks of differentiating in the CD3+-T-cell- differentiation media.
  • the concentration of TPO should be used such that it promotes the differentiation of hemogenic endothelium into a population of CD3+ T cells.
  • the concentration of TPO can range from 1 ng/mL to 200 ng/mL.
  • the concertation of TPO e.g., in the CD3+-T-cell-differentiation media
  • the concertation of TPO is 50 ng/ml.
  • the CD3+-T-cell-differentiation media (e.g., comprising IL-7 and/or FLT3) further comprises SCF for at least the first 2 weeks of differentiating in the CD3+-T-cell- differentiation media.
  • the CD3+-T-cell-differentiation media further comprises SCF for at least the first 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days of differentiating in the CD3+-T-cell-differentiation media.
  • CD3+-T-cell-differentiation media comprising SCF promotes differentiation into a population of CD5+ CD7+ ProT cells.
  • SCF, FLT3, IL7, and/or TPO are provided in the CD3+-T-cell- differentiation media at a concentration of at least 1 ng/mL, at least 2 ng/mL, at least 3 ng/mL, at least 4 ng/mL, at least 5 ng/mL, at least 6 ng/mL, at least 7 ng/mL, at least 8 ng/mL, at least 9 ng/mL, at least 10 ng/mL, at least 11 ng/mL, at least 12 ng/mL, at least 13 ng/mL, at least 14 ng/mL, at least 15 ng/mL, at least 16 ng/mL, at least 17 ng/mL, at least 18 ng/mL, at least 19 ng/mL, at least 20 ng/mL, at least 25 ng/mL, at least 30 ng/mL, at least 35 ng/mL, at least 40
  • the concentration of SCF, FLT3, IL7, and/or TPO can be the same or different.
  • CD3+ T cells can be detected after at least 5.0 weeks of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, CD3+ T cells can be detected after at least 1.5 weeks, 2 weeks, 2.5 weeks, 3.0 weeks, 3.5 weeks, 4.0 weeks, 4.5 weeks, or 5.0 weeks of differentiating in the CD3+-T-cell-differentiation media. In some embodiments, the population of CD3+ T cells comprises a population of CD4+CD8+ T cells, also referred to herein as double-positive or DP T cells.
  • Such CD4+CD8+ CD3+ T cells can be detected after at least 1.5 weeks, 2 weeks, 2.5 weeks, 3.0 weeks, 3.5 weeks, 4.0 weeks, 4.5 weeks, or 5.0 weeks of differentiating in the CD3+-T-cell-differentiation media.
  • the method further comprises differentiating the population of CD4+CD8+ T cells in a single-positive-T-cell-differentiation media for a sufficient time to promote differentiation into a population of CD4+ cells and a population of CD8+ cells.
  • the sufficient time to promote differentiation from the population of CD4+CD8+ T cells into a population of CD4+ T cells and a population of CD8+ cells is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
  • the sufficient time to promote differentiation from the population of CD34+ hemogenic endothelium into a population of CD4+ T cells and a population of CD8+ cells is at least 4.0 weeks, 4.5 weeks, 5.0 weeks, 5.5. weeks, or 6.0 weeks.
  • the single-positive-T-cell-differentiation media comprises 10 ng/ml IL- 15 and a T cell activator.
  • Interleukin- 15 IL- 15
  • IL-7 Interleukin 7
  • IL-2 interleukin 2
  • a variety of concentrations of IL- 15 can be used as long as it still promotes the differentiation of CD4+CD8+ T cells into single positive CD4+ cells and CD8+ cells.
  • the concentration of IL 15 can range from 1 ng/mL to 200 ng/mL, with a preferred concentration of 10 ng/ml.
  • the T cell activator comprises components (e.g., soluble tetrameric antibody complexes) that bind CD3 and CD28 (and optionally CD2) cell surface ligands. Binding of the T cell activator results in the cross-linking of CD3 and CD28 (and optionally CD2) cell surface ligands, thereby providing the required primary and co-stimulatory signals for T cell activation.
  • components e.g., soluble tetrameric antibody complexes
  • the T cell activator comprises a CD3/CD28 T cell activator (e.g., at a concentration of lOul/ml).
  • a CD3/CD28 T cell activator is available commercially (e.g., via StemCell TechnologyTM, item #10970).
  • the concentration of the CD3/CD28 T cell activator should be used such that it promotes the differentiation of CD4+CD8+ T cells into single positive CD4+ cells and CD8+ cells.
  • the concentration can range from 1 ul/mL to 200 ul/mL, with a preferred concentration of 10 ul/ml.
  • the T cell activator comprises CD3/CD28 T cell activator Dynabeads (e.g., used at one bead per cell).
  • CD3/CD28 T cell activator Dynabeads are available commercially (e.g., via ThermoFisherTM #11132D).
  • the concentrations of CD3/CD28 T cell activator Dynabeads should be used such that it promotes the differentiation of CD4+CD8+ T cells into single positive CD4+ cells and CD8+ cells.
  • the concentration can range from 1 bead/cell to 20 beads/cell, with a preferred concentration of 1 bead/cell.
  • the method further comprises, after at least 1 week (e.g., in the single-positive-T-cell-differentiation media), a step of CD4+ cell enrichment and/or CD8+ cell enrichment.
  • a step of CD4+ cell enrichment and/or CD8+ cell enrichment can occur at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days of culturing in the single-positive-T-cell-differentiation media.
  • the CD4+ or CD8+ cells can be enriched using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) with anti-CD4 or anti-CD8 antibodies accordingly.
  • MCS magnetic-activated cell sorting
  • FACS fluorescence-activated cell sorting
  • the entire T cell differentiation protocol described herein occurs in a stromal-free environment, e.g., the cells are cultured exposed to a non-stromal-derived Notch ligand (e.g., Notch ligand immobilized on a tissue culture plate).
  • At least a portion of the T cell differentiation protocol occurs in a stromal-free environment, e.g., the cells are cultured exposed to a non-stromal-derived Notch ligand (e.g., Notch ligand immobilized on a tissue culture plate).
  • a non-stromal-derived Notch ligand e.g., Notch ligand immobilized on a tissue culture plate.
  • the population of T cells derived using stromal -free methods as described herein, and in one embodiment, in combination with inhibition of an epigenetic regulator exhibits at least the following unexpected benefits compared to stromal co-culture methods: (1) increased potential for transplantation in humans; (2) decreased number of innate-like T cells; (3) increased number and/or percentage of resultant T cells (e.g., CD5+CD7+ Pro-T cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells; alpha-beta T cells); (4) gene expression profiles most similar to alpha beta T cells; (5) a more diverse TCR repertoire; and/or (6) increased TCR CDR length.
  • an epigenetic regulator e.g., an HMT; e.g., EZH1, G9a/GLP
  • the population of T cells e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells
  • an epigenetic regulator e.g., an HMT; e.g., EZH1, G9a/GLP
  • an HMT e.g., EZH1, G9a/GLP
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 500x, l,000x, or
  • TCRgd + i.e., innate-like gamma delta T cells.
  • Gamma delta T cells y5 T cells
  • TCR T-cell receptor
  • Most T cells are a[3 (alpha beta) T cells with a TCR composed of two glycoprotein chains called a (alpha) and (beta) TCR chains.
  • gamma delta (y5) T cells have a TCR that is made up of one y (gamma) chain and one 5 (delta) chain.
  • gamma delta T cells exhibit several characteristics that place them at the border between the more evolutionarily primitive innate immune system that permits a rapid beneficial response to a variety of foreign agents and the adaptive immune system, where B and T cells coordinate a slower but highly antigen-specific immune response leading to long-lasting memory against subsequent challenges by the same antigen.
  • Gamma delta T cells may be considered a component of adaptive immunity in that they rearrange TCR genes to produce junctional diversity and can develop a memory phenotype. However, the various subsets may also be considered part of the innate immunity in which a specific TCR can function as a pattern recognition receptor. See, e.g., Bom WK, Reardon CL, O'Brien RL (February 2006). "The function of gammadelta T cells in innate immunity". Current Opinion in Immunology. 18 (1): 31-8.
  • TCRgdt at most 10% of the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein are TCRgdt.
  • TCRgdt at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 14%, at most 15%, at most 16%, at most 17%, at most 18%, at most 19%, at most 20%, at most 21%, at most 22%, at most 23%, at most 24%, at most 25%, at most 26%, at most 27%, at most 28%, at most 29%, at most 30%, at most 31%, at most 32%, at most 33%, at most 34%, at most 35%
  • the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein comprises at least 10% more T cells than a population of T cells derived using a stromal method or without inhibition of an epigenetic regulator.
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% or more, or at least lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 500x, l,000x, or more
  • the population of T cells (e.g., CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells) derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein exhibits a gene expression profde that is more similar to a[3 T cells, than to other cells (e.g., y5 T cells; NK cells; iPSCs derived T cells using a OP9-DL4 co-culture system; T cells differentiated from cord blood CD34+ HSPCs), e.g., the gene profde of the derived T cells is at least 0.5% more similar to a a[3 T cells as compared to another cell type.
  • the gene profde of the derived T cells is at least 0.5% more similar to a a[3 T cells as compared to another cell type.
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits a gene expression profde of T cell signature genes and/or a[3 T cell signature genes that is at most 10% divergent from the gene expression profde of a[3 T cells.
  • the population of T cells derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein exhibits a gene expression profde of T cell signature genes and/or a[3 T signature cell genes that is at most 20% (e.g., at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 14%, at most 15%, at most 16%, at most 17%, at most 18%, at most 19%, or more) divergent from the gene expression profde of a[3 T cells.
  • a[3 T signature cell genes that is at most 20% (e.g., at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits a gene expression profde that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
  • the derived T cell has a greater percentage of similarity to the gene expression profde of an a[3 T cell than the gene profde of another cell type.
  • One skilled in the art can determine the similarity of gene expression in a T cell derived from stromal-free methods described herein and an a[3 T cell using standard methods, e.g., transcriptome sequencing of specific cell types (FACS-sorted cells).
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits a gene expression profde with a Pearson’s correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.75, 0.755, 0.76, 0.765, 0.77, 0.775, 0.78, 0.785, 0.79, 0.795, 0.8, 0.805, 0.81, 0.815, 0.82, 0.825, 0.83,
  • the population of CD3+ T cells exhibits a gene expression profile that is most similar to alpha beta T cells. In some embodiments, the population of CD3+ T cells exhibits a gene expression profile that is similar or substantially similar to alpha beta T cells. In some embodiments, the population of CD3 + T cells exhibits a gene expression profile that is at least 10%, 20%, 30%, 40% or more similar to alpha beta T cells. In some embodiments, the population of CD3+ T cells exhibits a gene expression profile with a Pearson’s correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.85.
  • the immune cell e.g., derived using stromal-free and/or inhibition of an epigenetic regulator as described herein, exhibits a gene expression profile that is most similar to alpha beta T cells. In some embodiments, the immune cell exhibits a gene expression profile that is similar or substantially similar to alpha beta T cells. In some embodiments, the immune cell exhibits a gene expression profile that is at least 10%, 20%, 30%, 40% or more similar to alpha beta T cells. In some embodiments, the immune cell exhibits a gene expression profile with a Pearson’s correlation coefficient compared to peripheral blood alpha beta T cells that is at least 0.85.
  • the population of T cells derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least
  • the derived T cell expresses a greater number of signature genes from an a[3 T cell than signature genes from another cell type.
  • signature gene refers to a gene that exhibits a characteristic expression pattern in a specific cell type (e.g., T cell, a[3 T cell); a signature gene can be required for the function of a specific cell type.
  • T cell signature genes and a T cell signature genes are described further herein.
  • a specific cell type e.g., T cell, aP T cell
  • the population of T cells derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein expresses at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least
  • T cell signature genes include GRB2 (Growth Factor Receptor Bound Protein 2); NFATC3 (Nuclear Factor Of Activated T Cells 3); ZAP70 (Zeta Chain Of T Cell Receptor Associated Protein Kinase 70); RAFI (Raf-1 Proto-Oncogene, Serine/Threonine Kinase); PIK3CG (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Gamma); PIK3R1 (Phosphoinositide-3-Kinase Regulatory Subunit 1); CALM3 (Calmodulin 3); PTPN7 (Protein Tyrosine Phosphatase Non-Receptor Type 7); LAT (Linker For Activation Of T Cells); NFKBIA (NFKB Inhibitor Alpha); VAV1 (Vav Guanine Nucleotide Exchange Factor 1); S
  • Non-limiting examples of a[3 T cell signature genes include ATP1 IB (ATPase Phospholipid Transporting 11B); PPP4R3A (Protein Phosphatase 4 Regulatory Subunit 3A); CAB39 (Calcium Binding Protein 39); GLS (Glutaminase); UBE2Z (Ubiquitin Conjugating Enzyme E2 Z); INPP4A (Inositol Polyphosphate-4-Phosphatase Type I A); RAB22A (Ras-Related Protein Rab-22A, Member Ras Oncogene Family); SMARCD2 (SWI/SNF (SWItch/Sucrose Non-Fermentable) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily D, Member 2); VPS26B (VPS26, Retromer Complex Component B, Vacuolar Protein Sorting-Associated Protein 26B); CERK (Ceramide Kinase);
  • the population of T cells derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein exhibits a more diverse TCR repertoire compared to T cells not derived using such stromal-free methods or without inhibition of an epigenetic regulator.
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits a Productive Simpson Clonality value of about 0.000-0.025. A value closer to 0 represents a higher level of diversity compared to clonality. A value closer to 1 represents a higher level of clonality compared to diversity.
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits a Productive Simpson Clonality value of at most 0.01, at most 0.015, at most 0.02, at most 0.025, at most 0.03, at most 0.035, at most 0.04, at most 0.045, at most 0.05, at most 0.055, at most 0.06, at most 0.065, at most 0.07, at most 0.075, at most 0.08, at most 0.085, at most 0.09, at most 0.095, or at most 0. 1.
  • the population of T cells derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein exhibits a Productive Simpson Clonality value of about 0.025.
  • variable domain of both the T-cell receptor (TCR) a-chain and [3-chain each have three hypervariable or complementarity-determining regions (CDRs; e.g., CDR1, CDR2, CDR3).
  • CDRs hypervariable or complementarity-determining regions
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits an increased CDR (e.g., CDR1, CDR2, CDR3) length compared to T cells derived using stromal methods or without inhibition of an epigenetic regulator.
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits CDR (e.g., CDR1, CDR2, CDR3) length that is, on average, about 3 nucleotides (nt), 6 nt, 9 nt, or 12 nt or more longer than the CDRs of T cells derived using stromal methods or without inhibition of an epigenetic regulator.
  • CDR e.g., CDR1, CDR2, CDR3
  • the population of T cells derived using stromal -free methods and/or inhibition of an epigenetic regulator as described herein exhibits CDR (e.g., CDR1, CDR2, CDR3) length that is, on average, about 27 nt, 30 nt, 33 nt, 36 nt, 39 nt, 42 nt, 45 nt, 48 nt, 51 nt, 54 nt, 57 nt, or 60 nt or longer.
  • CDR e.g., CDR1, CDR2, CDR3
  • the population of T cells derived using stromal-free methods and/or inhibition of an epigenetic regulator as described herein exhibits a CDR3 length that is, on average, about 42 nt long, compared to 39 nt on average for control iPSC-derived T cells, or 45 on average for peripheral blood mononuclear cell (PBMC)-derived T cells.
  • PBMC peripheral blood mononuclear cell
  • the resultant population of CD34+ hemogenic endothelium or another population as described herein are genetically modified.
  • the native T cell receptor locus can be removed and/or replaced to enhance targeted specificity.
  • an endogenous HLA e.g., class I and/or class II major histocompatibility complexes
  • the genetic modification can comprise introduction and expression of non-canonical HLA-G and HLA-E to prevent NK cell-mediated lysis (see e.g., Riolobos L et al. 2013), which can provide a source of universal T cells for immunotherapy, e.g., cancer immune therapy.
  • the genetic modification comprises expressing a chimeric antigen receptor (CAR).
  • CARs also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors
  • CARs are receptor proteins that have been engineered to give T cells the new ability to target a specific protein.
  • the receptors are chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor.
  • Methods of engineering chimeric antigen receptor T cells also known as CAR T cells are known in the art.
  • methods of genetically modifying a cell to express a CAR can comprise but are not limited to: transfection or electroporation of a cell with a vector encoding a CAR; transduction with a viral vector (e.g., retrovirus, lentivirus) encoding a CAR; gene editing using zin finger nucleases (ZFNs), transcription activator-like effector nucleases (TAEENs), meganuclease- TALENs, or CRISPR-Cas; or any other methods known in the art of genetically modifying a cell to express a CAR.
  • a viral vector e.g., retrovirus, lentivirus
  • ZFNs zin finger nucleases
  • TAEENs transcription activator-like effector nucleases
  • TALENs meganuclease- TALENs
  • CRISPR-Cas CRISPR-Cas
  • a population of cells at an early stage of differentiation e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs
  • a population of cells at an early stage of differentiation e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs
  • ESCs e.g., ESCs; PSCs; iPSCs; hemogenic endothelium; HSCs
  • the antigen-binding region of the CAR is directed against an antigen involved in a disease or disorder, such as but not limited to cancer, autoimmune disease, or heart disease (e.g., cardiac fibrosis).
  • a disease or disorder such as but not limited to cancer, autoimmune disease, or heart disease (e.g., cardiac fibrosis).
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems.
  • Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
  • Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • the cancer is a primary cancer.
  • the cancer is a malignant cancer.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (z. e.
  • metastasize refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term “benign” or “non-malignanf ’ refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • neoplasm refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues.
  • a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastases. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphom
  • CD19-specific CAR T cell therapies have achieved dramatic objective responses for a high percent of patients with CD 19-positive leukemia or lymphoma.
  • the antigen-binding region of the CAR is directed against CD19; see e.g., US patents US10221245, US10357514; US patent publication US20160152723; PCT publication W02016033570; the contents of each of which are incorporated herein by reference in their entireties.
  • Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses.
  • the selection of the antigen binding domain of the invention will depend on the particular type of cancer to be treated.
  • Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-l lRa, IL-13Ra, EGFR, B7H3, Kit, CA-IX, CS-1, MUC1, BCMA, bcr-abl, HER2, [3-human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, CD123, cyclin B 1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3,
  • the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof; see e.g., US Patent publications 20170209492 and 20180022795, the contents of each of which are incorporated herein by reference in their entireties.
  • the population of engineered immune cells comprises an immune cell differentiated using methods described herein, including but not limited to: PSCs; iPSCs; hemogenic endothelium; HSCs; CD5+CD7+ ProT cells; CD3+ T cells; CD4+CD8+ T cells; CD4+ T cells; CD8+ T cells.
  • the immune cell exhibits a gene expression profile that is most similar to alpha beta T cells.
  • the population of cells further comprises a pharmaceutically acceptable carrier.
  • These engineered immune cells can be culture expanded to increase the number of cells for use.
  • the engineered immune cells described herein are useful in the laboratory for biological studies.
  • these cells can be derived from an individual having a genetic disease or defect, and used in the laboratory to study the biological aspects of the disease or defect, and to screen and test for potential remedy for that disease or defect.
  • the engineered immune cells described herein are useful in cellular replacement therapy and other medical treatment in subjects having the need. For example, patients who have undergone chemotherapy or irradiation or both, and manifest deficiencies in immune function and/or lymphocyte reconstitution, or in cancer immune therapy.
  • the engineered immune cells described herein are administered (i.e., implanted or transplanted) to a subject in need of cellular replacement therapy.
  • a method of cellular replacement therapy or for the treatment of cancer, autoimmune disorders, hematological diseases, or other genetic diseases and disorders in a subject, comprising (a) providing a somatic cell from a donor subject, (b) generating multilineage hematopoietic progenitor cells (e.g., hemogenic endothelium, HSPCs) from pluripotent stem cells derived from the somatic cell as described in any of the preceding paragraphs; (c) optionally inhibiting a histone methyltransferase in the resultant population of multilineage hematopoietic progenitor cells as described in any of the preceding paragraphs; (d) differentiating the resultant population of multilineage hematopoietic progenitor cells in the presence of a notch ligand to promote differentiation into the lymphoid lineage (e.g., T cells) as described in any of the preceding paragraphs, and (e) implanting
  • Hematological diseases are disorders which primarily affect the blood.
  • Non-limiting such diseases or disorders include myeloid derived disorders such as hemoglobinopathies (congenital abnormality of the hemoglobin molecule or of the rate of hemoglobin synthesis), examples, sickle-cell disease, thalassemia, and methemoglobinemia; Anemias (lack of red blood cells or hemoglobin), Pernicious anemia; disorders resulting in decreased numbers of cells, such as myelodysplastic syndrome, neutropenia (decrease in the number of neutrophils), and thrombotic thrombocytopenic purpura (TTP), thrombocytosis, hematological malignancies such as lymphomas, myelomas, and leukemia.
  • myeloid derived disorders such as hemoglobinopathies (congenital abnormality of the hemoglobin molecule or of the rate of hemoglobin synthesis), examples, sickle-cell disease, thalassemia, and methemoglobinemia; Anemias (lack of
  • Lymphomas such as Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma, Anaplastic large cell lymphoma, Splenic marginal zone lymphoma, Hepatosplenic T-cell lymphoma, and Angioimmunoblastic T-cell lymphoma (AILT); myelomas such as Multiple myeloma, Waldenstrom macroglobulinemia, Plasmacytoma; leukemias that increases defect WBC such as Acute lymphocytic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenous leukemia (AML), Chronic Idiopathic Myelofibrosis (MF), Chronic myelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), Chronic neutrophilic leukemia (CNL), Hairy cell leukemia (HCL
  • autoimmune disease refers to a class of diseases in which a subject's own antibodies react with host tissue or in which immune effector T cells are autoreactive to endogenous self-peptides and cause destruction of tissue.
  • self-antigens refers to an antigen of a normal host tissue. Normal host tissue does not include neoplastic cells.
  • Non-limiting examples of autoimmune diseases that can be treated include pemphigus (pemphigus vulgaris, pemphigus foliaceus or paraneoplastic pemphigus), Crohn's disease, idiopathic thrombocytopenic purpura (ITP), heparin induced thrombocytopenia (HIT), thrombotic thrombocytopenic purpura (TTP), Myasthenia Gravis (MG), and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP).
  • pemphigus pemphigus vulgaris, pemphigus foliaceus or paraneoplastic pemphigus
  • Crohn's disease idiopathic thrombocytopenic purpura (ITP), heparin induced thrombocytopenia (HIT), thrombotic thrombocytopenic purpura (TTP), Myasthenia Gravis (MG), and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP).
  • autoimmune diseases include autoimmune thrombocytopenia, immune neutropenia, antihemophilic FVIII inhibitor, antiphospholipid syndrome, Kawasaki Syndrome, ANCA-associated disease, polymyositis, bullous pemphigoid, multiple sclerosis (MS), Guillain-Barre Syndrome, chronic polyneuropathy, ulcerative colitis, diabetes mellitus, autoimmune thyroiditis, Graves' opthalmopathy, rheumatoid arthritis, ulcerative colitis, primary sclerosing cholangitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, Hashimoto's thyroiditis, Goodpasture's syndrome, autoimmune hemolytic anemia, scleroderma with anticollagen antibodies, mixed connective tissue disease, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis (e.g., crescentic glomerul
  • the autoimmune diseases include hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune uveoretinitis, glomerulonephritis, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, autoimmune urticarial neuropathy, autoimmune axonal neuropathy, Balo disease, Behcet's disease, Castleman disease, celiac disease, Chagas disease, chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome,
  • administering introducing
  • transplanting are used interchangeably in the context of the placement of described cells, e.g. hematopoietic progenitor cells, into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced.
  • the cells e.g. hematopoietic progenitor cells, or their differentiated progeny (e.g., T cells) can be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.
  • the engineered immune cells described herein are optionally expanded ex vivo prior to administration to a subject. In other embodiments, the engineered immune cells are optionally cryopreserved for a period, then thawed prior to administration to a subject.
  • the engineered immune cells used for cellular replacement therapy can be autologous/autogenic ("self) or non-autologous ("non-self,” e.g., allogeneic, syngeneic or xenogeneic) in relation to the recipient of the cells.
  • autologous refers to cells from the same subject.
  • Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
  • “Syngeneic,” as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison.
  • "Xenogeneic,” as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells of the invention are allogeneic.
  • the engineered immune cell described herein that is to be implanted into a subject in need thereof is autologous or allogeneic to the subject.
  • the engineered immune cell described herein can be derived from one or more donors, or can be obtained from an autologous source. In some embodiments, the engineered immune cells are expanded in culture prior to administration to a subject in need thereof. [00377] In various embodiments, the engineered immune cell described herein can be derived from one or more donors, or can be obtained from an autologous source.
  • the recipient subject prior to implantation, is treated with chemotherapy and/or radiation.
  • the chemotherapy and/or radiation is to reduce endogenous stem cells to facilitate engraftment of the implanted cells.
  • the engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein are treated ex vivo with prostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) to promote subsequent engraftment in a recipient subject.
  • NAC N-acetyl-L-cysteine
  • the recipient subject is a human.
  • the subject has been previously diagnosed with HIV or other viral disease, a hematological disease, or undergoing a cancer treatment.
  • a subject is selected to donate a somatic cell which would be used to produce iPSCs and an engineered immune cell described herein.
  • the selected subject has a genetic disease or defect.
  • the donor subject is a human, non-human animal, rodent or nonrodent.
  • the subject can be any mammal, e.g., a human, other primate, pig, rodent such as mouse or rat, rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep or goat, or a non-mammal such as a bird.
  • the donor has been previously diagnosed with HIV, a hematological disease or cancer.
  • a biological sample a population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells is obtained from the donor subject.
  • the biological sample, a population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells described herein can be derived from one or more donors, or can be obtained from an autologous source.
  • the embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, hematopoietic progenitor cells are isolated from the donor subject, transfected, cultured (optional), and transplanted back into the same subject, i.e. an autologous cell transplant.
  • the donor and the recipient subject is the same individual.
  • the embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor who is an HLA- type match with a subject (recipient).
  • HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. These represent the minimum number of cell surface antigen matching required for transplantation. That is the transfected cells are transplanted into a different subject, i.e., allogeneic to the recipient host subject.
  • the donor’s or subject’s embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells can be transfected with a vector or nucleic acid comprising the nucleic acid molecule(s) described herein, the transfected cells are cultured, inhibited, and differentiated as disclosed, optionally expanded, and then transplanted into the recipient subject.
  • the transplanted engineered immune cells engraft in the recipient subject.
  • the transplanted engineered immune cells reconstitute the immune system in the recipient subject.
  • the transfected cells can also be cryopreserved after transfected and stored, or cryopreserved after cell expansion and stored.
  • the engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein may be administered as part of a bone marrow or cord blood transplant in an individual that has or has not undergone bone marrow ablative therapy.
  • genetically modified cells contemplated herein are administered in a bone marrow transplant to an individual that has undergone chemoablative or radioablative bone marrow therapy.
  • a dose of cells is delivered to a subject intravenously.
  • the cells are intravenously administered to a subject.
  • patients receive a dose of the modified cells described herein, e.g., engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein, of about 1 x 10 5 cells/kg, about 5 x 10 5 cells/kg, about 1 x 10 6 cells/kg, about 2 x 10 6 cells/kg, about 3 x 10 6 cells/kg, about 4 x 10 6 cells/kg, about 5 x 10 6 cells/kg, about 6 x 10 6 cells/kg, about 7 x 10 6 cells/kg, about 8 x 10 6 cells/kg, about 9 x 10 6 cells/kg, about 1 x 10 7 cells/kg, about 5 x 10 7 cells/kg, about 1 x 10 8 cells/kg, or more in one single intravenous dose.
  • the modified cells described herein e.g., engineered immune cells or the histone methyltransferase inhibite
  • patients receive a dose of the modified cells described herein, e.g., engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein, of at least 1 x 10 5 cells/kg, at least 5 x 10 5 cells/kg, at least 1 x 10 6 cells/kg, at least 2 x 10 6 cells/kg, at least 3 x 10 6 cells/kg, at least 4 x 10 6 cells/kg, at least 5 x 10 6 cells/kg, at least 6 x 10 6 cells/kg, at least 7 x 10 6 cells/kg, at least 8 x 10 6 cells/kg, at least 9 x 10 6 cells/kg, at least 1 x 10 7 cells/kg, at least 5 x 10 7 cells/kg, at least 1 x 10 8 cells/kg, or more in one single intravenous dose.
  • the modified cells described herein e.g., engineered immune
  • patients receive a dose of the modified cells described herein, e.g., engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein, of about 1 x 10 5 cells/kg to about 1 x 10 8 cells/kg, about 1 x 10 6 cells/kg to about 1 x 10 8 cells/kg, about 1 x 10 6 cells/kg to about 9 x 10 6 cells/kg, about 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, about 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, about 2 x 10 6 cells/kg to about 8 x 10 6 cells/kg, about 2 x 10 6 cells/kg to about 5 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 5 x 10 6 cells/kg, about 3 x 10 6 cells/kg to about 4 x 10 8 cells/kg, or
  • the engineered immune cells or the histone methyltransferase inhibited, multilineage hematopoietic progenitor cell described herein or T cells differentiated using a stroma- free method as described herein are administered as a suspension with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, as therapeutic compositions.
  • Therapeutic compositions contain a physiologically tolerable carrier together with the cell composition and optionally at least one additional bioactive agent as described herein, dissolved or dispersed therein as an active ingredient.
  • the therapeutic composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired.
  • a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject.
  • a formulation comprising cells can include e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and/or can be adapted for use with the cells as described herein using routine experimentation.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
  • the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation.
  • compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the preparation can also be emulsified or presented as a liposome composition.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • an active agent used in the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field of art.
  • a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
  • the “pharmaceutically acceptable” carrier does not include in vitro cell culture media.
  • composition of engineered immune cells described further comprises a pharmaceutically acceptable carrier.
  • a second or subsequent dose of cells is administered to the recipient subject.
  • a second administration can be given between about one day to 30 weeks from the previous administration.
  • Two, three, four or more total subsequent administrations can be delivered to the individual, as needed, e.g., determined by a skilled clinician.
  • a cell composition can be administered by any appropriate route which results in effective cellular replacement treatment in the subject, i.e. administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, i.e. at least 1 x 10 4 cells are delivered to the desired site for a period of time.
  • Modes of administration include injection, infusion, or instillation, “Injection” includes, without limitation, intravenous, intra-arterial, intraventricular, intracardiac injection and infusion.
  • injection or infusion is generally preferred.
  • Efficacy testing can be performed during the course of treatment using the methods described herein. Measurements of the degree of severity of a number of symptoms associated with a particular ailment are noted prior to the start of a treatment and then at later specific time period after the start of the treatment.
  • a pharmaceutical composition comprising an immune as described herein or a population thereof can be used for cellular replacement therapy in a subject.
  • compositions of modified (also referred to as engineered) cells for use in in vivo cellular replacement therapy, medical therapy such as cancer immune therapy, and for the in vitro studies of disease modeling, drug screening, and hematological diseases.
  • the advantage of the disclosure protocols is the methods permit semi-permanent bulk production of desired immune cells or other types of hematopoietic cells (i.e. cells differentiated from multipotent HSCs,) from a variety of types of cell source, from stem cells, hematopoietic progenitor cells, and mature and differentiated somatic cells, all of which can be readily collected from the patient's body.
  • desired immune cells or other types of hematopoietic cells i.e. cells differentiated from multipotent HSCs, from a variety of types of cell source, from stem cells, hematopoietic progenitor cells, and mature and differentiated somatic cells, all of which can be readily collected from the patient's body.
  • the produced engineered immune cells or engineered histone methyltransferase- inhibited, CD34 + /CD 38 lo/_ hematopoietic progenitor cells (e.g., hemogenic endothelium) or T cells differentiated using a stroma-free method as described herein can be transplanted into a patient for various medical treatments such as immune system reconstruction therapy (e.g., after bone marrow ablation) or immunotherapy (e.g., in cancer therapy or autoimmune diseases).
  • immune system reconstruction therapy e.g., after bone marrow ablation
  • immunotherapy e.g., in cancer therapy or autoimmune diseases.
  • One added advantage is that if the donor of the source cells and recipient of the engineered immune cells are the same person, the produced engineered immune cells have HLA that are identical to the recipient and this avoids host-graft immune rejection after the transplantation. For recipient patients that are HLA allogeneic to the donor person of the source cells, host-graft immune rejection is greatly reduced.
  • the produced engineered immune cells or engineered histone methyltransferase- inhibited, CD34+/CD 38- hematopoietic progenitor cells or T cells differentiated using a stroma-free method as described herein can also be cryopreserved till needed in the future.
  • HSC hematopoietic stem cell
  • HSCs human pluripotent stem cells
  • a method of cellular replacement therapy comprising administering an immune cell as described herein or population thereof, or a composition comprising said immune cell or population thereof, or a pharmaceutical composition comprising said immune cell or population thereof to a recipient subject in need thereof.
  • the recipient subject has undergone chemotherapy and/or irradiation.
  • the recipient subject has deficiencies in immune function and/or lymphocyte reconstitution.
  • the immune cell or population thereof is treated ex vivo with prostaglandin E2 and/or antioxidant N-acetyl-L-cysteine (NAC) to promote subsequent engraftment in a recipient subject.
  • NAC N-acetyl-L-cysteine
  • kits for differentiating T cells using a stroma-free method as described herein relate to kits for differentiating T cells using a stroma-free method as described herein, among others. Described herein are kit components that can be included in one or more of the kits described herein.
  • the kit comprises an effective amount of CD3+ T-cell differentiation factors (e.g., IL-7, SCF, FLT3, and/or TPO); or an effective amount of iPSC differentiation factors (e.g., OCT4, SOX2, KLF4, c-MYC, nanog, and/or LIN28); or an effective amount of hemogenic endothelium differentiation factors (e.g., BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL-11, IGF-1, SCF, and EPO); or an effective amount of single-positive T-cell differentiation factors (e.g., IL- 15 and/or a T cell activator such as a CD3/CD28 T cell activator); or an effective amount of an inhibitor of an epigenetic regulator (e.g., MC1568; CAY10591; UNC0224; UNC0638; A366; BRD4770; BIX01294; UNC0642
  • an epigenetic regulator e
  • such cell differentiation factors can be supplied in a lyophilized form or a concentrated form that can diluted prior to use with cultured cells.
  • Preferred formulations include those that are nontoxic to the cells and/or does not affect growth rate or viability etc.
  • T-cell differentiation factors can be supplied in aliquots or in unit doses.
  • the kit comprises a soluble Notch ligand or composition thereof. In some embodiments, the kit does not comprise stromal cells as described herein.
  • the kit further comprises a vector comprising a nucleic acid encoding a CAR.
  • kits can optionally include one or more agents that permit the detection of markers for T cell maturation (e.g., CD5, CD7, CD3, CD4, CD8, TCRgd, TCR alpha or beta, etc.) or a set thereof.
  • markers for T cell maturation e.g., CD5, CD7, CD3, CD4, CD8, TCRgd, TCR alpha or beta, etc.
  • kits can optionally include one or more agents that permit the detection of markers for T cell activation (e.g., CD 107a, CD69, CD25, HLA-DR, IFNg, TNFa, etc.) or a set thereof.
  • Such kits can optionally include one or more agents that permit the detection of markers for hemogenic endothelium (e.g., CD34, CD38, CD45, KDR, CD235, CD43, etc.).
  • the kit optionally comprises informational material.
  • the kit can also contain a substrate for coating culture dishes, such as laminin, fibronectin, Poly-L-Lysine, or methylcellulose.
  • the compositions in the kit can be provided in a watertight or gas tight container which in some embodiments is substantially free of other components of the kit.
  • a cell differentiation reagent can be supplied in more than one container, e.g., it can be supplied in a container having sufficient reagent for a predetermined number of differentiation assays, e.g., 1, 2, 3 or greater.
  • One or more components as described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the components described herein are substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of soluble Notch ligand; or the production of T cells differentiated using a stroma-free method as described herein; or the concentration, date of expiration, batch or production site information, and so forth of reagents used herein such as cell differentiation factors.
  • the informational material relates to methods for using or administering the components of the kit.
  • the kit can include a component for the detection of a marker for cell differentiation.
  • the kit can include one or more antibodies that bind a cell marker, or primers for an RT-PCR or PCR reaction, e.g., a semi-quantitative or quantitative RT-PCR or PCR reaction.
  • Such components can be used to assess the activation of cell maturation markers or the loss of undifferentiated or immature cell markers.
  • the detection reagent is an antibody, it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the antibody or other detection reagent can be linked to a label, e.g., a radiological, fluorescent (e.g., GFP) or colorimetric label for use in detection.
  • the detection reagent is a primer, it can be supplied in dry preparation, e.g., lyophilized, or in a solution.
  • the kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a Styrofoam box.
  • the enclosure can be configured so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.
  • the term “monomer” refers to one of the basic structural units of an oligomer described herein, i.e., a single DLL4 ligand.
  • oligomer or “oligomeric complex” is used interchangeably herein and refers to a molecule possessing at least two monomers.
  • the architecture of an oligomer can vary.
  • Specific oligomers for use in the invention include a dimeric, trimeric, tetrameric, pentameric, hexameric, octameric, icosahedral or any other higher oligomeric state.
  • a single repeating structural unit forms the oligomer (i.e., only DLL4 monomers).
  • two or more structural units are repeated — either in a pattern or randomly — to form the oligomer (i.e., at least DLL1 and DLL4).
  • scaffold refers to a structure, either naturally occurring or synthetically produced, capable of immobilizing at least to monomers to form a oligomeric complex.
  • the term “cell” refers to a single cell as well as to a population of (i.e., more than one) cells.
  • the population may be a pure population comprising one cell type, such as a population of pluripotent stem cells or a population of differentiated T cells.
  • the term “population” refers to a pure population or to a population comprising a majority (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%) of one cell type.
  • the population may comprise more than one cell type, for example a mixed cell population. It is not meant to limit the number of cells in a population; for example, a mixed population of cells may comprise at least one differentiated cell. In the present invention, there is no limit on the number of cell types that a mixed cell population may comprise.
  • the term “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and also give rise to all the blood cell types of the three hematopoietic lineages, erythroid, lymphoid, and myeloid. These cell types include the myeloid lineages (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and the lymphoid lineages (T-cells, B-cells, NK-cells).
  • myeloid lineages monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • T-cells, B-cells, NK-cells lymphoid lineages
  • Human HSCs are determined as CD34 + , CD59 + , CD90/Thyl + , CD38 low/ ", c-kit/CDl 17" /low , and Lin".
  • Mouse HSC- are considered CD34 low/ ", SCA-U, CD90/Thyl +/low , CD38 + , c-Kit/CDl 17 + , and Lin". Detecting the expression of these marker panels allows separation of specific cell populations via techniques like fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the term “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and that have the following cell surface markers: CD34+, CD59+, Thyl/CD90 + , CD38 lo/ ’, CD133+, c-Kit/CDl 17 /10 , and Lin’.
  • the term “hematopoietic stem cell” or “HSC” refers to a stem cell that is at least CD34+.
  • the term “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and that is at least CD34 + and c-kit/CDl 17 lo/_ .
  • the term “hematopoietic stem cell” or “HSC” refers to a stem cell that has self-renewal capacity and that is at least CD38 low/_ , c-kit/CDl 17 /low .
  • HSC can be used interchangeably with the term “hematopoietic stem and progenitor cell” (HSPC).
  • iPS cell As used herein, the terms “iPS cell”, “iPSC”, and “induced pluripotent stem cell” are used interchangeably and refers to a pluripotent cell artificially derived by the transfection of the following reprogramming factors OCT4, SOX2, KLF4, and optionally c-MYC or nanog and LIN28, from a differentiated cell, e.g., a somatic cell.
  • reprogramming factors include OCT4, SOX2, NANOG, and LIN28.
  • hPSC refers to a human pluripotent stem cell.
  • the term “lineage” when used in the context of stem and progenitor cell differentiation and development refers to the cell differentiation and development pathway, which the cell can take to becoming a fully differentiated cell.
  • a HSC has three hematopoietic lineages, erythroid, lymphoid, and myeloid; the HSC has the potential, i.e., the ability, to differentiate and develop into those terminally differentiated cell types known for all these three lineages.
  • multilineage used, it means the cell is able to, in the future, differentiate and develop into those terminally differentiated cell types known for more than one lineage.
  • the HSC has multilineage potential.
  • limited lineage means the cell can differentiate and develop into those terminally differentiated cell types known for one lineage.
  • CMP myeloid progenitor cell
  • MEP megakaryocyte -erythroid progenitor
  • Terminally differentiated cells of the myeloid lineage include erythrocytes, monocytes, macrophages, megakaryocytes, myeloblasts, dendritic cells, and granulocytes (basophils, neutrophils, eosinophils, and mast cells); and terminally differentiated cells of the lymphoid lineage include T lymphocytes/ T cells, B lymphocytes/B cells, dendritic cells, and natural killer cells.
  • a progenitor cell refers to an immature or undifferentiated cell that has the potential later on to mature (differentiate) into a specific cell type (a fully differentiated or terminally differentiated cell), for example, a blood cell, a skin cell, a bone cell, or hair cells.
  • a specific cell type a fully differentiated or terminally differentiated cell
  • Progenitor cells have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell, which it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential.
  • Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate. A progenitor cell also can proliferate to make more progenitor cells that are similarly immature or undifferentiated.
  • differentiated cell is meant any primary cell that is not, in its native form, pluripotent as that term is defined herein.
  • the term a “differentiated cell” also encompasses cells that are partially differentiated, such as multipotent cells (e.g. adult somatic stem cells).
  • the term “differentiated cell” also refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., from an undifferentiated cell or a reprogrammed cell) where the cell has undergone a cellular differentiation process.
  • a reprogrammed cell as this term is defined herein, can differentiate to lineage-restricted precursor cells (such as a mesodermal stem cell or a endodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as an tissue specific precursor, for example, a cardiomyocyte precursor, or a pancreatic precursor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • lineage-restricted precursor cells such as a mesodermal stem cell or a endodermal stem cell
  • precursor cells such as a mesodermal stem cell or a endodermal stem cell
  • other types of precursor cells such as an tissue specific precursor, for example, a cardiomyocyte precursor, or a pancreatic precursor
  • end-stage differentiated cell which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • multipotent when used in reference to a “multipotent cell” refers to a cell that is able to differentiate into some but not all of the cells derived from all three germ layers. Thus, a multipotent cell is a partially differentiated cell. Multipotent cells are well known in the art, and examples of multipotent cells include adult somatic stem cells, such as for example, hematopoietic stem cells and neural stem cells, hair follicle stem cells, liver stem cells etc. Multipotent means a stem cell may form many types of cells in a given lineage, but not cells of other lineages.
  • a multipotent blood stem cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons; cardiovascular progenitor cell (MICP) differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types; pancreas-derived multipotent progenitor (PMP) colonies produce cell types of pancreatic lineage (cells that produces insulin, glucagon, amylase or somatostatin) and neural lineage (cells that are morphologically neuron-like, astrocytes-like or oligodendrocyte-like).
  • MIMP cardiovascular progenitor cell
  • a "reprogramming gene” refers to a gene whose expression, contributes to the reprogramming of a differentiated cell, e.g. a somatic cell to an undifferentiated cell (e.g. a cell of a pluripotent state or partially pluripotent state, multipotent state).
  • a reprogramming gene can be, for example, genes encoding master transcription factors Sox2, Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like.
  • the term "reprogramming factor” refers to the protein encoded by the reprogramming gene.
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein refers to a nucleic acid (e.g. a nucleic acid encoding a reprogramming transcription factor, e.g. Sox2, Oct3/4, Klf4, Nanog, Lin-28, c-myc and the like) or a protein (e.g., a transcription factor polypeptide) that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in lower amounts.
  • a substance e.g.
  • nucleic acid encoding a sox2 transcription factor, or a protein, e.g., a SOX2 polypeptide will be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance.
  • isolated signifies that the cells are placed into conditions other than their natural environment.
  • isolated does not preclude the later use of these cells thereafter in combinations or mixtures with other cells.
  • expanding refers to increasing the number of like cells through cell division (mitosis).
  • proliferating and “expanding” are used interchangeably.
  • a “cell-surface marker” refers to any molecule that is expressed on the surface of a cell.
  • Cell-surface expression usually requires that a molecule possesses a transmembrane domain.
  • Some molecules that are normally not found on the cell-surface can be engineered by recombinant techniques to be expressed on the surface of a cell.
  • Many naturally occurring cell-surface markers are termed “CD” or “cluster of differentiation” molecules.
  • Cell-surface markers often provide antigenic determinants to which antibodies can bind to.
  • a cell-surface marker of particular relevance to the methods described herein is CD34.
  • the useful hematopoietic progenitor cells e.g., hemogenic endothelium
  • the useful hematopoietic progenitor cells e.g., hemogenic endothelium
  • the useful hematopoietic progenitor cells e.g., hemogenic endothelium
  • CD34 preferably express CD34 or in other words, they are CD
  • a cell can be designated “positive” or “negative” for any cell-surface marker, and both such designations are useful for the practice of the methods described herein.
  • a cell is considered “positive” for a cell-surface marker if it expresses the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker, and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell. It is to be understood that while a cell may express messenger RNA for a cell-surface marker, in order to be considered positive for the methods described herein, the cell must express it on its surface.
  • a cell is considered “negative” or “negative/low” (abbreviated as “-/lo” or “lo/-”) for a cell-surface marker if the cell does not express the marker on its cell surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell.
  • the agents can all comprise the same label or tag, such as fluorescent tag, and thus all cells positive for that label or tag can be excluded or removed, to leave uncontacted hematopoietic stem or progenitor cells for use in the methods described herein.
  • a histone methyltransferase inhibitor or “inhibitor” is any molecule that inhibits of expression of a histone methyltransferase (e.g., G9a, GLP, EZH1), or inhibits the catalytic activity of the enzyme to methylate lysine resides on the substrate histone protein.
  • a histone methyltransferase inhibitor can be an siRNA or dsRNA that inhibits of expression of G9a, GLP, or EZH1 in the inhibited cell, or a gRNA that promotes the degradation of the mRNA of G9a, GLP, or EZH1 in the inhibited cell.
  • a histone methyltransferase inhibitor is a small molecule that antagonizes the enzyme activity.
  • examples include but are not limited to small molecules AMI-1, A-366, BIX-01294, BIX01338, BRD4770, chaetocin, UNC0224, UNC0631, UNC0638, UNC0642, UNC0646, EPZ5676, EPZ005687, GSK343, EPZ-6438, 3-deazaneplanocin A (DZNeP) HC1, UNC1999, MM-102, SGC 0946, Entacapone, EPZ015666, UNC0379, EIl, MI-2 (Menin-MLL Inhibitor), MI-3 (Menin-MLL Inhibitor), PFI-2, GSK126, EPZ004777, BRD4770, and EPZ-6438 as described herein.
  • DZNeP 3-deazaneplanocin A
  • small molecule refers to a chemical agent including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • the small molecule is a heterorganic compound or an organometallic compound.
  • inhibitory RNA is meant to include a nucleic acid molecule that contains a sequence that is complementary to a target nucleic acid (e.g., a target microRNA) that mediates a decrease in the level or activity of the target nucleic acid.
  • a target nucleic acid e.g., a target microRNA
  • Non-limiting examples of inhibitory RNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, and antisense oligonucleotides. Methods of making inhibitory RNAs are described herein. Additional methods of making inhibitory RNAs are known in the art.
  • the G9a/GLP or EZH1 microRNA described herein is an inhibitory RNA that causes a decrease in the activity of G9a/GLP or EZH1 mRNA.
  • an interfering RNA refers to any double stranded or single stranded RNA sequence, capable - either directly or indirectly (i.e., upon conversion) of inhibiting or downregulating gene expression by mediating RNA interference.
  • Interfering RNA includes, but is not limited to, small interfering RNA ("siRNA”) and small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • RNA interference refers to the selective degradation of a sequence-compatible messenger RNA transcript.
  • an shRNA small hairpin RNA refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem.
  • the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer is a member of the RNase III family.
  • post-transcriptional processing refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and/or Drosha.
  • a "small interfering RNA” or “siRNA” as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner.
  • the small RNA can be, for example, about 18 to 21 nucleotides long.
  • Each siRNA duplex is formed by a guide strand and a passenger strand.
  • the endonuclease Argonaute 2 (Ago 2) catalyzes the unwinding of the siRNA duplex. Once unwound, the guide strand is incorporated into the RNA Interference Specificity Complex (RISC), while the passenger strand is released.
  • RISC uses the guide strand to find the mRNA that has a complementary sequence leading to the endonucleolytic cleavage of the target mRNA.
  • Retroviruses are RNA viruses that utilize reverse transcriptase during their replication cycle.
  • the term "retrovirus” refers to any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus.
  • type c retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus.
  • the retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase.
  • This double-stranded DNA form of the virus is capable of being integrated into the chromosome of the infected cell; once integrated, it is referred to as a “provirus.”
  • the provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules, which encode the structural proteins and enzymes needed to produce new viral particles.
  • LTRs Long terminal repeats
  • LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication.
  • the LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome.
  • the viral LTR is divided into three regions called U3, R and U5.
  • the U3 region contains the enhancer and promoter elements.
  • the U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence.
  • the R (repeat) region is flanked by the U3 and U5 regions.
  • the promoter within the LTR, including the 5' LTR is replaced with a heterologous promoter.
  • heterologous promoters examples include, for example, a spleen focus-forming virus (SFFV) promoter, a tetracycline-inducible (TET) promoter, a P-globin locus control region and a [3-globin promoter (LCR), and a cytomegalovirus (CMV) promoter.
  • SFFV spleen focus-forming virus
  • TET tetracycline-inducible
  • LCR [3-globin promoter
  • CMV cytomegalovirus
  • lentivirus refers to a group (or genus) of retroviruses that give rise to slowly developing disease.
  • Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephal
  • HIV human immuno
  • viruses Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes, i.e., T-cells.
  • R region refers to the region within retroviral LTRs beginning at the start of the capping group (i.e., the start of transcription) and ending immediately prior to the start of the poly A tract.
  • the R region is also defined as being flanked by the U3 and U5 regions. The R region plays an important role during reverse transcription in permitting the transfer of nascent DNA from one end of the genome to the other.
  • promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
  • the enhancer/promoter may be “endogenous,” “exogenous,” or “heterologous.”
  • An “endogenous” enhancer/promoter is one which is naturally linked with a given gene in the genome.
  • An “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA, iRNA, miRNA, siRNA, etc.
  • the nucleic acid can be selected, for example, from a group including: nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), and locked nucleic acid (LNA).
  • PNA peptide-nucleic acid
  • pc-PNA pseudo-complementary PNA
  • LNA locked nucleic acid
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, microRNAi (miRNA), and antisense oligonucleotides.
  • the term “engraftment” in reference to a recipient host is when the new blood-forming cells start to grow and which are derived from the implanted cells and make healthy blood stem cells that show up in recipient’s blood after a minimum period of 10 days after implantation. Engraftment can occur as early as 10 days after transplant but is more common around 14-20 days.
  • substitution with respect to the immune system or the blood system in a recipient host refers to the rebuilding the innate reservoir or working system, or part thereof within the body of recipient host to a natural or a functionally state.
  • a functionally state For example, such as bone marrow after chemotherapy had obliterated the bone marrow stem cells.
  • the absence of a given treatment or agent can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cellular replacement therapy.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. hematologic disease, cancer, etc.) or one or more complications related to such a condition, and optionally, have already undergone treatment for a hematologic disease or the one or more complications related to a hematologic disease.
  • a subject can also be one who has not been previously diagnosed as having a hematologic disease or one or more complications related to a hematologic disease.
  • a subject can be one who exhibits one or more risk factors for a hematologic disease or one or more complications related to a hematologic disease or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • a variant amino acid or DNA sequence can be at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
  • Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al.
  • Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.
  • expression refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing.
  • Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
  • the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic.
  • “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.
  • the term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.
  • the gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • a polypeptide, nucleic acid, or cell as described herein can be engineered.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • the differentiated and/or engineered T cell described herein is exogenous. In some embodiments, the differentiated and/or engineered T cell described herein is ectopic. In some embodiments, the differentiated and/or engineered T cell described herein is not endogenous.
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • endogenous refers to a substance that is native to the biological system or cell.
  • ectopic refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
  • Nucleic acids encoding a polypeptide as described herein can be comprised by a vector.
  • the term "vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non-viral.
  • the term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • the vector can be recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments, the vector comprises sequences originating from at least two different species. In some embodiments, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
  • non-native e.g., heterologous
  • the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system.
  • the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism).
  • the vector and/or nucleic acid sequence described herein is codon- optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments, the vector and/or nucleic acid sequence described herein is codon- optimized for expression in a human cell. In some embodiments, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.
  • sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • Non-limiting examples of a viral vector of this invention include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector a baculovirus vector, and a chimeric virus vector.
  • a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. a hematological disease or cancer.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a hematological disease or cancer.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • administering refers to the placement of any therapeutic described herein, e.g., a cell population, into a subject by a method or route which results in at least partial delivery of the therapeutic at a desired site.
  • Pharmaceutical compositions comprising the compounds or cells disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid.
  • Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • a soluble notch ligand oligomer complex comprising at least two Notch ligand monomers and a scaffold.
  • Notch ligand monomer is selected from the group consisting of Delta-like- 1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged 1 (JAG1), and Jagged 2 (JAG2).
  • oligomer complex of any one of the preceding paragraphs, wherein the complex is dimeric, trimeric, tetrameric, pentameric, hexameric, octameric, icosahedral or any other higher oligomeric state.
  • composition comprising the oligomer complex of any one of the preceding paragraphs.
  • a method of producing a soluble Notch ligand oligomer complex comprising contacting a plurality of Notch ligand monomers with a population of scaffolds for a time sufficient to promote formation of a complex.
  • the plurality of Notch ligand monomers further comprises a GS linker. 14. The method of any one of the preceding paragraphs, wherein the plurality of Notch ligand monomers is fused to SpyTag via the GS linker.
  • a soluble Notch ligand oligomer complex produced from the method of any one of the preceding paragraphs.
  • a method of activating Notch signaling in a population of cells comprising contacting a population of cells with the soluble Notch ligand oligomer complex of any one of the preceding paragraphs, or the composition of any one of the preceding paragraphs.
  • a method comprising: a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; b) inhibiting a histone methyltransferase in the resultant population of CD34 + hemogenic endothelium; and c) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + - T-cell differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3 + T cells.
  • a method comprising: a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; b) inhibiting an epigenetic regulator in the resultant population of CD34 + hemogenic endothelium; and c) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + - T-cell differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3 + T cells.
  • a method comprising: a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; b) inhibiting G9a and/or GLP in the resultant population of CD34 + hemogenic endothelium; and c) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + - T-cell differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3 + T cells.
  • a method comprising: a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + - T-cell-differentiation media in the presence of the soluble Notch ligand for a sufficient time to promote differentiation into a population of CD3 + T cells.
  • soluble Notch ligand is soluble Delta-like-1 (DLL1), Delta-like-3 (DLL3), Delta-like-4 (DLL4), Jagged 1 (JAG1), or Jagged 2 (JAG2).
  • the CD3 + -T-cell- differentiation media comprises 15 ng/ml FLT3 and 25 ng/ml IL7.
  • the CD3 + -T-cell- differentiation media further comprises 5 ng/mL thrombopoietin (TPO) and/or 30 ng/ml SCF for at least the first 2 weeks of differentiating in the CD3 + -T-cell- differentiation media.
  • TPO thrombopoietin
  • SCF 30 ng/ml SCF
  • the population of CD3 + T cells comprises a population of CD4 + CD8 + T cells.
  • the method of any one of the preceding paragraphs further comprising differentiating the population of CD4 + CD8 + T cells in a single-positive-T-cell- differentiation media for a sufficient time to promote differentiation into a population of CD4 + cells and a population of CD8 + cells.
  • the method of any one of the preceding paragraphs, wherein the sufficient time to promote differentiation from the population of CD4 + CD8 + T cells into a population of CD4 + T cells and a population of CD8 + cells is at least 1 week.
  • the method of any one of the preceding paragraphs wherein the sufficient time to promote differentiation from the population of CD34 + hemogenic endothelium into a population of CD4 + T cells and a population of CD8 + cells is at least 5 weeks.
  • the single-positive-T- ce 11 -differentiation media comprises 10 ng/mL IL- 15 and a T cell activator.
  • the T cell activator comprises a lOul/ml CD3/CD28 T cell activator.
  • the T cell activator comprises one bead of CD3/CD28 T cell activator dynabeads per cell.
  • the method of any one of the preceding paragraphs further comprising, after at least 1 week, a step of CD4 + cell enrichment and/or CD8 + cell enrichment.
  • the population of pluripotent stem cells comprises induced pluripotent stem cells (iPS cells) or embryonic stem cells (ESC).
  • iPS cells induced pluripotent stem cells
  • ESC embryonic stem cells
  • the induced pluripotent stem cells are produced by introducing only reprogramming factors OCT 4, S0X2, KLF4 and optionally c-MYC or nanog and LIN28 into mature cells.
  • the induced pluripotent stem cells are produced by introducing the reprogramming factors two or more times into the mature cells.
  • the method of any one of the preceding paragraphs, wherein the sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium is at least 8 days.
  • the aggregation media comprises BMP4, SB-431542, CHIR99021, bFGF, VEGF, IL-6, IL- 11, IGF-1, SCF, and EPO.
  • the method of any one of the preceding paragraphs, wherein the aggregation media comprises 10 ng/ml BMP4, 6 mM SB-431542, 3 mM CHIR99021, 5 ng/ml bFGF, 15 ng/ml VEGF, 10 ng/ml IL-6, 5 ng/mL IL-11, 25 ng/mL IGF-1, 50 ng/mL SCF, and 2 U/ml EPO.
  • the method of any one of the preceding paragraphs further comprising selecting or isolating the resultant population of CD34 + hemogenic endothelium using expression of surface markers on the population of CD34 + hemogenic endothelium.
  • the method of any one of the preceding paragraphs wherein the population of CD34 + hemogenic endothelium is CD45 negative/low.
  • the method of any one of the preceding paragraphs, wherein the population of CD34 + hemogenic endothelium is CD38 negative/low.
  • the method of any one of the preceding paragraphs further comprising the step of genetically modifying the resultant population of CD34 + hemogenic endothelium or the resultant population of CD3 + T cells.
  • the genetic modification is editing an endogenous HLA, removing an endogenous TCR, and/or expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the histone methyltransferase catalyzes the addition of methyl group to the histone 3 lysine residue 9 (H3K9) and/or histone 3 lysine residue 27 (H3K27).
  • H3K9 and/or H3K27 is inhibited by a small molecule inhibitor or a nucleic acid inhibitor.
  • the histone methyltransferase H3K9 and/or H3K27 small molecule inhibitor is a heterorganic compound or an organometallic compound.
  • the method of any one of the preceding paragraphs, wherein the histone methyltransferase H3K9 and/or H3K27 small molecule inhibitor is selected from the group consisting of BIX-01294, UNC0638, E72, BRD4770, A-366, chaetocin, UNC0224, UNC0631, UNC0646, EPZ005687, EPZ-6438 (E7438), 3- deazaneplanocin A (DZNep), Ell, GSK343, GSK126, and UNC1999.
  • nucleic acid inhibitor is a nucleic acid targeting the expression of histone methyltransferase.
  • nucleic acid inhibitor is a RNA interference inhibitor or agent.
  • nucleic acid inhibitor is a EZH1 specific nucleic acid that is selected from the group consisting of an aptamer that binds EZH1, a EZH1 specific RNA interference agent, and a vector encoding a EZH1 specific RNA interference agent, wherein the RNA interference agent comprises one or more of the nucleotide sequences selected from SEQ ID NO: 11-19.
  • the epigenetic regulator is a DNA-methyltransferase (DNMT); a methyl-CpG-binding domain (MBD) protein; a DNA demethylase; a histone methyl transferase (HMT); a methyl-histone binding protein; a histone demethylase; a histone acetyl transferase (HAT); an acetyl- binding protein; or a histone deacetylase (HDAC).
  • DNMT DNA-methyltransferase
  • MBD methyl-CpG-binding domain
  • HMT histone methyl transferase
  • HAT histone acetyl transferase
  • HDAC histone deacetylase
  • G9a and/or GLP inhibitor is selected from the group consisting of: UNC0224; UNC0638; A366; BRD4770; BIX01294; UNC0642; UNC0631; UNC0646; UNC0321; E72; BIX- 01338; BRD9539; Chaetocin; and DCG066.
  • a method comprising: a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + - T-cell-differentiation media comprising 15 ng/ml LLT3 and 25 ng/ml IL7 in the presence of a soluble Notch ligand for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises 5 ng/mL TPO and 30 ng/ml SCL for at least the first two weeks.
  • a method comprising: a) differentiating a population of pluripotent stem cells in aggregation media for a sufficient time to promote differentiation into a population of CD34 + hemogenic endothelium; and b) differentiating the resultant population of CD34 + hemogenic endothelium in a CD3 + - T-cell-differentiation media comprising 15 ng/ml LLT3 and 25 ng/ml IL7 in the presence of a soluble Notch ligand for at least 4 weeks to promote differentiation into a population of CD3 + T cells; wherein the CD3 + -T-cell-differentiation media further comprises 5 ng/mL TPO, 30 ng/ml SCL, and a G9a/GLP inhibitor for at least the first two weeks.
  • TCR T cell receptor
  • CDR complementarity-determining region
  • the immune cell of any one of the preceding paragraphs wherein the immune cell exhibits a gene expression profde that is at least 10%, 20%, 30%, 40% or more similar to alpha beta T cells.
  • the immune cell of any one of the preceding paragraphs wherein the immune cell exhibits a T cell receptor (TCR) complementarity-determining region (CDR) that is at least 3 nucleotides longer than an immune cell differentiated without inhibition of methyltransferase, using stromal cells.
  • TCR T cell receptor
  • CDR complementarity-determining region
  • a composition comprising an immune cell of any one of the preceding paragraphs or population thereof.
  • the composition of any one of the preceding paragraphs further comprising a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising an immune cell of any one of the preceding paragraphs or population thereof, and a pharmaceutically acceptable earner. 90.
  • the pharmaceutical composition of any one of the preceding paragraphs for use in cellular replacement therapy in a subject.
  • a method of cellular replacement therapy comprising administering an immune cell of any one of the preceding paragraphs or population thereof, or a composition of any one of the preceding paragraphs, or a pharmaceutical composition of any one of the preceding paragraphs to a recipient subject in need thereof.
  • Notch expression in the cell increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or greater as compared a suitable control.
  • composition comprising a cell of any one of the preceding paragraphs or population thereof.
  • a pharmaceutical composition comprising a cell of any one of the preceding paragraphs or population thereof, and a pharmaceutically acceptable carrier.
  • a method comprising a) inhibiting a histone methyltransferase in a population of CD34 + hemogenic endothelium; and b) differentiating the population of CD34 + hemogenic endothelium in Natural Killer (NK)-ce 11 -differentiation media in the presence of a soluble Notch ligand for a sufficient time to promote differentiation into a population of CD56 + NK cells.
  • NK Natural Killer
  • DLL4 Delta-like ligands
  • Notch activation requires mechanical pulling of the DLL4 ligand by the Notch receptors, and therefore for signaling to occur, the DLL4 ligand needs to be tethered either to a cell surface or in a plate-bound format 11 .
  • Soluble Notch ligand-oligomeric protein complex engineering [00496] It was specifically hypothesized by the inventors that multivalent soluble Notch DLL4 ligands can bring cells together via receptor-ligand interactions across cell surfaces ( Figure IB) and when cells move, they can exert a physical pulling on the receptor, resulting in the activation of Notch signaling pathway.
  • the inventors used a library of computationally designed de novo proteins featuring diverse valencies, sizes, and geometries that could be used as scaffolds to engineer multivalent oligomeric DLL4 complexes 13, 14 . Protein scaffolds that can represent different multivalencies, geometries, and topologies were chosen for ligand presentation.
  • the Icos protein cage is a heteromeric protein complex assembled from the C3 and C5 proteins mentioned above, featuring 20x C3 and 12x C5, thus a total of 120 single chains.
  • oligomeric scaffolds were genetically fused to SC through a flexible GGSGGS linker and expressed and purified in E. coli, whereas the DLL4 protein was fused to ST through a GS linker and expressed and purified in mammalian Expi293F cells.
  • the total number of DLL4 ligands on the cage surface could be either 60 (when either 20x C3 or 12x C5 are conjugated to DLL4) or 120 (when both 20x C3 and 12x C5 are conjugated to DLL4).
  • the resultant protein complexes were fully characterized by using biochemical techniques, namely, native SDS-PAGE and negative staining electron microscopy (EM) before using them for Notch activation experiments.
  • Notch activation using soluble oligomeric DLL4 in a reporter adherent cell line [00499] Once the oligomer-DLL4 protein complexes were formed and characterized, their ability to activate the Notch pathway was investigated. An adherent U2OS reporter cell line engineered to express Notchl fused with Gal4 (U2OS-Notchl-Gal4) was used as a readout of Notch activation. Upon extracellular Notch activation through binding to oligomer-DLL4 complex, Gal4 is released from the Notch 1 to activate the Luciferase gene downstream of a UAS promoter.
  • Notch activation was screened for all the oligomer-DLL4 complexes C3 through Icosl20 at different concentrations and observed that soluble C3-DLL4, Icos60-DLL4, and Icosl20-DLL4 mediated differential Notch pathway activation despite equimolar equivalents of ligand, indicated a geometric specificity for receptor activation ( Figure 2 and 7A-7B). Moreover, the soluble trimeric C3-DLL4 was comparable to plate-bound DLL4, and showed an interesting inverse dose response (Figure 2), the highest activation being at 10 nM concentration. Taken together, C3-DLL4 exhibited the highest activation in the adherent reporter U2OS-Notchl-Gal4 cell system in a concentration-dependent way.
  • Notch signaling is indispensable for the differentiation of T lymphoid cells, and plays a critical role in HSC emergence from hemogenic endothelium.
  • human iPSCs can be efficiently differentiated into hemogenic endothelium, followed by culture on immobilized-DLL4 plates (2D-DLL4) to produce T cells 7 .
  • 2D-DLL4 were replaced with protocols incorporating soluble oligomeric DLL4 complexes to generate T cells.
  • CE - derived T cell progenitors were subjected to different compositions of the oligomer-DLL4 complexes and monitored the production of CD4 and CD8 positive T cells. Confirming the activation of Notch signaling observed in the U2OS reporter line, CD4+/CD8+ T cells were detected in suspension cultures supplemented with trimeric C3-DLL4 complexes, with T cell production dependent on the concentration of C3-DLL4 complex ( Figure 3).
  • Engineered oligomeric DLL4 facilitates cell-cell interaction and subsequent cell clustering
  • C3-DEE4 was added at different concentrations into growing K562 reporter cells and looked for possible cell-cell interaction.
  • C3-DEE4 1 uM
  • cells looked homogenously dispersed in the culture media, however, as the concentration of the protein went down, cells started interacting to form large clumps, each clump containing tens of cells, and the highest clump formation was observed at 10 nM of the protein concentration ( Figure 4A).
  • Figure 4B As the concentration of C3-DLL4 decreased, cells became homogeneously distributed again.
  • Cells that did not receive any C3-DLL4 treatment Figure 4B
  • cells grown on immobilized DLL4 Figure 4C
  • K562 reporter cells were incubated with C3-DLL4 at various concentrations for 1 hour. Prior to the formation of clusters, the binding of C3-DLL4 on the cell surface was assessed using an antibody that stained the 6xHis tag associated with DLL4. It was found that 1 uM concentration of C3-DLL4 stained all the cells and showed the highest fluorescence intensity (Figure 4D). As the concentration of C3-DLL4 decreased, the fluorescence intensity of the cells decreased. At a concentration of 10 nM, two cell populations were observed, one bound to C3-DLL4 and the other not.
  • the bound population had lower fluorescence intensity compared to the one incubated with 1 uM concentration, indicating that the cells at lower C3-DLL4 concentration were not fully saturated with the ligand.
  • free Notch 1 receptors from one cell can interact with a neighboring cell that has unoccupied DLL4 ligands of the C3-DLL4 complex ( Figure 4F).
  • C3-DLL4 can bridge cells to induce cell-cell interaction between K562 reporter cells which finally lead to the formation of cellular clusters.
  • Endogenous Notch activation mediated by engineered soluble oligomeric DLL4 [00509] After demonstrating C3-DLL4 mediated Notch activation in engineered cell lines, the inventors set out to activate Notch in endogenous cell system. Towards this end, two distinct cell lines, breast cancer line MDA-MB-231 and astrocytes line SVG-A, that express endogenous Notch receptors and are known to respond to Notch ligand were selected. The upregulation of Notch targeting genes upon C3-DLL4 (10-50 nM) treatment in both the cell line was investigated. The transcript level of Notch targeting genes, Hesl, Heyl, Hey2, HeyL and NRARP was estimated using RT-qPCR.
  • Notch signaling is indispensable for the development of long-term, definitive Hematopoietic Stem and Progenitor Cells (HSPCs) and T-lymphocytes (T-cells) 1.
  • HSPCs definitive Hematopoietic Stem and Progenitor Cells
  • T-cells T-lymphocytes
  • the inventors have been focused on in vitro generation of therapeutic HSPCs and T-cells from human induced Pluripotent Stem Cells (iPSCs) using various cytokines and growth factors including Notch ligands.
  • Notch ligands, DLL4 or DLL1 have been presented trans to the differentiating cells either in a plate-bound format or from a stromal cell.
  • a soluble Notch ligand in lieu of a plate-bound or stromal presentation, would enable bioreactor scale-up and facilitate T-cell manufacture for clinical studies. Moreover, engineered forms of Notch ligand can be highly informative tools for probing mechanisms of Notch receptor function.
  • Described herein is the development of a soluble oligomeric DLL4 protein complex system that can activate Notch signaling through induced cell-cell interaction. Efficient activation of Notch was demonstrated using soluble DLL4 complexes in both adherent reporter cell lines as well as in manufacturing T-cells from cord blood (CB) derived proT cells. Taken together, the approach described herein is a major step forward to realize streamlined manufacturing of T-cells for adoptive cell therapy.
  • CB cord blood

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